Omics Technologies and Bio-engineering: Volume 2: Towards Improving Quality of Life [1 ed.] 0128158700, 9780128158708

Table of contents :
Dedication
Dedication
List of contributors
List of Contributors
About the editors
About the Editors
Contents 1-5
Contents 5-10
Contents 11-15
Contents 16-20
Chapter 01
1 Microbial Omics: Applications in Biotechnology
1.1 Introduction
1.2 Structural Genomics
1.2.1 Comparative and Pan-Genomics
1.2.2 Immunogenomics
1.2.3 Post-Genomics
1.3 Functional Genomics
1.3.1 Transcriptomics
1.3.2 Proteomics (Interactomics)
1.3.3 Metabolomics
1.4 Conclusions and Perspectives
References
Chapter 02
2 Omics Approaches in Viral Biotechnology: Toward Understanding the Viral Diseases, Prevention, Therapy, and Other Applicat...
2.1 Introduction
2.2 The History of Identification of Viruses at a Molecular Level and Metagenomics
2.3 Advancements in Techniques to Study Virus–Host Interactions
2.3.1 Hybridization
2.3.1.1 Microarray Techniques
2.3.1.2 Subtractive Hybridization
2.3.2 Methods Based on PCR
2.3.2.1 Degenerate PCR
2.3.2.2 Random PCR
2.3.3 Metatranscriptomics Analysis
2.3.3.1 Metagenomics Joined With Metatranscriptomic Analyses
2.3.4 Viral Screening for Development of Therapeutics
2.3.5 Therapy
2.3.5.1 Omics Approach for Elucidating Host and Virus Interaction
2.4 Applications
2.4.1 Cell-Based Screenings
2.4.2 Gain-of-Function Method
2.4.3 Loss-of-Function Method
2.4.4 Comparative Genome Profiling
References
Further Reading
Chapter 03
3 Algal Biotechnology: An Update From Industrial and Medical Point of View
3.1 Microalgae—An Introduction
3.1.1 Biological Importance of Microalgae
3.1.1.1 Foods
3.1.1.2 Feed
3.1.1.3 Fatty Acids
3.1.1.4 Cosmetics
3.1.1.5 Biofertilizers
3.1.1.6 Anticancer Activity
3.1.1.7 Antiviral
3.1.1.8 Antibacterial
3.1.1.9 Antifungal
3.1.1.10 Biofuel
3.1.1.11 CO2 Sequestration
3.1.1.12 Wastewater Treatment
3.1.1.13 Bioremediation/Phycoremediation
3.2 Seaweeds (Macroalgae)—An Introduction
3.2.1 Seaweed Phycocolloids
3.2.2 Phycocolloids of Red Seaweeds
3.2.2.1 Agar and Its Structure
3.2.2.1.1 Carrageenan
3.2.2.2 Polysaccharides of Brown Seaweeds
3.2.2.2.1 Alginate
3.2.2.2.2 Laminarin
3.2.2.2.3 Fucoidan
3.2.2.3 Polysaccharides of Green Seaweeds
3.2.2.3.1 Ulvan
3.2.2.3.2 Food
3.2.2.4 Prebiotic Potential of Polysaccharides Present in Seaweeds
3.2.2.5 Mechanism of Action of Prebiotics
3.2.2.6 Nutraceuticals
3.2.2.7 Cosmetics and Cosmeceuticals
3.2.2.8 Pharmaceuticals
3.2.2.9 Seaweeds as Biological Control Against Animal and Plant Pathogens
3.2.2.10 Bioremediation
3.2.2.11 Pigment Extraction and Production
3.2.2.12 Seaweeds Tissue Culture
3.2.2.13 Drug Delivery
3.2.2.14 Bioenergy
3.2.2.15 Biofuel
3.2.2.16 Biomineralization
3.2.2.17 Bionanocrystallization
3.3 Conclusion
References
Further Reading
Chapter 04
4 Omics Approaches in Fungal Biotechnology: Industrial and Medical Point of View
4.1 Introduction
4.2 Insights Into Fungal Genomics
4.3 Insights Into Fungal Transcriptomics
4.4 Insights Into Fungal Proteomics
4.5 Insights Into Fungal Metabolomics
4.6 Bioinformatics
4.7 Sample Preparation Challenges
4.8 Fungal Omics—A Medical Perspective
4.8.1 Role of Fungi on Immunocompromised Patients
4.8.2 Fungi and the Gut Microflora
4.9 Fungal Omics—An Industrial Perspective
4.10 Conclusion
References
Further Reading
Chapter 05
5 Genetic Engineering for Plant Transgenesis: Focus to Pharmaceuticals
5.1 Introduction
5.2 Plants as Bioreactors
5.2.1 Recombinant Protein Production From Plants
5.2.1.1 Plants for Open-Field and Greenhouse Production of Pharmaceuticals
5.2.1.2 Plant-Based Expression Systems
5.2.1.3 Bioreactor-Based Plant Systems
5.2.1.4 Increasing Heterologous Protein Accumulation in Plants
5.2.1.5 Purification of Recombinant Proteins From Plants
5.3 Plant-Made Pharmaceuticals
5.3.1 Plantibodies
5.3.2 Edible Vaccines
5.3.3 PMPs: Commercial Status
5.4 Chloroplast Genome Engineering for Pharmaceuticals
5.5 Future Directions
References
Chapter 06
6 Agricultural Biotechnology: Engineering Plants for Improved Productivity and Quality
6.1 Introduction of Agricultural Biotechnology
6.1.1 Origin and Definition of Agricultural Biotechnology
6.1.2 Plant Breeding Program
6.1.2.1 Conventional Plant Breeding
6.1.2.2 Modern Plant Breeding
6.1.3 Application of Modern Agriculture
6.1.3.1 Yield Increase
6.1.3.2 Enhancement of Compositional Traits
6.1.3.3 Crop Adaptation
6.2 Genetic Engineering Strategies for Crop Improvement
6.2.1 Introduction of Plant Genetic Modification
6.2.2 Plant Transformation Techniques
6.2.2.1 Physicochemical Methods
6.2.2.2 Biological Methods
6.2.2.2.1 Agrobacterium-Mediated Plant Transformation
6.2.2.2.2 Virus-Mediated Plant Transformation
6.2.2.2.3 In Planta Transformation
6.3 Applications of Genetically Modified Crops
6.3.1 Resistance to Biotic Stress
6.3.1.1 Insect Resistance
6.3.1.1.1 Resistance Gene From Microorganisms
6.3.1.1.2 Resistance Genes From Higher Plants and Animals
6.3.1.2 Disease Resistance
6.3.1.3 Virus Resistance
6.3.2 Resistance to Abiotic Stresses
6.3.2.1 Herbicide Resistance
6.3.2.2 Tolerance to Water-Deficit Stresses
6.4 Genetic Manipulation for Crop Quality
6.4.1 Transgenic for Improved Fruit Storage
6.4.2 Golden Rice
6.4.3 Eco-Social Impact of Genetically Modified Crops
6.4.4 Current Status of GM Plants
6.4.5 Goals of Genetic Engineering in Crop Improvement
6.4.6 Concerns About Transgenic Plants
6.5 Genetic Assisted Plant Breeding
6.5.1 Introduction to Molecular Markers
6.5.1.1 Prerequisites and General Activities of MAB
6.5.2 Variety Identification and Seed Purity Analysis
6.5.2.1 Genetic Distance Analysis
6.5.3 MABC Breeding
6.5.3.1 Nearly Isogenic Strategies
6.5.4 Molecular Markers for Hybrid Vigor
6.6 Future Prospects
References
Chapter 07
7 Functional Food Biotechnology: The Use of Native and Genetically Engineered Lactic Acid Bacteria
7.1 Introduction
7.2 Definitions
7.3 Lactic Acid Bacteria
7.4 Nutraceutical Production by LAB
7.4.1 Vitamins
7.4.2 Bioactive Peptides
7.4.3 Exopolysaccharides
7.4.4 Antioxidant Enzymes
7.4.5 Other Beneficial Enzymes
7.5 Probiotic Effects of LAB
7.5.1 Probiotics in Intestinal Inflammation
7.5.1.1 Probiotics and Their Effects on Host’s Immunity and the Prevention of Infections
7.5.1.2 Probiotics for Obese Hosts
7.5.1.3 Probiotics and Reduction of Cardiovascular Risk
7.5.1.4 Probiotics in Cancer Prevention
7.5.1.5 Probiotics in Healthy Host
7.6 Concluding Remarks
References
Chapter 08
8 Omics and Edible Vaccines
8.1 Introduction: An Overview of Edible Vaccines
8.1.1 Production of Edible Vaccines Using Genomics
8.1.2 Production of Edible Vaccines Using Transcriptomics
8.1.3 Production of Edible Vaccines Using Proteomics
8.1.4 Production of Edible Vaccines Using Metabolomics
8.2 Edible Vaccines
8.2.1 Plasmid/Vector Mediated
8.2.2 Gene Gun or Biolistic Method
8.2.3 Electroporation/Electrotransfection
8.2.4 Lipofection
8.3 Mode of Action of Edible Vaccines
8.4 Conventional Vaccines Versus Edible Vaccines
8.5 Disadvantages of Edible Vaccines
8.6 Applications of Edible Vaccines
8.6.1 Autoimmune Diseases
8.6.2 Gastrointestinal Disorders
8.6.3 Malaria
8.6.4 Measles
8.6.5 Hepatitis B
8.7 Clinical Trials and Research Studies
8.8 Second-Generation Edible Vaccines
8.9 Current Developments
8.9.1 Banana, Tomato, and Potato
8.10 Patents on Edible Vaccines
8.11 Future Prospects
References
Further Reading
Chapter 09
9 Plant Metabolic Engineering
9.1 Introduction
9.1.1 Metabolites
9.1.1.1 Types of Metabolites
9.1.1.2 Importance of Secondary Metabolites
9.2 Metabolic Engineering—A Tool for Creating Desired Diversity
9.3 Approaches and Strategies
9.3.1 Systems for Metabolic Engineering
9.3.1.1 Plant Systems
9.3.1.2 In vitro Cultures
9.3.1.3 Microbial Cells
9.3.2 Management and Modulation of Metabolic Flux
9.3.2.1 Identification of Key Genes
9.3.2.2 Redirecting Flux by Overexpression and Silencing of Genes
9.3.2.3 Diverting Whole Pathway Flux by Regulation of Transcription Factors
9.3.2.4 Diverting Whole Pathway Flux by Using Cis-regulatory Elements
9.3.3 Systems Biology in Plant Metabolic Engineering
9.3.3.1 Strategies of Systems Biology
9.3.3.2 Integration of High-Throughput Omics Experiments
9.3.3.2.1 Genome Based Analysis
9.3.3.2.2 Transcriptome Based Analysis
9.3.3.2.3 Proteome Based Analysis
9.3.3.2.4 Metabolome Based Analysis
9.3.3.3 In silico Modeling and Simulation of Plant Metabolism
9.3.3.3.1 Kinetic Model-Based Analysis
9.3.3.3.2 Flux Model-Based Analysis
9.3.3.3.3 Genome-Scale Model-Based Analysis
9.3.3.4 Tools and Databases for In silico Modeling and Simulation
9.3.3.4.1 Integrated Metabolic Database System
9.3.3.4.2 Integrated Metabolic Networks
9.4 Applications of Metabolic Engineering
9.4.1 In Industry
9.4.2 In Food and Neutraceuticals
9.4.3 In Pharmacy and Medicine
9.4.4 In Agriculture
9.5 Current Status and Limitations
9.6 Future Aspects of Metabolic Engineering
9.7 Conclusions
References
Chapter 10
10 Biocontrol Technology: Eco-Friendly Approaches for Sustainable Agriculture
10.1 Biopesticides Versus Chemical Pesticide: Face to Face
10.2 Biocontrol: Therapy in Organic Farming
10.3 Mechanisms Employed by Biocontrol Agents for Plant Disease Management
10.3.1 Antibiosis
10.3.2 Mycoparasitism
10.3.3 Competition
10.3.4 Induced Resistance in Host Plants
10.4 Strain Improvement of Biocontrol Agents
10.4.1 Mutagenesis
10.4.2 Protoplast Fusion
10.4.3 Transformation
10.5 Omics in Biocontrol Technology
10.5.1 Genomics
10.5.2 Proteomics
10.5.3 Metabolomics
10.5.4 Secretomics
10.6 Conclusion and Future Prospects
10.7 Summary
References
Further Reading
Chapter 11
11 Bioengineering Towards Fighting Against Superbugs
11.1 Introduction
11.1.1 Global Perspective of Microbial Drug Resistance
11.1.2 Human Actions Contributing Towards MDR Development
11.2 Molecular Basis of Resistance
11.2.1 Acquired Resistance
11.2.1.1 Biochemical Inactivation of Drugs
11.3 Industrially Important Drug-Resistant Pathogens
11.3.1 MDR in Tuberculosis
11.3.1.1 Group 1
11.3.1.2 Group 2 (Streptomycin/Capreomycin)
11.3.1.3 Group 3 (FQ)
11.3.1.4 Group 4 (Ethionamide/Prothionamide, and Thiomides)
11.3.1.5 Group 5 (Linezolid and Clofazimine)
11.4 MDR in Pseudomonas aeruginosa
11.5 Drug Resistance in Candida albicans
11.6 MDR in Malaria
11.7 MDR in Herpes Simplex Virus (HSV)
11.8 Strategies to Control AMR
11.8.1 Infection Prevention and Control at Personal and Community Level
11.8.2 Policy, Cost, and Surveillance of MDR Pathogens: Political Commitment
11.8.3 Fostering Innovations
11.9 Biotechnological Interventions to Counter MDR
11.9.1 Nano-Silver: Antimicrobial Agents
11.9.2 Zinc Oxide Nanoparticles as Synergic Antimicrobials
11.10 The Antibacterial Mechanism of Nanoparticles
11.11 Conclusion and Future Perspectives
References
Chapter 12
12 Nanotechnology in Bioengineering: Transmogrifying Plant Biotechnology
12.1 Introduction
12.1.1 What is Plant/Crop Bioengineering?
12.2 Where Nanotechnology Can Help in PB?
12.2.1 Nanocides: NMs as Explant Sterilants in Plant Tissue Culture
12.2.2 Nanovehicles: NMs as Gene/Protein Delivery Vehicles
12.2.2.1 Plant Gene Transformation: What Are the Techniques?
12.2.2.2 Nano-enabled Plant Gene Transformation
12.2.2.3 Nano-enabled Vectorless or Direct Physical Methods
12.2.2.3.1 NM-enabled Transformation
12.2.2.3.2 Nanobiolistics or Nanoprojectile-based Gene Gun Technique
12.2.2.4 Other Nano-enabled Direct Techniques
12.2.2.5 Nano-enabled Chemical Techniques
12.2.2.6 Nano-enabled Vector-mediated or Indirect Gene Transformation Techniques
12.2.3 Nanosequencing: Nanopore-based Gene or Protein Sequencing Tools/Techniques
12.2.4 Nanobioimaging: NMs for High-Resolution Real-Time Imaging
12.2.5 Nanotheranostics: Nano-based Therapy and Diagnostic Products for Plant Pests and Pathogens
12.2.6 Nanobarcoding: Naming and Sorting the GM Crops
12.2.7 Nanogrowth Enhancers: NMs to Enhance Seed Germination and Plant Growth
12.2.8 Bioinspired/Nano-enabled Plants
12.3 Conclusions
References
Further Reading
Chapter 13
13 Techniques in Biotechnology: Essential for Industry
13.1 Brief History of Biotechnology
13.2 Fermentation
13.2.1 Fermentation Method
13.2.2 Inoculum (Microorganisms)
13.2.3 Substrate
13.2.4 Fermentors
13.2.5 Culture Conditions
13.2.6 Product
13.3 Biocatalysts/Enzymes
13.4 Industrial Production of Biocatalysts/Enzymes
13.4.1 Enzyme Definition
13.4.2 Enzyme Production Methods
13.4.3 Purification of Enzyme
13.4.4 Advances in Enzyme Industry
13.4.5 Application of Enzyme
13.5 Paper and Pulp Industry
13.6 Biofuels
13.7 Environmental Biotechnology
13.7.1 Application
13.8 Food Process Technology
13.8.1 Advances and Applications
13.9 Biorefinery
13.9.1 Applications
13.10 Bioreactors
13.11 Future Techniques
13.11.1 CRISPR/Cas9
13.11.2 Microbiome and Personalized Medicine
13.11.3 Sequencing
13.11.4 Mass Spectrometry
References
Further Reading
Chapter 14
14 Omics Approaches in Industrial Biotechnology and Bioprocess Engineering
14.1 Introduction
14.2 The Omics Revolution: Implications for Industrial Biotechnology
14.3 Omics Tools in Industrial Biotechnology and Bioprocess Engineering
14.3.1 Next-Generation Sequencing
14.3.2 Mutagenesis
14.3.3 Reverse Genetics
14.3.4 Cell Line Development
14.3.5 Synthetic Biology
14.3.6 Data Depository and Bioinformatics Tools
14.4 Combined Omics Approaches
14.5 Challenges in Omics-for-Industry
14.6 Conclusion and Future Perspectives
References
Chapter 15
15 Omics Approaches and Applications in Dairy and Food Processing Technology
15.1 Introduction
15.1.1 Historical Perspective
15.1.2 Biotechnological Developments in Dairy and Food Processing
15.1.2.1 Cheese
15.1.2.1.1 Microbial Rennet and Recombinant Chymosin
15.1.3 Bio Yogurt
15.2 Omics: From Farm to Fork
15.3 Proteomics: General Strategies and Analytical Methods
15.3.1 Protein Extraction
15.3.2 Protein Separation
15.3.2.1 Gel-Based Proteomic Approach
15.3.2.2 Gel-Free Proteomic Approach
15.3.3 Protein Identification
15.3.3.1 Mass Spectrometry
15.3.3.1.1 Ionization Techniques
15.3.3.2 Mass Analyzers
15.3.4 Comprehensive Data Analysis
15.4 Proteomics of Milk and Milk Products
15.4.1 Proteomics of Milk Proteins
15.5 Proteomics of Food Technology
15.5.1 Postharvest Processing
15.5.2 Cereal and Other Crops
15.6 Proteomics in Assessing
15.6.1 Quality of Foods
15.7 Transcriptomics in Food Safety
15.8 Future Prospects
15.8.1 Transcriptomics, Proteomics, and Metabolomics
15.8.2 Integrating Omics
15.9 Challenges and Opportunities in Food Omics
15.10 Conclusion
References
Chapter 16
16 Omics Approaches in Enzyme Discovery and Engineering
16.1 Introduction
16.2 Novel Enzymes Discovery for Industrial Applications
16.3 Molecular Engineering of Available Industrial Enzymes
16.4 Industrial Applications of Enzymes and Examples of Bioengineered Enzymes Currently in Common Use
16.4.1 Enzymes in the Food Industry
16.4.2 Enzymes in the Animal Feed Industry
16.4.3 Corn and Cellulose Processing
16.4.4 Enzymes in Surfactants and Detergents
16.4.5 Enzymes in Organic Bio-Synthesis
16.4.6 Other Promising Applications for Enzymes Within the Textile and Carbon Capture Industries
16.5 Conclusion and Future Perspectives
References
Chapter 17
17 Biomedical Engineering: The Recent Trends
17.1 Introduction
17.2 Areas of BME
17.2.1 Bioinstrumentation
17.2.2 Biomechanics
17.2.2.1 Sports Biomechanics
17.2.2.2 Continuum Mechanics
17.2.3 Biotribology
17.2.4 Computational Biomechanics
17.2.5 Biofluid Mechanics
17.2.6 Biomaterials
17.2.7 Tissue Engineering
17.2.8 Biorobotic
17.2.9 Biosensors
17.2.10 Neuroengineering
17.3 Future Directions
References
Further Reading
Chapter 18
18 Omics Approaches in Biofuel Technologies: Toward Cost Effective, Eco-Friendly, and Renewable Energy
18.1 Introduction
18.2 Brief Overview of the First-Generation Biofuel Technologies
18.3 Second-Generation Biofuel Technologies
18.4 Third-Generation Biofuel Technologies
18.4.1 Microalgae Cultivation
18.4.2 Microalgae Biomass Harvesting
18.4.3 Lipid Extraction and Biodiesel Production
18.5 Practical Challenges Ahead in Biofuel Technologies
18.6 Omics Advancement and Approaches for Cost-Effective Production of Renewable Energy
18.7 Conclusion and Future Perspectives
References
Further Reading
Chapter 19
19 Omics-Based Bioengineering in Environmental Biotechnology
19.1 Introduction
19.2 Application of Omics in Soil Microbial Ecology
19.2.1 Metagenomics and Soil Function
19.2.2 Metatranscriptomics and Soil Function
19.2.3 Extracting Value From Metatranscriptomics
19.2.4 Niche Specialization and Differentiation
19.3 Application of Omics in Controlling Pollution
19.4 Application of Omics-Based Bioengineering for Chemical Toxicity Screening
19.5 Omics Applications in Environmental Stress-Related Gene and Protein Modifications
19.6 Conclusion and Future Perspective
References
Further Reading
Chapter 20
20 Biochar for Carbon Sequestration: Bioengineering for Sustainable Environment
20.1 Introduction
20.1.1 What Is Environmental Sustainability?
20.1.2 Why There Are Increasing Concerns?
20.1.3 How to Address ES-Related Issues?
20.2 What Is Biochar?
20.2.1 What Are Its Types?
20.2.2 How Biochar Can be Produced?
20.2.3 Why Biochar Can be a Possible Solution for ES?
20.3 Biochar-Based Bioengineering Technologies
20.3.1 Biochar and Various Use Efficiency Strategies
20.3.1.1 Biochar as Nutrient Delivery Vehicle
20.3.1.2 Biochar Amendments Affecting Soil Nutrient Status and Enhancing Nutrient Use Efficiency
20.3.1.3 Biochar for Enhancing Water Use Efficiency
20.3.2 Biochar and Climate Change Abatement: Curbing Greenhouse Gas Emissions
20.3.3 Biochar-Based Bioengineering of Ecological Niches
20.3.3.1 Heavy Metal Removal
20.3.3.2 Organic Pollutant Removal
20.3.3.3 Sorption of Excess N or P From Wastewater
20.3.4 Biochar–Soil Microbial Community Interactions: Possible Implications
20.4 Agronomic Effects of Biochar Amendments in Vegetables
20.4.1 Biochar-Plant Growth Effects and Yield Impacts
20.5 Conclusion
References
Further Reading
Index
Index

Citation preview

OMICS TECHNOLOGIES AND BIO-ENGINEERING

OMICS TECHNOLOGIES AND BIO-ENGINEERING Towards Improving Quality of Life VOLUME 2 Microbial, Plant, Environmental and Industrial Technologies Edited by

Debmalya Barh Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, India Laborato´rio de Gene´tica Celular e Molecular, Departamento de Biologia Geral, Instituto de Cieˆncias Biolo´gicas (ICB), Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Vasco Azevedo Laborato´rio de Gene´tica Celular e Molecular, Departamento de Biologia Geral, Instituto de Cieˆncias Biolo´gicas (ICB), Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2018 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-815870-8 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: John Fedor Acquisition Editor: Rafael Teixeira Editorial Project Manager: Mariana Kuhl Production Project Manager: Kiruthika Govindaraju Cover Designer: Vicky Pearson Esser Typeset by MPS Limited, Chennai, India

Dedication We dedicate this book to our next generation.

List of Contributors Hasan Afzaal Riphah International University, Islamabad, Pakistan Amjad Ali National University of Science and Technology (NUST), Islamabad, Pakistan; Polish Academy of Sciences, Warsaw, Poland Zeeshan Ali Shifa Tameer-e-Millat University, Islamabad, Pakistan Jaspreet S. Arora Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India Kulanthaiyesu Arunkumar Central University of Kerala, Kasaragod, India Marcela S.P. Azevedo Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Vasco Azevedo Institute of Integrative Omics and Applied Biotechnology, Nonakuri, West Bengal, India; Universidade Federal de Minas Gerais (ICB/UFMG), Belo Horizonte, Brazil Mustafeez M. Babar Shifa Tameer-e-Millat University, Islamabad, Pakistan Debmalya Barh Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Institute of Integrative Omics and Applied Biotechnology, Nonakuri, West Bengal, India Kartikay Bisen Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Isabel S. Carvalho University of Algarve, Faro, Portugal Rekha Chawla Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India Karthik Chinnannan Periyar University, Salem, Tamil Nadu, India Ana Coelho University of Algarve, Faro, Portugal Sintia S. De Almeida Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Siomar De Castro Soares Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Alejandra de Moreno de LeBlanc Centro de Referencia para Lactobacilos (CERELACONICET), San Miguel de Tucuma´n, Argentina Cassiana S. De Sousa Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Rajesh K. Dubey Punjab Agricultural University, Ludhiana, India Atul Grover Defence Institute of Bio-Energy Research, Haldwani, India Alvina Gul National University of Sciences and Technology (NUST), Islamabad, Pakistan; Polish Academy of Sciences, Warsaw, Poland Sanjay M. Gupta Defence Institute of Bio-Energy Research, Haldwani, India Syed S. Hassan Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

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LIST OF CONTRIBUTORS

Yousef I. Hassan Guelph Research and Development Centre, Guelph, ON, Canada Shahid Iqbal University of Agriculture Faisalabad, Faisalabad, Pakistan Rija Irfan National University of Sciences and Technology (NUST), Islamabad, Pakistan Jyoti S. Jadaun CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India Anu Kalia Punjab Agricultural University, Ludhiana, India Chetan Keswani Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Faria Khan National University of Science and Technology (NUST), Islamabad, Pakistan; Polish Academy of Sciences, Warsaw, Poland Md Gulam M. Khan University of Sherbrooke, Sherbrooke, QC, Canada Surender Khatodia Amity University Haryana, Gurgaon, India Sathiya Kumar Periyar University, Salem, Tamil Nadu, India Sekar Kumaran Muthayammal College of Arts and Science, Rasipuram, Tamil Nadu, India Jean Guy LeBlanc Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucuma´n, Argentina Tessalia D. Luerce Universidade Federal de Minas Gerais (ICB/UFMG), Belo Horizonte, Brazil Lekha C. Meher Defence Institute of Bio-Energy Research, Haldwani, India Mohammad F. Miah Queen’s University, Kingston, ON, Canada Zujaja T. Misbah National University of Sciences and Technology (NUST), Islamabad, Pakistan; Shifa Tameer-e-Millat University, Islamabad, Pakistan Bhawana Mishra CSIR-Central Institute for Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow, Uttar Pradesh, India Anderson Miyoshi Universidade Federal de Minas Gerais (ICB/UFMG), Belo Horizonte, Brazil Chandra S. Mukhopadhyay Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India Anjana Munshi Central University of Punjab, Bathinda, India Malik G. Mustafa University of Sherbrooke, Sherbrooke, QC, Canada Lokesh K. Narnoliya CSIR-Central Institute for Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow, Uttar Pradesh, India Mohammed Nasim Defence Institute of Bio-Energy Research, Haldwani, India Duy Nguyen University of Sherbrooke, Sherbrooke, QC, Canada Ricardo Nunes University of Algarve, Faro, Portugal Indra A. Padikasan Periyar University, Salem, Tamil Nadu, India Sajida Parveen National University of Sciences and Technology (NUST), Islamabad, Pakistan Vikas Y. Patade Defence Institute of Bio-Energy Research, Haldwani, India S.M. Paul Khurana Amity University Haryana, Gurgaon, India Anne C. Pinto Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Venkata R. Pothineni Stanford University, Palo Alto, CA, United States Raja Rathinam University of Algarve, Faro, Portugal

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Ratul M. Ram Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Clarissa S. Rocha Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Pavel Samuleev Royal Military College of Canada, Kingston, ON, Canada Neelam S. Sangwan CSIR-Central Institute for Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow, Uttar Pradesh, India Rajender S. Sangwan CSIR-Central Institute for Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow, Uttar Pradesh, India Ramaraj Sathasivam Sangmyung University, Seoul, South Korea Kanwal Shaheen Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan Hemaiswarya Shanmugam Anna University, MIT Campus, Chennai, Tamil Nadu, India Manju Sharma Amity University Haryana, Gurgaon, India S.P. Sharma Punjab Agricultural University, Ludhiana, India Vandana Sharma Indraprastha Apollo Hospital, New Delhi, India Wanderson M. Silva Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Harikesh B. Singh Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Surya P. Singh Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Govindaraju Subramaniyan Periyar University, Salem, Tamil Nadu, India Natesan Sudhakar Muthayammal College of Arts and Science, Rasipuram, Tamil Nadu, India Tehreem Tanveer Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan Ruchi Tripathi Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Sandhya Tripathi CSIR-Central Institute for Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow, Uttar Pradesh, India Daria Trofimova Queen’s University, Kingston, ON, Canada Kinza Waqar National University of Sciences and Technology (NUST), Islamabad, Pakistan Muhammad A. Zahid Shifa Tameer-e-Millat University, Islamabad, Pakistan Najam-us-Sahar S. Zaidi National University of Sciences and Technology (NUST), Islamabad, Pakistan Ting Zhou Guelph Research and Development Centre, Guelph, ON, Canada

About the Editors Vasco Azevedo is graduated from veterinary school of the Federal University of Bahia in 1986. He obtained his Master (1989) and Ph.D. (1993) degrees in microbial genetics from the Institut National Agronomique Paris-Grignon (INAPG) and Institut National de la Recherche Agronomique (INRA), France, respectively. He did his Postdoctoral research (1994) at the Department of Microbiology, School of Medicine, University of Pennsylvania, United States. Since 1995, he is a professor at the Federal University of Minas Gerais (UFMG), Brazil. In 2004, Prof. Azevedo won the Livre-doceˆncia contest at the University of Sa˜o Paulo, which is considered as the best university in Brazil. Livre-doceˆncia is a degree awarded by the Higher Education Department of Brazil through a public examination open only to the doctoral degree holders and is a recognition to a superior quality of teaching and research. In 2017, Prof. Azevedo defended his third thesis to become a Doctorate in Bioinformatics from the UFMG. He is a also a Fellow of Brazilian Academy of Sciences. He has published 380 research articles, 3 books, and 29 book chapters. Prof. Azevedo is expert in bacterial genetics, genomics, transcriptome, proteomics, and development of new vaccines and diagnostics against infectious diseases. He is pioneer in genetics of Lactic Acid Bacteria and Corynebacterium pseudotuberculosis in Brazil.

Debmalya Barh, M.Sc. (applied genetics), M.Tech. (biotechnology), M.Phil. (biotechnology), Ph.D. (biotechnology), Ph.D. (bioinformatics), Post-Doc (bioinformatics), PGDM, is a honorary Principal Scientist at the Institute of Integrative Omics and Applied Biotechnology (IIOAB), India—a virtual global platform of multidisciplinary research and advocacy. He is blended with both academic and industrial research and has more than 12 years bioinformatics and personalized diagnostic/medicine industry experience where his main focus is to translate academic research into high value commercial products for common mans’ reach. He has published more than 150 articles in reputed international journals and has edited 15 cutting-edge omics related reference books published by Taylor & Francis, Springer, Elsevier, etc. He has also coauthored 301 book chapters. He also frequently reviews articles for international journals like Nature Publications, Elsevier, BMC Series, PLoS One, etc. Due to his significant contribution in the field, he has been recognized by Who’s Who in the World and Limca Book of Records.

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C H A P T E R

1 Microbial Omics: Applications in Biotechnology Cassiana S. De Sousa1, Syed S. Hassan1, Anne C. Pinto1, Wanderson M. Silva1, Sintia S. De Almeida1, Siomar De Castro Soares1, Marcela S.P. Azevedo1, Clarissa S. Rocha1, Debmalya Barh1,2 and Vasco Azevedo2 1

Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 2Institute of Integrative Omics and Applied Biotechnology, Nonakuri, West Bengal, India

1.1 INTRODUCTION To better understand the biology of a microorganism some technologies are necessary, with the aim to elucidate and enlarge this knowledge for more detail. Thus, the omics studies add up to support this search and create a significant number of information essential to analyze the molecules and their role in cell function. Among the different areas that contribute to better study the cell the omics can be highlighted: genomics, transcriptomics, proteomics, metabolomics, and interactomics that in association with bioinformatics can reveal the microorganism’s biology. These areas assist in a better uptake of the biologic mechanisms (Palsson, 2002), may provide greater reliability in studies related to biotechnology, and can reduce costs in the industry (Dry et al., 2000), since biotechnology is a technology which handles the cellular and molecular biologic processes to develop products or even technologies that may improve the quality of life (Chisti and Moo-Young, 1999). With the aid of the omics studies, the microbial biotechnology can lead to an improvement in the vaccines and diagnostic tools, modification of pathogens to reduce their virulence, improved microbial agents for pest control, and development of biologic agents to solve environmental problems, recovering water or contaminated soils, among others.

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00001-2

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Copyright © 2018 Elsevier Inc. All rights reserved.

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Moreover, omics can aid crop breeding programs, as they can make these programs faster and improve next-generation crop production (Eldakak et al., 2013). Omics can act together with breeding/plant genetic engineering for the development of more productive energy crops (Ndimba et al., 2013).

1.2 STRUCTURAL GENOMICS Structural genomics is defined as the sequence and structure analysis of genome elements: genes, regulators, and mobile elements (Aburjaile et al., 2014). Structural genomics aims to grow our basic knowledge of biologic macromolecules while lessening the average costs of structure determination. Alongside, the structural bioinformatics is related to the analyses and prediction of the three-dimensional (3D) structure of biologic macromolecules like proteins, RNA, and DNA. The term structural is the same as in structural biology, and structural bioinformatics is considered as a part of computational structural biology, whereas, structural genomics tends to describe the 3D structure of every protein encoded by a given genome. This approach provides a high-throughput method for structure determination including experimental and modeling approaches. The first omics that will be reported here is the genomics, which by itself does not tell us about the biologic mechanisms without the aid of other strategies. However, it is a fundamental step to reveal the sequence of genomes, allow the study of genes, predict their function and location, and reveal operons and other essential information of the organism. Whereas the term bioinformatics refers to describe an interdisciplinary approach that analyzes biologic questions by applying methods and algorithms beginning from mathematics and informatics, bioinformatics and microbial genomics concentrate on the large amounts of newly available data for meaningful interpretation, within the background of existing knowledge. The cost per sequenced base pair has reduced drastically, and currently, many research institutions have access to the latest sequencing technology. Among them are GS FLX by Roche/454 Life Technologies (Margulies et al., 2005), SOLiD by Applied Biosystems (Smith et al., 2010; Valouev et al., 2008), Genome Analyzer by Solexa/Illumina (Bentley et al., 2008), Ion Torrent PGM by Life Technologies (Bragg et al., 2013), CGA Platform by Complete Genomics (Drmanac et al., 2010), and PacBio RS/Single-Molecule Real-Time (SMRT) by Pacific Biosciences (Eid et al., 2009). These modern sequencing technologies emerge rapidly, making a bacterial genome affordable and to be sequenced quickly, that also enables the analysis of bacterial genomes and their expression with more depth and accuracy; the analysis of the resulting omics data remains the real challenge. In genomic studies, after the detection of an essential gene in a given genome, such as an antigen, for example, this specific gene can be isolated and inserted into the DNA of a microorganism so that it can be transcribed, thus obtaining large quantities of this molecule, which is useful in the production of more efficient vaccines (Liew, 1990). The function of a protein is inferred from the protein structure. Additionally, structural genomics can identify novel protein folds and potential targets for drug discovery. There are two types of experimental methods for structure determination: X-ray crystallography and ˚ resolution and 100% model accuracy. Both are nuclear magnetic resonance (NMR), with 1.0 A

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expensive and time consuming. Sometimes it is even difficult to obtain a structure but structures provide more compromising results, and much more useful information is inferred from the protein structure. The structures obtained are mainly used for studying catalytic mechanisms and for designing and improving ligands. There are computational methods for structure prediction based on sequence or structural homology with a protein of known structure or the chemical and physical principles for a protein with no homology to any known structure. They include ab initio, comparative homology modeling, and threading (fold recognition) structure prediction methods with a structure resolution ranging from RMSD value of 1.5 to 4.8 and with a model accuracy of # 95%. The best quality structure models arising from these approaches have important applications like docking of macromolecules and prediction of protein partners, virtual screening, and docking of small compounds, defining antibody epitopes, and molecular replacement in X-ray crystallography experiments, among others. The DNA sequencing and the information technology allowed to understand the genetic variation and to make a correlation between this variation and the plant performance. This way, this technology, which can be applied on food crops and other plant species, can fasten and cheapen the process of plant breeding. Moreover, genomics can be applied to breeding programs in a great variety of ways (Bore´m and Fritsche-Neto, 2013). Genomics is a valuable tool considering a new revolution in the plant genetic improvement, as this approach opened a new horizon to breeders when the study of the genome as a whole was allowed. The methods of high-throughput DNA sequencing, for example, next-generation sequencing (NGS), are among the major bases of genetic improvement (Pe´rez-de-Castro et al., 2012). In agrigenomics (omics studies applied on agriculture), the main objective is to solve problems related to climate variation. The challenge is to produce enough amounts of food as the world population is growing over the years. It is important to identify favorable genomic characteristics, in plant and cattle, which can confer increased food production, always considering the harvest and healthy animals and, moreover, direct the research toward sustainability. Advances in genomic studies, for example, have contributed to the identification of potential genetic targets for developing genetically modified strains of microalgae to offer increased production of lipids and become alternative sources of sustainable energy (Misra et al., 2013). In the medical area, the new sequencing generation technology, through genomics, has contributed to the clinical diagnostics research, for example. Genomic medicine helped, together with bioinformatics analyses, to achieve improved and accelerated diagnosis of monogenic diseases, facilitating emergency tests in newborns, since these diseases are indistinguishable at birth. The diagnosis must be fast because the disease progresses in an accelerated way and can lead to death. Also, the early diagnostic accelerates the treatment, preventing the progression of the pathology (Saunders et al., 2012).

1.2.1 Comparative and Pan-Genomics Due to many genomes being deposited in databases, the comparative genomics emerged and allowed the comparison between the DNA content of different genomes and

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unveiled their phylogeny. From comparing the genomes, important differences involved with phenotypic and evolutive characteristics between the organisms can be discovered. Moreover, this information can be used to apply the microorganisms into the industry field. Comparative genomics is a relatively new science of comparing these similarities and differences across all living organisms, the genome sequences of different species, for example, human, mouse, and a wide variety of other organisms ranging from bacteria to chimpanzees (Touchman, 2010). It helps in providing access to the core (intra- and an inter-species conserved set of genes), accessory (conserved set of genes in more than one strain), and strain-specific genes, hence helping the researchers at the molecular level to distinguish different life forms from each other (Fig. 1.1). Acidithiobacillus ferrooxidans was the first microorganism of the biomining area to have the genome sequenced. The main aim of the research was to obtain information on energy metabolism, responsible for bioleaching. From these results, genes related to the metabolism of phosphate, sulfur, and iron or involved in the biosynthesis of resistance to metals, amino acids, or precursors to the formation of extracellular polysaccharides could be studied (Sugio et al., 2005). Another study involving the genomics used streptomycetes as a target, as they present features of high commercial interest to a variety of antibiotics, antiparasitic agents, metabolites with pharmacologic activities, and enzymes of great importance to the industry (Demain, 1999). Thus, the complete sequence of Streptomyces avermitilis and the comparison of their genome sequence and secondary metabolic products against different genomes of the Actinobacteria group were presented, which produced valuable information to the use of Streptomyces in the industrial field and improved the secondary metabolites production, including antibiotics (Ikeda et al., 2003). Comparing the fruit fly and human genome reveals that about 60% of genes are conserved (Adams et al., 2000), that is, the two organisms share a core set of genes. It is a powerful tool for studying evolutionary changes among organisms. Dramatic results have emerged that provided breakthroughs in biomedical and agricultural sciences linked to human disease and diagnosis, organ and bone marrow transplantation, human and animal vaccine candidate identification against harmful pathogens, and the fast breeding of new crop plant varieties.

FIGURE 1.1 Representation of interspecies pan, core, accessory, and singletons/strain-specific genome contents.

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Pan-genomics, on the other hand, is a relatively new area of comparative genomics that deals with the characterization of genomes by comparison, of related species. It provides access to the pan, core, accessory, and strain-specific genes. The “pan-genome” is the complete inventory of genes found in any member of the species; the “core genome” is composed of the genes that are present in all the species strains and that are thus necessary for basic life processes; accessory genes make part of the genome that are shared by, in minimum, more than one strain; and the “singletons/strain-specific genome” represent a set of genes found only in a given strain.

1.2.2 Immunogenomics Immunity is a protective mechanism used by the body primarily for protection against disease, that is, immune cells like neutrophils, macrophages, dendritic, T, and B cells recognize and eliminate pathogens. It is known to have an innate, adaptive component, which acts in different ways to defend the body. The unique characteristics of the immune system, their structural and functional diversity, and their ability to interact with and recognize molecules suggest that the immune system can be compared to a network of thousands of molecules. There emerged a need to know more about the system, thus raising a new field, immunoinformatics. This new area encompasses different areas within computational biology like immunogenomics, immunoproteomics, predicting epitopes, and reverse vaccinology. About reverse vaccinology, computer techniques are used to identify molecules of the immune system, including the study and design of algorithms for epitope mapping of B and T cells, that aim to assist in the designing of vaccines by the prediction of immunogenic epitopes. This type of prediction requires evaluating the complete genome of the pathogen for the identification of antigens. An advantage of the reverse vaccinology for obtaining antigens over nonconventional methods is that one does not need to cultivate the pathogen for extraction and subsequent identification of proteins. Additionally, a logic evolution is the structural vaccinology, which is based on a combination of genomics and structural biology, where the information used is derived from the 3D structure of protein. This methodology aims to optimize and facilitate the production of antigens and on an industrial scale, with greater security. Another advantage is the reduction of antigenic variation between related species just like the rapid development in vaccines design for protection against all microbial protein variants captured in proteins of hybrid vaccines. The limitation of this technology is mainly focused on gene products that generate carbohydrates or glycolipids. Since the interest in vaccine production is the protein products, these unexpected elements are ineffective to induce an immune response. The essential tools for the development of vaccines epitopes are mapping and prediction of the epitope that allows an excellent approximation, as it can eliminate effects from the pathogen. Through prediction, it is also possible to use the epitopes avoiding problems such as hypervariable sequences using combine epitopes of B and T cells. There are several online tools for the prediction of T-cell epitopes by major histocompatibility complex (MHC) class I and class II (Table 1.1). A widely used tool for the prediction

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TABLE 1.1 Immunologic Databases Database

Characteristic

URL

AntiJen

Database on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology

http://www.ddg-pharmfac.net/ antijen/AntiJen/antijenhomepage. htm

BCIpep

Database of B-cell epitopes from a wide range of pathogens

http://crdd.osdd.net/raghava/ bcipep/

BepiPred

Predicts the location of linear B-cell epitopes using a combination of a hidden Markov model and a propensity scale method

http://www.cbs.dtu.dk/services/ BepiPred/

dbMHC

Platform for DNA and clinical data related to the human major histocompatibility complex (MHC)

http://www.ncbi.nlm.nih.gov/ gv/mhc/main.cgi?cmd 5 init

DiscoTope

Predicts discontinuous B-cell epitopes from protein 3D structures

http://www.cbs.dtu.dk/services/ DiscoTope/

Epitome

Database of antigenic residues and the antibodies that interact with them

https://www.rostlab.org/ services/epitome/

GPX-Macrophage

Online resource for expression-based studies of a range of macrophage cell types following treatment with pathogens and immune modulators

http://gpxmea.gti.ed.ac.uk/

HaptenDB

Database of haptens, has 2D and 3D structures of most of haptens

http://www.imtech.res.in/ raghava/haptendb/

HLArestrictor

Is a tool for patient-specific predictions of HLA restriction http://www.cbs.dtu.dk/services/ elements and optimal epitopes within peptides HLArestrictor/

HPtaa

Database of potential tumor-associated antigens

http://www.bioinfo.org.cn/hptaa/

IEDB-3D

3D structural component of the Immune Epitope Database (IEDB)

http://www.iedb.org/

IL2Rgbase

Database of mutations causing human X-linked SCID

http://www.ncbi.nlm.nih.gov/ lovd/home.php?select_db 5 IL2RG

IMGT/HLA

Database for sequences of the human major histocompatibility complex (HLA)

http://www.ebi.ac.uk/ipd/ imgt/hla/

IMGT/LIGM-DB

Database of immunoglobulin (IG) and T-cell receptor (TCR) nucleotide sequences, from human and other vertebrate species

http://www.imgt.org/ligmdb/

IMGT

Common access to immunogenetics data, including IGs, http://www.imgt.org/ TCRs, MHC, and related proteins of the immune system of human and other vertebrate species

IMGT-GENE-DB

Genome database for IGs and TCR genes from human and http://www.imgt.org/genedb/ mouse, and, in development, from other vertebrate species queryPage

IEDB

Catalog of experimentally characterized B- and T-cell epitopes, as well as data on MHC binding and MHC ligand elution experiments

http://www.immuneepitope.org/

(Continued)

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TABLE 1.1 (Continued) Database

Characteristic

URL

IPD-ESTDAB

Provides access to the European Searchable Tumour Cell-Line Database (ESTDAB), a cell bank of immunologically characterized melanoma cell lines

http://www.ebi.ac.uk/ipd/ estdab/

IPD-HPA— Human Platelet Antigens

Database for the data which define the human platelet antigens (HPA)

http://www.ebi.ac.uk/ipd/hpa/

IPD-KIR - Killercell IG-like Receptors

Database for human KIR sequences. Killer-cell IG-like receptors (KIR)

http://www.ebi.ac.uk/ipd/kir/

IPD-MHC

Database for sequences of the MHC from a number of different species

http://www.ebi.ac.uk/ipd/mhc

MHCcluster

Is a tool to functionally cluster MHC class I (MHCI) molecules based on their predicted binding specificity

http://www.cbs.dtu.dk/services/ MHCcluster/

MHCBN

Database contain MHC-binding peptides

http://crdd.osdd.net/raghava/ mhcbn/

MHC-Peptide Interaction Database

Highly curated database for sequence structure function information on MHC peptide interactions

http://variome.bic.nus.edu.sg/ mpidt/

MUGEN Mouse Database

Database of murine models of immune processes and immunologic diseases

http://www.mugen-noe.org/ database/

NetChop

Produces neural network predictions for cleavage sites of the human proteasome

http://www.cbs.dtu.dk/services/ NetChop/

NetCTL

Predicts CTL epitopes in protein sequences

http://www.cbs.dtu.dk/services/ NetCTL/

NetCTLpan

Predicts CTL epitopes in protein sequences

http://www.cbs.dtu.dk/services/ NetCTLpan/

NetMHC

Predicts binding of peptides to a number of different HLA alleles using artificial neural networks (ANNs)

http://www.cbs.dtu.dk/services/ NetMHC/

NetMHCcons

Predicts binding of peptides to any known MHC class I molecule

http://www.cbs.dtu.dk/services/ NetMHCcons/

NetMHCII

Predicts binding of peptides to HLA-DR, HLA-DQ, HLA-DP, and mouse MHC class II alleles using ANNs

http://www.cbs.dtu.dk/services/ NetMHCII/

NetMHCIIpan

Predicts binding of peptides to MHC class II molecules

http://www.cbs.dtu.dk/services/ NetMHCIIpan/

NetMHCpan

Predicts binding of peptides to any known MHC molecule using ANNs

http://www.cbs.dtu.dk/services/ NetMHCpan/

NetMHCstab

Predicts the stability of peptide binding to a number of different MHC molecules using ANNs

http://www.cbs.dtu.dk/services/ NetMHCstab-1.0/

NNAlign

Identifying sequence motifs in quantitative peptide data http://www.cbs.dtu.dk/services/ NNAlign/ (Continued) I. MICROBIAL AND PLANT TECHNOLOGIES

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TABLE 1.1 (Continued) Database

Characteristic

URL

Protegen

Protective antigen database and analysis system

http://www.violinet.org/protegen

SuperHapten

Manually curated hapten database integrating information from literature and web resources

http://bioinformatics.charite.de/ superhapten

SYFPEITHI

Database of MHC ligands and peptide motifs

http://www.syfpeithi.de/

TMAD

The Stanford Tissue Microarray Database

https://tma.im/cgi-bin/home.pl

VBASE2

Integrative database of germ-line V genes from the IG loci of human and mouse

http://www.vbase2.org

VDJsolver

Analysis of human IG VDJ recombination

http://www.cbs.dtu.dk/services/ VDJsolver/

VIOLIN

Is an integrated vaccine literature data mining

http://www.violinet.org/index. php

of T-cell epitopes is NetMHC 3.2 server which predicts binding of peptides to some different HLA alleles using artificial neural networks (ANNs) and weight matrices. Already, the NetMHCII 2.2 server predicts peptide ligands in mice for MHC class II allele HLA-DR, HLA-DQ, and HLA-DP using ANNs. The prediction of B-cell epitopes can be made through the databases “Immune Epitope Database and Analysis Resource IEDB.” The use of parameters such as hydrophilicity, flexibility, accessibility, turns, exposed surface, polarity, and the antigenic propensity of polypeptides chains has been correlated with the location of continuous epitopes, or by “BepiPred” that is a method for linear prediction of B-cell epitopes, which combines hidden Markov model and propensity scale methods. Other databases also play an important role as a source of information for the development of new tools and algorithms. The Nucleic Acids Research Molecular Biology Database Collection (http://www.oxfordjournals.org/nar/database/c/) had 33 immunologic databases in March 2014, some of which are listed in Table 1.1. The International Immunogenetics Information System (IMGT), which is the database of comprehensive reference in immunogenetics and immunoinformatics, specializes in immunoglobulins (IGs) or antibodies, T-cell receptors (TCRs), MHC, among others. As aforementioned, the main goal of immunoinformatics is the development of algorithms that can play a role both in the creation of vaccines and in the analysis of gene products generated by pathogens like viruses and bacteria. There are a wide variety of predictors that play a real role in the prediction of epitopes. However, it is still of great importance to have a prior knowledge of protein sequence and structure.

1.2.3 Post-Genomics Bioinformatics is involved in many ways in microbial biotechnology from the last decade like computationally analyzing the wet-lab data, genome sequencing, identification

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of protein coding sequences, and genome comparison to identify the function of a gene, the development of genomic and proteomics databases, and interpretation of phenotypes from genotypes. It encompasses the identification of regulatory pathways, identification of protein protein, protein DNA, and protein RNA interactions, simulations analyses of metabolic reactions to report the effect of reaction rates, and the analysis of available experimental microarray data to study the association between the gene expressions and stress conditions. In a broad sense, bioinformatics has been applied in (1) the study of protein protein and protein DNA interactions to understand regulatory pathways, (2) the automated reconstruction and comparison of metabolic pathways and so the regulatory mechanisms, (3) inferring microbial evolution on a large scale (phylogenomics) from NGS data, (4) modeling 2D and 3D structure of proteins, and (5) the docking of 3D models of proteins with potential ligands of therapeutic nature in pathogenic microorganisms. The understanding of 3D structure of proteins has a major impact in understanding protein protein and protein DNA functions and interactions. The techniques developed in bioinformatics have the potential to expedite (1) the discovery of causes of diseases, (2) vaccine and rational drug design, and (3) improved cost-effective agents for bioremediation by eliminating the dead ends (Bansal, 2005). Most of the bioinformatics techniques, however, are critically dependent upon the knowledge derived from wet laboratories and the available computational algorithms and tools. Till date, unfortunately, both the resources have limited capability of handling a vast amount of data to interpret genomics and proteomics with so many mysteries. A more rapid progress in bioinformatics and wet-lab techniques are required, and it had to remain interdependent and focused towards harmonizing each other for their progress and the progress of biotechnology in the future, manipulating the microbial cells at a systemic level.

1.3 FUNCTIONAL GENOMICS Functional genomics is defined as the use of genome data to study genome-wide gene and protein expression and their functions. For that, high-throughput methods are used to understand gene transcription and translation and protein protein interaction (PPI) better. The functional genomics studies are related to the genes function in the genome. The role of the gene and its modulation in a given condition is crucial to understand the biology of an organism better. This study involves studies of the transcripts and proteins that play an essential role in the functioning of biochemical processes of cells, associated with the regulation.

1.3.1 Transcriptomics Another branch of omics that has been explored are the studies of gene transcripts. Transcriptomics is the area of functional genomics which studies more deeply the molecular mechanisms of the transcripts expressed under a certain environmental condition. From the analysis of transcripts, the gene function can be suggested.

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The most used tools to study the transcriptional profile are the microarrays and the new generation technologies (NGS), due to a great variety of available platforms, which can differ in sensitivity, specificity, and genome coverage. RNA-Seq, a high-throughput technology, is another good option for transcriptome sequencing and is advantegious to microarray (Sharma and Vogel, 2014). One study (Bischof et al., 2013) performed a comparison between the transcriptomes of the fungus Trichoderma reesei grown on lactose or wheat straw (cellulose) as differentiated sources for carbon production. It was possible to verify the difference in enzymes production and in the molecular physiology of fungus grown on both carbon sources, being bigger when grown on wheat straw. Thus, the genes that were induced are involved in several important biologic processes that can be manipulated to improve the T. reesei strain to increase the production of enzymes necessary to degrade polymers into soluble monosaccharides. Another contribution of transcriptomics to biotechnology was about the increase in the biofuel production. The microbial production of alkane, a fossil fuel, has been demonstrated but the toxicity to microorganism can be a hindrance to a greater production of this hydrocarbon. Therefore, further studies are needed to understand the molecular mechanisms of alkanes microorganisms interaction and to develop a more tolerant host to this element. Thus, through transcriptomic studies with Saccharomyces cerevisiae, it was possible to unveil the response of this microorganism against alkane biofuel and from these data, it is possible to establish strategies to make this species more tolerant, increasing the production of this biofuel (Ling et al., 2013). In the medical area, relevant studies have been conducted aided by transcriptomics to unravel the genetic regulation, to better understand the development of diseases, aiming for a satisfactory health and prevention of different diseases. Jiang et al. (2015) demonstrated a different response that was believed to be about neutrophils. Previously, it was thought that these cells operated in nonspecific responses, but with the transcriptional studies using RNA-Seq, the researchers observed specificity considering pathology. Thus they published a gene catalog that may contribute to the expansion of biomarkers in rheumatic diseases which can also be promising for identification of other diseases. The application of transcriptomics is growing in different areas such as, in the field of sustainable environment, among others. Regarding the environment, studies of the influence of heavy metals on health are poorly reported. To describe the heavy metals such as cadmium, which bring considerable environmental damage and hence to human and animal health, researchers checked the influence of cadmium on corn roots and conducted transcriptional profile studies. In these studies, analysis of differentially expressed genes was performed between two different genotypes. They identified genes that may contribute to a satisfactory corn response against this heavy metal and, eventually, possibly create a tolerant corn against this type of stress (Peng et al., 2015).

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1.3.2 Proteomics (Interactomics) Although the area of transcriptomics provides valuable data for new studies, mRNA levels do not always reflect protein expression, which may be directly related to mRNA lifetime and the availability of nontranslated RNAs. Besides, measure of mRNA may not always be correlated to the cellular phenotype, since translated proteins may undergo posttranslational modifications (PTMs) such as oxidation, phosphorylation, and glycosylation, which cannot be predicted from genomics and transcriptomics analyses. Given those limitations, the proteomics field arises as a new approach to investigate protein expression under different situations, like host pathogen interactions, mixed microbial communities, and, also, microbial metaproteomics (Moxon et al., 2009). The systematical study of all cell proteins and their functional annotation can bring up novel research pathways (Schmidt et al., 2014). By definition, proteomics is the study of entire pool of proteins in a cell or organism, their characteristics, interaction, and it is essential to understand the cell physiology to better understand molecular processes (Schmidt et al., 2014). In proteomics studies, two approaches may be highlighted as the most prominently used: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) based proteomics, which can be coupled to liquid chromatography (LC). Briefly, in 2D-PAGE, researchers take advantage of two orthogonal physicochemical properties of the proteins, the isoelectric point (pI) and the molecular weight (mw), to separate the whole proteome of a given organism or the differentially expressed proteome under a given situation (Otto et al., 2014). 2D-PAGE is combination of qualitative and a quantitative technique as it tracks down the presence/absence and the intensity of spots, respectively. Moreover, proteins that have undergone PTM change their migration patterns in electrophoresis and may be easily evidenced from the whole dataset. However, 2D-PAGE also presents some disadvantages, which include low reproducibility and inability to analyze proteins with extreme pH and mw (Moxon et al., 2009; Otto et al., 2014). To circumvent these problems, the second approach, the MS based proteomics, conducts mass spectrometer measurements of the mass-to-charge ratio of the ions and, then, detects the frequency of ions at each m/z ratio. The approach may be divided into topdown or bottom-up measurements. While the top-down strategy uses intact proteins for the investigation of sequence structure and posttranslational determinations, bottom-up or shotgun proteomics is performed in ions achieved from intact or enzymatically digested proteins aiding in high-resolution quantification and identification of a vast number of proteins (Becker and Bern, 2011). Beyond these techniques, others can be highlighted in the context of proteomics, such as one-dimensional (1D) PAGE, difference in gel electrophoresis, matrix-assisted laser desorption ionization time of flight mass spectrometer (MALDI-TOF MS), stable isotope labeling with amino acid in cell culture, peptide or spectral counting, shaving (proteolytic enzyme digestion to expose bacterial surface proteins), and proteome fractionation (van de Guchte et al., 2012). Proteomics approaches may be actively applied to pathogenic bacteria research, for instance, in target selection, through the comparison of differentially expressed proteins during normal and disease-causing states of pathogenic organisms, which unravels putative virulence factors; and, drug discovery, through identification of secreted proteins

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followed by 3D modeling and docking of antibiotics that may be posteriorly assessed in vivo (Bull et al., 2000). Proteome analyses are considered one of the major appropriate tools to evaluate phenotypic responses in the starter and probiotic strains, considering the environment they are exposed to. These tools can reveal new perspectives to food industries and human health (Mangiapane et al., 2015). Some researchers, in the last years, are demonstrating an increased interest in developing proteomic analyses to study microorganisms better. Berlec et al. (2011) performed in silico and experimental analyses with the surface proteins of two strains of Lactococcus lactis: MG1363 and NZ9000 with the objective to find new candidates to act as carrier proteins. Proteomic studies of starter strains of Oenococcus oeni demonstrated that they are safe to be used in wine fermentation (Bourdineaud, 2006). Concerning bacterial pathogenesis, many proteomic studies have shown the importance of this approach for the study of virulence factors in a wide variety of pathogenic bacteria: Neisseria meningitides, Escherichia coli, Bacillus anthracis, Salmonella typhimurium, and Staphylococcus aureus, among others. In addition to identifying virulence factors, it is important to know their role in the pathogenesis and their interaction with host cells. In this context, high-throughput structural analyses can aid (Wu et al., 2008). With both proteomics and comparative proteomics, an enormous number of proteins can be identified and quantified for therapeutic intention. For example, using comparative proteomics between normal and diseased cells, high targets and biomarkers can be elucidated (Heffner et al., 2014). In a comparative proteomics study between healthy tissues and human pulmonary adenocarcinoma, 32 differentially expressed proteins were identified. Among them, Pkm2 and cofilin-1 were significantly upregulated in adenocarcinoma. As they were correlated with the severity of epithelial dysplasia, they can be key molecules for potential diagnosis, prognosis, and therapeutics (Peng et al., 2011). Moreover, proteomics can complement traditional hybridoma and phage display on the discovery of novel monoclonal antibodies (Heffner et al., 2014). Cheung et al. (2012) used proteomics to identify sequences of antigen-specific antibody directly from circulating polyclonal antibodies using immunized rabbits and mice. Proteomics can aid the field of vaccinology and antibody therapeutics. Also, proteomic approaches can be applied to the optimization of bioprocess development and, consequently, lowering drug costs. It is due, for example, to the possibility of identifying factors responsible for enhancing the capability of cells to the production of therapeutic proteins (Heffner et al., 2014). Although identification, quantification, and characterization of some isolated proteins in normal conditions or during pathogenesis are still desirable, proteomics is moving forward into deeper analyses based on PPIs or interactomes. Interactomics analyses may unravel vital processes in living bacteria, such as cellular communication and gene expression control, and also host pathogen interactions in the way of action of virulence factors and the host immunologic response (Grosdidier et al., 2009). Despite the use of robust and reliable algorithms, international rules for the processing, storage, and data interpretation should be well established. Then, the full potential of proteomics research can be achieved (Schmidt et al., 2014).

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1.3.3 Metabolomics The identification and quantification of the metabolites collection (the metabolome) produced and/or modified by an organism with the objective to understand how the metabolite levels can influence phenotype is called metabolomics (Goodacre et al., 2004; Mapelli et al., 2008). The other omics studies can be complemented by metabolomics, and that is one of the motifs that made it a valuable tool for a great variety of disciplines, such as functional genomics, systems biology, pharmacogenomics, and others. To use the metabolome data with high-efficiency well-curated databases, high-quality data and useful algorithms are needed (Goodacre et al., 2004). Moreover, as the metabolites can be part of different reactions it is harder to analyze metabolome data than the transcriptome and the proteome ones (Mapelli et al., 2008). The levels of each metabolite depend on the context in which the cell is inserted. An important observation about the metabolome analysis is that it is hard to establish a direct connection between genes and metabolites. Moreover, one metabolite can be part of diverse metabolic pathways, which makes it even harder to interpret the metabolomic data (Villas-Boˆas and Gombert, 2006). The metabolome comprises ionic species like hydrophilic carbohydrates, alcohols and volatile ketones, amino acids and organic acids, hydrophobic lipids, and complex natural products. Such complexity, associated with the significant concentration variation and the short half-life of many metabolic intermediates, makes the simultaneous analysis of the whole metabolome of a cell an almost impossible task. As mentioned here, the metabolome has been studied by the application of efficient methods of preparing samples coupled to a combination of various analytical techniques (Villas-Boˆas and Gombert, 2006). The metabolome analysis comprises two main approaches: the directed analysis and the metabolic profile, as described in Fig. 1.2.

FIGURE 1.2 The metabolome analysis.

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TABLE 1.2 Steps of the Analysis of the Metabolome Step

Description/substeps

1. Sample preparation

Interruption of cellular metabolism—“quenching”;extraction of metabolites;sample concentration

2. Analysis of metabolites

By mass spectrometry (MS), high-performance liquid chromatography, liquid chromatography (LC), nuclear magnetic resonance (NMR), or such techniques coupled

3. Processing of data Converting data into useful and interpretable information (by the use of algorithms, data mining, inference, and prediction)

To realize the metabolome analysis it is necessary to follow some important steps. They are sample preparation, analysis of metabolites, and processing of data. These steps and their description are presented in Table 1.2. Some authors have described the steps used for analyzing the metabolome and can be consulted for a more profound reading (van der Werf et al., 2007; Garcia et al., 2008; van Gulik, 2010; Reaves and Rabinowitz, 2011). Several important information about a microbial system can be obtained from metabolic footprinting and fingerprinting analyses, and both of them can be applied to functional genomics and biotechnology. For example, they can be applied (1) to differentiate strains in a fast, trustful, and better way than other approaches (Allen et al., 2003); (2) to elucidate quorum sensing circuits (mechanisms used by the bacterias to communicate, colonize, and attack their hosts by intracellular metabolites) that can be critical to the development of new drugs (Cegelski et al., 2008); (3) to monitor the physiologic status of a cell culture (Winder et al., 2008); (4) to monitor industrial bioprocess (Barrera-Martı´nez et al., 2011); and (5) to do metabolic modeling in microbial systems (Tang, 2011), among others. Due to the facility to manipulate and their importance to human health and biosphere, microorganisms can be used to study biologic systems. The microbial metabolism is a powerful tool to integrate the biologic information to the microbiology systems with the aim to understand microbial interactions, cell functions, and the interactions with the host. Among the areas that the microbial metabolism studies contribute, the human ecosystems (gut microbiota), environmental ecosystems, and microbial metabolism reconstruction can be highlighted (Tang, 2011). To understand the infant gut colonization, a gnotobiotic mice model was used to investigate the metabolome progression. To do that, ex-germ-free mice were colonized with Bacteroides thetaiotaomicron and Bifidobacterium longum and the urine of these mice was analyzed by high-throughput MS. The metabolome data showed gradual and punctual changes on the metabolites production. Moreover, early colonization events affect the nature of small circulating molecules in the host. It was identified that molecules related to carbohydrate and amino acid metabolic processes can provide clues about the dynamic changes that happen during the bacterial colonization (Marcobal et al., 2015). In another study, the metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii were identified. In this study, an acute colitis model using gnotobiotic mice was used to evaluate the inflammatory colitis scores, and a gas

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1.4 CONCLUSIONS AND PERSPECTIVES

chromatography-time of flight MS was used to monitor the metabolomic profile in blood, ileum, cecum, colon, and feces. As a result, some metabolites were overrepresented along the GIT and in serum, and they were correlated with the protective effect of F. prausnitzii against colitis (Miquel et al., 2015). Metabolomics is being increasingly used in the study of infectious diseases. For example, to learn more about tuberculosis, a study was carried out to analyze the metabolic profile of plasma from patients by using untargeted MS. This study offers insights into pathogenesis and host response as it revealed novel diagnosis biomarkers. Despite all the progress reached by metabolomics, it is still in an emerging stage. However, its importance to biologic systems and to complement other omics is undeniable (Tang, 2011).

1.4 CONCLUSIONS AND PERSPECTIVES Studies involving omics present exceptional results that contribute to the development of biotechnology thus, assist with an adequate amount of data and their meaningful interpretation. The interaction of all omics areas takes us to the term systems biology, which is increasingly contributing to the understanding of biology in the context of exchanges of these different areas and how they work in a particular environment. Fig. 1.3 illustrates how all omics interrelate and the systems biology embraces the tools of all of them. In conclusion, the areas of omics may be complementarily used in bacterial studies. For instance, we can track down genes organized in operons through genomic sequencing (genomics), which are co-regulated, thus, transcribed (transcriptomics) and translated FIGURE 1.3 approaches.

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(proteomics) in a given condition, and taking part in the same metabolic pathway (metabolomics and interactomics). Those proteins involved in the same metabolic pathway may be suitable targets for industrial biotechnology. Additionally, interactomics may also be used to unravel virulence factors that are in strict contact with proteins from the host, playing a putative role in pathogenesis, which are useful drug targets for disease control. The next decade of genomics will continue to emphasize on the functional analyses and promote a systematic and integrated approach for life science studies. Microbiologic research has already adopted this perspective, yielding results and insights not possible with the traditional methodology. Microbes are no longer regarded as isolated organisms existing in a system, but rather as an integrated component for understanding functional biology (Tang, 2011).

References Aburjaile, F.F., Santana, M.P., Viana, M.V.C., Silva, W.M., Folador, E.L., Silva, A., Azevedo, V., 2014. In tech: Genomics, SMGroup. Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., et al., 2000. The genome sequence of Drosophila melanogaster. Science 287 (5461), 2185 2195. Allen, J., Davey, H.M., Broadhurst, D., Heald, J.K., Rowland, J.J., Oliver, S.G., et al., 2003. High-throughput classification of yeast mutants for functional genomics using metabolic footprinting. Nat. Biotechnol. 21 (6), 692 696. Bansal, A.K., 2005. Bioinformatics in microbial biotechnology—a mini review. Microb. Cell Fact. 4, 19. Barrera-Martı´nez, I., Gonza´lez-Garcı´a, R.A., Salgado-Manjarrez, E., Aranda-Barradas, J.S., 2011. A simple metabolic flux balance analysis of biomass and bioethanol production in Saccharomyces cerevisiae fed-batch cultures. Biotechnol. Bioprocess. Eng. 16 (1), 13 22. Becker, C.H., Bern, M., 2011. Recent developments in quantitative proteomics. Mutat. Res. 722 (2), 171 182. Bentley, D.R., Balasubramanian, S., Swerdlow, H.P., Smith, G.P., Milton, J., Brown, C.G., et al., 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456 (7218), 53 59. ˇ Berlec, A., Zadravec, P., Jevnikar, Z., Strukelj, B., 2011. Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome. Appl. Environ. Microbiol. 77 (4), 1292 1300. Bischof, R., Fourtis, L., Limbeck, A., Gamauf, C., Seiboth, B., Kubicek, C.P., 2013. Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose. Biotechnol. Biofuels 6 (1), 127. Bore´m, A., Fritsche-Neto, R., 2013. Biotecnologia Aplicada ao Melhoramento de Plantas. Suprema, Visconde do Rio Branco. Bourdineaud, J.-P., 2006. Both arginine and fructose stimulate pH-independent resistance in the wine bacteria Oenococcus oeni. Int. J. Food Microbiol. 107 (3), 274 280. Bragg, L.M., Stone, G., Butler, M.K., Hugenholtz, P., Tyson, G.W., 2013. Shining a light on dark sequencing: characterising errors in Ion Torrent PGM data. PLoS Comput. Biol. 9 (4), e1003031. Bull, A.T., Ward, A.C., Goodfellow, M., 2000. Search and discovery strategies for biotechnology: the paradigm shift. Microbiol. Mol. Biol. Rev. 64 (3), 573 606. Cegelski, L., Marshall, G.R., Eldridge, G.R., Hultgren, S.J., 2008. The biology and future prospects of antivirulence therapies. Nat. Rev. Microbiol. 6 (1), 17 27. Cheung, W.C., Beausoleil, S.A., Zhang, X., Sato, S., Schieferl, S.M., Wieler, J.S., et al., 2012. A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nat. Biotechnol. 30 (5), 447 452. Chisti, Y., Moo-Young, M., 1999. In: Moses, V., Cape, R.E., Springham, D.G. (Eds.), Biotechnology: The Science and the Business, second ed. Harwood Academic Publishers, New York, pp. 177 222. Demain, A.L., 1999. Pharmaceutically active secondary metabolites of microorganisms. Appl. Microbiol. Biotechnol. 52 (4), 455 463.

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Peng, X., Gong, F., Zhao, Y., Zhou, L., Xie, Y., Liao, H., et al., 2011. Comparative proteomic approach identifies PKM2 and cofilin-1 as potential diagnostic, prognostic and therapeutic targets for pulmonary adenocarcinoma. PLoS One 6 (11), e27309. Pe´rez-de-Castro, A.M., Vilanova, S., Can˜izares, J., Pascual, L., Blanca, J.M., Dı´ez, M.J., et al., 2012. Application of genomic tools in plant breeding. Curr. Genomics 13 (3), 179 195. Reaves, M.L., Rabinowitz, J.D., 2011. Metabolomics in systems microbiology. Curr. Opin. Biotechnol. 22 (1), 17 25. Saunders, C.J., Miller, N.A., Soden, S.E., Dinwiddie, D.L., Noll, A., Alnadi, N.A., et al., 2012. Rapid whole-genome sequencing for genetic disease diagnosis in neonatal intensive care units. Sci. Transl. Med. 4 (154), 154ra135. Schmidt, A., Forne, I., Imhof, A., 2014. Bioinformatic analysis of proteomics data. BMC Syst. Biol. 8 (Suppl. 2), S3. Sharma, C.M., Vogel, J., 2014. Differential RNA-seq: the approach behind and the biological insight gained. Curr. Opin. Microbiol. 19, 97 105. Smith, A.M., Heisler, L.E., St Onge, R.P., Farias-Hesson, E., Wallace, I.M., Bodeau, J., et al., 2010. Highlymultiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Res. 38 (13), e142. Sugio, T., Miura, A., Parada, V.P.A., Badilla, O.R., 2005. Bacteria strain wenelen DSM 16786, use of said bacteria for leaching of ores or concentrates containing metallic sulfide mineral species and leaching processes based on the use of said bacteria or mixtures that contain said bacteria [Internet]. [cited Dec. 10, 2015]. Available from: http://www.google.com/patents/US20060094094. Tang, J., 2011. Microbial metabolomics. Curr. Genomics 12 (6), 391 403. Touchman, J., 2010. Comparative genomics. Nat. Educ. Knowl. 3 (10), 13. van de Guchte, M., Chaze, T., Jan, G., Mistou, M.-Y., 2012. Properties of probiotic bacteria explored by proteomic approaches. Curr. Opin. Microbiol. 15 (3), 381 389. van Gulik, W.M., 2010. Fast sampling for quantitative microbial metabolomics. Curr. Opin. Biotechnol. 21 (1), 27 34. van der Werf, M.J., Overkamp, K.M., Muilwijk, B., Coulier, L., Hankemeier, T., 2007. Microbial metabolomics: toward a platform with full metabolome coverage. Anal. Biochem. 370 (1), 17 25. Valouev, A., Ichikawa, J., Tonthat, T., Stuart, J., Ranade, S., Peckham, H., et al., 2008. A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Res. 18 (7), 1051 1063. Villas-Boˆas, S.G., Gombert, A.K., 2006. Ana´lise do metaboloma Uma ferramenta biotecnolo´gica emergente na era po´s-genoˆmica. Biotecnol. Cieˆnc. Desenvolv. Ano IX, no 36, janeiro/junho. Winder, C.L., Dunn, W.B., Schuler, S., Broadhurst, D., Jarvis, R., Stephens, G.M., et al., 2008. Global metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites. Anal. Chem. 80 (8), 2939 2948. Wu, H.-J., Wang, A.H.-J., Jennings, M.P., 2008. Discovery of virulence factors of pathogenic bacteria. Curr. Opin. Chem. Biol. 12 (1), 93 101.

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C H A P T E R

2 Omics Approaches in Viral Biotechnology: Toward Understanding the Viral Diseases, Prevention, Therapy, and Other Applications in Human Betterment Kinza Waqar, Rija Irfan and Alvina Gul National University of Sciences and Technology (NUST), Islamabad, Pakistan

2.1 INTRODUCTION Viruses possess the ability to change the machinery of the living host body for the benefit of the continuity of the survival and are thus categorized as living organisms (parasites). The virus categorized as invasive is capable of infection and showing pathogenicity (Mcfadden, 2006). The host cells possess the ability to evolve and carry out various network of defensive measures against the pathogens but some microbial pathogens have the ability to neutralize the defensive measures of the host cells, rendering them useless (Mcfadden, 2006). It is a challenging and puzzling job to identifying the complex host responses that occur when a viral infection is encountered by the living host. It requires finding and categorizing the various host factors which could be involved actively in the cycle of virus infection. Moreover, there is also a need to categorize the identified host responses into a qualitative and a quantitative category through viral pathogenesis (Zheng et al., 2012).

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00002-4

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2.2 THE HISTORY OF IDENTIFICATION OF VIRUSES AT A MOLECULAR LEVEL AND METAGENOMICS There are multiple existing techniques to identify viruses. Some of the traditional techniques used to discover many of the well-known viruses consist of electron microscopy, inoculation studies, cell culture, and serology (Storch, 2007). These techniques have a lot of limitations such as the fact that viruses, because of being so small in size, cannot be cultured in a laboratory but rather have to be characterized by utilizing molecular methods. However, there have been numerous technological advances in the field of virology. This advancement in technology has set the precedent for metagenomics, a study of a collection of microbial populations called the microbiome. It is a culture-independent study which analyzes the content of the nucleotide sequence in the sample (Petrosino et al., 2009). The study of the viral microbiome, also known as the virome, is conducted on different samples of biological and environmental applications on humans, animals, water, soil, and plants (Breitbart et al., 2003; Zhang et al., 2006; Finkbeiner et al., 2008; Schoenfeld et al., 2008; Fierer et al., 2007). Metagenomics sequencing makes it possible to find the functional potential hidden in the microbiome code. Moreover, metagenomics analyses are being utilized in discovering novel enzymatic functions as well as microorganisms and the genes which could possibly be utilized in bioremediation (Russell et al., 2011; Lovley, 2003) for better know-how of the host
pathogen interactions and also for creating novel therapeutic strategies which could be used for human diseases. However, metagenomics has some limitations in the variation of DNA according to the site of the body and the type of sample; for example, the sputum samples or samples of lung tissue in cystic fibrosis contain a large quantity of human DNA which is released by the neutrophils when the body shows an immune response. It can even amount to a total of 99% of the DNA (Breitenstein et al., 1993).

2.3 ADVANCEMENTS IN TECHNIQUES TO STUDY VIRUS
HOST INTERACTIONS In the present years, virology is undergoing rapid advancement as a result of “highthroughput genome sequencing and proteome screening technologies.” Progress in proteome screening technologies and proteomics based on mass spectrometry have largely encouraged research inquiry into viral proteomes and also response of the host related to viral infections (Zheng et al., 2011). Mass spectrometry is one of the most core and emergent part of the most important discoveries made in the field of virology. The virus
host cell has formed a complex evolutionary relation because of their dynamic interactions that work to either sponsoring virus replication or help the host to carry out its defense against the pathogens invading the host cell and the body. The viral cell promotes multiple proteome changes which makes the understanding of the virus at a molecular level, a difficult task requiring both breadth and an in-depth analysis of the infection. Mass spectrometry is the solution to bridge the gap between experimental dichotomies. Its use in unbiased

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system analysis and hypothesis-run studies has fastened the pace of discoveries in the area of virus pathogenicity and defense of the host (Greco et al., 2014). Mass spectrometry
based techniques are being used to investigate the hosts of viruses and the viruses themselves. Following are the mass spectrometry
based techniques and their applications in virology: 1. Tandem affinity purification (TAP)
based mass spectrometry: This technique takes its role in explaining protein
protein interactions. It is used to study the proteins and the proteome and its structure which is very important when studying the evolution of viral proteins when viruses replicate within the host cell (Jorba et al., 2008). It uses a dual epitope tag for continued immunoaffinity isolations and it has been used in cells infected by viruses. This technique was used to isolate the vaccinia virus called A56 as well as K2 proteins. This was done by using a streptavidin and a calmodulin-attaching TAP system. The virus and the proteins were separated as they localized on to the surface of the cell and were then needed for interaction with the entry fused complex (Moss, 2007). 2. Co-immunoprecipitation
based mass spectrometry: This technique is used to identify the components that bind to a target protein. Co-immunoprecipitation is performed either in a single-step process or as a component of tandem purification. To obtain the target from the solution, an antibody which targets a known member of the complex is attached to a bead and then incubated with lysate. This antibody which is used for co-immunoprecipitation recognizes the protein of our interest or tagged protein which has been made by genetically fusing a tag with our protein of concern. An alternate way of performing co-immunoprecipitation is by using a molecular tag and binding it with our protein of interest which is expressed from a transgene and then purified by using an antibody against the added tag. This tag can then further on be used for a tandem purification or a multi- or single-step purification (Schaffer et al., 2010). The technique was used to define the protein localization in Caenorhabditis elegans. In this experimental design, several tags were constructed and used in C. elegans. The reason behind this was that the functional effect of adding a single tag is not usually known and, therefore, different tags are attached at the C or the N terminus. The functionality of the tagged protein can be determined if it is able to extract the phenotype of a null mutant. Moreover, it is common to use green fluorescent protein
tagged proteins to govern protein localization in C. elegans and this can also be conducted as a single-step process of purification (Zhang et al., 2007). 3. HLA peptidome scanning chip
based mass spectrometry approach: This technique is used to look for peptides associated with disease as well as mutant or differently expressed proteins in a host cell after a viral infection. An example of the use of this technique is in the defense mechanisms employed by the cytotoxic T lymphocytes within the human body against the intracellular pathogens that attack the body. HLA class 1 associated immunopeptides by mass spectrometry has become a very important tool to increase our understanding of the T-cell responses against the pathogens, for example, HIV-1 (Ternette et al., 2015).

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There are several other approaches that are being used to discover the pathogen and they include hybridization and PCR-based methods. The effectiveness of the methods mentioned earlier which are based on PCR and hybridization is established when the virus which interests us is first purified from the host cell and other nucleic acid contaminants. These amplified products are then identified by sequence. In the present day, the advances in sequencing have led us to the era of second-generation sequencing in which huge chunks of data can be sequenced in one go (Petrosino et al., 2009). Sections 2.3.1
2.3.5 provide a description of the current hybridization and PCR techniques for detection of viruses affecting animals.

2.3.1 Hybridization 2.3.1.1 Microarray Techniques Microarrays are made up of probes of oligonucleotides which are of high density; in other words, these are portions of DNA immobilized on a solid surface. The sequences made up of complementary strands present in a test sample are tagged with fluorescent nucleotides which then hybridize with a probe present on a microarray. This hybridization can then be observed and followed by quantification using an observation method based on fluorescence to establish the result. This way the relative abundance or profusion of the sequences of the nucleic acid in a sample is found (Clewley, 2004). There are two types of techniques of microarray which are utilized for the identification of the virus. The first utilizes short oligonucleotide probes and the other utilizes long oligonucleotide probes. The technique utilizing short oligonucleotide probes has been used to detect the human herpes virus (Foldes-Papp et al., 2004). Applications of microarray have been utilized in discovering viruses in animals, for example, beluga whale was found to have the coronavirus (Mihindukulasuriya et al., 2008). The technology used in microarray is considered as a very important tool in viral screening as it is used in screening for a huge amount of pathogens concurrently. 2.3.1.2 Subtractive Hybridization The principle of subtractive hybridization is the removal of the sequences of nucleic acid which are common in the two related samples, leaving behind the different sequences. This differentiates between the sequences in two related samples. There are two sources of nucleic acid called the “tester and the driver”; the tester consists of the pathogenic sequence (Ambrose and Clewley, 2006). Restriction enzymes are used to digest the DNA present in the tester and the driver. It should be noted that only the DNA fragments of tester sample are ligated with adapters. Next the two populations of DNA are first mixed, then they are further denatured into segments, and in the end they are annealed to form four molecules which are “tester/ tester,” the “hybrids of tester,” “driver,” and the “driver/driver.” Through this method, the virus is then sufficiently enriched and the sample of tester is then sequenced to identify the pathogen.

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2.3.2 Methods Based on PCR 2.3.2.1 Degenerate PCR It utilizes primers which are specifically designed and manufactured to anneal to the “highly conserved sequence regions,” which are mutual in related viruses. When the primers are designed, they include some degeneracy that allows them to attach to all or the commonly recognized variants on the conserved sequences because these regions are usually not entirely conserved. Degenerate primers are utilized in the detection of viruses which include novel viruses from existent viral homologous families. An example of the use of the primers is in rabbits; they were used to detect an alphaherpesvirus that was linked with their deaths (Jin et al., 2008). 2.3.2.2 Random PCR Random PCR is used to conduct microarray analysis by amplification and labeling of the probes using fluorescent dyes. It is also associated with identifying novel viruses. Random PCR utilizes two different PCR reactions and two different primers. A singular primer is utilized in initial PCR and has a distinct 50 sequence with a degenerate hexamer at 30 end. After this, the next PCR reaction is done with another different specific primer which is complementary to the 50 region of the initial primer which allows for the amplification of the first products created in the initial reaction. Random PCR is used for finding RNA and DNA viruses and is used for identification of unknown viruses. Examples of a virus which is found through random PCR include a circular DNA virus which was found in the feces of wild chimpanzees (Blinkova et al., 2010) and a seal picornavirus (Kapoor et al., 2008).

2.3.3 Metatranscriptomics Analysis A general metatranscriptomic experiment includes seclusion of the complete RNA from the microbiome followed with the enrichment of RNA which depends on the type of RNA which has to be sequenced, for example, mRNA, lincRNA, and microRNA. The RNA is then further fragmented into small pieces and then cDNA is synthesized by using reverse transcriptase and random oligo dT primers. The next step includes the repair of the 50 and 30 ends or just the 50 or the 30 end of the cDNA followed by ligation with adapters. After this, the library is cleaned up, amplification and quantification is carried out, and lastly, the library is sequenced (Liu and Graber, 2006; Ozsolak and Milos, 2011; Ozsolak et al., 2009; Hickman et al., 2013). However, large-scale use of metatranscriptomics has its limitations in the following: storage and collection of the RNA samples, problems with obtaining sufficient amounts of high-quality RNA from human microbiome samples, the enrichment of mRNA by removal of rRNA, short life of the mRNA, limited transcriptome database, contaminations in the host RNA, and lastly, the poly-A RNA collection kits which are required to store the mRNA population is not usable in prokaryotes (Bikel et al., 2015).

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2.3.3.1 Metagenomics Joined With Metatranscriptomic Analyses Metatranscriptomics is becoming a practical tool to analyze the regulation and dynamics of transcriptionally active microbes. By using metagenomics in combination with metatranscriptomics has enabled us to observe gut microbiome and the results show that it has unique groups of active microorganism in different individuals (Benitez-Paez et al., 2014; Franzosa et al., 2014). The potential of this combination of metagenomics and metatranscriptomics can be observed by looking into the Franzosa study (Franzosa et al., 2014) which states that a fraction of almost 59% of the microbial transcripts are differentially regulated relative to their genomic abundances. Furthermore, it depicted the quantity of gene families, with many that were less abundant at the metagenomics level turning out to be quite active at the level of metatranscriptomics and vice versa (Franzosa et al., 2014). It can be concluded that performing one of the two techniques alone could lead to underestimations or overestimations of the functional relevance of the genes which are encoded in the metagenomes. The author also adds that the functional diversity at the level of transcription displays a subject-specific metagenome regulation pattern which is measured by the function of the top 10 gene families in the specific analyzed individual (Franzosa et al., 2014).

2.3.4 Viral Screening for Development of Therapeutics The large portion of the antiviral therapeutics which are available in the market involves inhabitation of a viral protein which shows catalytic ability. Viruses encode necessary enzymes which are unique from cellular genes which may make them easier to be targeted with specific therapeutics. Scientists can utilize this property by applying highthroughput small molecular screening technologies or enzyme assays which are target based to determine specific drugs which could inhibit the viral proteins. It is easier to apply this technique on viruses that have a limited coding ability as it is easier to identify if a specific compound is targeting the viral protein or not. This technique is still in research and has recently been employed by utilizing a small molecule library called REDD1 and tested on influenza virus and vesicular stomatitis virus (VSV) infection and by using the Niemann-Pick C1 as a cellular receptor for the Ebola virus (Chen et al., 2010).

2.3.5 Therapy 2.3.5.1 Omics Approach for Elucidating Host and Virus Interaction Viruses, by virtue of their structure, are dependent on their host for the cellular processes such as DNA replication, RNA capping and splicing, translation, and transport of messenger RNA to accomplish the replication of their genome for invading host machinery and survival (Cherry, 2009). During the process, the viral proteins interact with the host cell proteins to assist the process. Multiple sophisticated technologies have been used to understand the complete process of viral genome interaction with the host cell genome, using the host cell factors, but the complete identification had not been possible due to the absence of systematic methodologies. The recent progress in the development of novel techniques to study cellular factors involved in host
virus interaction has now made it

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possible to develop a detailed understating (Watanabe et al., 2010). The development of RNA interference (RNAi) methods has brought the possibilities to study the loss-offunction screenings (Meliopoulos et al., 2012). Some of the recent advances in the high-throughput screening using virus as the examples are listed in Section 2.4.

2.4 APPLICATIONS 2.4.1 Cell-Based Screenings The antiviral drugs work by inhibiting the viral proteins, therefore hampering the completion of the molecular mechanism needed to complete the viral genome replication. The viruses encode special enzymes which are distinct from the host cell enzymes. Screening for such enzymes helps developing the drugs that inhibit the viral replication cycle. Previously the screening of target proteins led to the development of libraries which were used by the pharmaceutical industry to develop the antiviral drug of interest, but the exact target protein of interest is still marked unknown in most of the disease. Therefore there is a room for improvement and exploration for the development of target-based biochemical assays. Cell-based assays are used to pinpoint the target compounds used against the viral proteins. Since viruses have limited coding capacities, the screening can provide straightforward evidence whether a specific compound hampers the viral replication process by targeting the viral proteins. Compounds can be targeted this way but the identification of the cellular pathway is not possible with the compound screening technique. New targets for some of the viruses have been discovered using this technique. Some examples have been shared in Table 2.1. The method of developing therapeutic molecules using the small molecules or target molecule/compound screening has worked successfully for various diseases but the limitations of this method can be overcome by the use of reverse genetic screening methods which directly reveal the sequence of the target molecule (Mata et al., 2011; Coˆte´ et al., 2011; Filone et al., 2010).

2.4.2 Gain-of-Function Method One of the basic techniques that are used in genetic screening is the gain-of-function method, the other being the loss-of-function approach. In the gain-of-function approach, the function of a specific gene can be probed with the help of the ectopic cDNA TABLE 2.1 Examples of Target Proteins in Viral Replication Cycle Compound

Function

Protein kinase Cε (PKCε)

A factor required to assist the infection by Rift Valley virus

REDD1

A factor that restricts the infection by VSV and the influenza virus

Niemann-Pick C1 (NPC1)

Ebola virus cellular receptor

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expression. Previously, the technique used the libraries of shotgun cloned cDNA. The MCG collection has enhanced the applicability of this technique.

2.4.3 Loss-of-Function Method The RNAi methodology has been used for the development of the loss-of-function screening. The different RNAi tools such as the short hairpin RNAs, small interfering RNA, and the long double-stranded RNA are some of the commercially available tools used for this purpose.

2.4.4 Comparative Genome Profiling The comparative genome profiling helps to find out the potential viruses in the same family or between different families that target the same enzymes, proteins, or cellular factors. The screening for the proteins gives only the modest target proteins by the RNAi screens because of a minimal overlap against the same virus (Stertz and Shaw, 2011). The comparison between the viruses of the same group has been made; for example, the comparative screening for coxsackievirus B and polio virus infection in polarized endothelial cells was performed and more than 70% host factor dependency overlap was observed (Coyne et al., 2011). Genome profiling study revealed a high level of dependency in the flaviviruses (Krishnan et al., 2008). A comparative genome profiling performed on the gene set of lymphocytic choriomeningitis virus, varicella zoster virus, and human parainfluenza virus type 3 revealed that all three viruses had 35% similarity of the validated genes (Panda et al., 2011).

References Ambrose, H.E., Clewley, J.P., 2006. Virus discovery by sequence-independent genome amplification. Rev. Med. Virol. 16 (6), 365
383. Benitez-Paez, A., Belda-Ferre, P., Simon-Soro, A., Mira, A., 2014. Microbiota diversity and gene expression dynamics in human oral biofilms. BMC Genomics 15, 311. Bikel, S., Valdez-Lara, A., Cornejo-Granados, F., Rico, K., Canizales-Quinteros, S., Sobero´n, X., et al., 2015. Combining metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems-level understanding of human microbiome. Comput. Struct. Biotechnol. J. 13, 390
401. Blinkova, O., Victoria, J., Li, Y., Keele, B.F., Sanz, C., Ndjango, J.B., et al., 2010. Novel circular DNA viruses in stool samples of wild-living chimpanzees. J. Gen. Virol. 91, 74
86. Breitbart, M., Hewson, I., Felts, B., Mahaffy, J.M., Nulton, J., Salamon, P., et al., 2003. Metagenomic analyses of an uncultured viral community from human faeces. J. Bacteriol. 185 (20), 6220
6223. Breitenstein, S., Tummler, B., Romling, U., 1993. Pulsed field gel electrophoresis of bacterial DNA isolated directly from patients’ sputa. Nucleic Acids Res. 23 (4), 722
723. Chen, T., Yu, W.H., Izard, J., Baranova, O.V., Lakshmanan, A., Dewhirst, F.E., 2010. The human oral microbiome database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database 2010 (1), baq013. Cherry, S., 2009. What have RNAi screens taught us about viral
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452. Clewley, J.P., 2004. A role for arrays in clinical virology: fact or fiction? J. Clin. Virol. 29, 2
12. Coˆte´, M., Misasi, J., Ren, T., Bruchez, A., Lee, K., Filone, C.M., et al., 2011. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection. Nature 477 (7364), 344
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Coyne, C.B., Bozym, R., Morosky, S.A., Hanna, S.L., Mukherjee, A., Tudor, M., et al., 2011. Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers. Cell Host Microbe 9 (1), 70
82. Fierer, N., Breitbart, M., Nulton, J., Salamon, P., Lozupone, C., Jones, R., et al., 2007. Metagenomic and smallsubunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl. Environ. Microbiol. 73, 7059
7066. Filone, C.M., Hanna, S.L., Caino, M.C., Bambina, S., Doms, R.W., Cherry, S., 2010. Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon. PLoS One 5 (11), e15483. Finkbeiner, S.R., Allred, A.F., Tarr, P.I., Klein, E.J., Kirkwood, C.D., Wang, D., 2008. Metagenomic analysis of human diarrhea: viral detection and discovery. PLoS Pathog. 4, e1000011. Foldes-Papp, Z., Egerer, R., Birch-Hirschfeld, E., Striebel, H.M., Demel, U., Tilz, G.P., et al., 2004. Detection of multiple human herpes viruses by DNA microarray technology. Mol. Diagn. 8, 1
9. Franzosa, E.A., Morgan, X.C., Segata, N., Waldron, L., Reyes, J., Earl, A.M., 2014. Relating the metatranscriptome and metagenome of the human gut. Proc. Natl Acad. Sci. USA 111 (22), E2329
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604. Hickman, S.E., Kingery, N.D., Ohsumi, T.K., Borowsky, M.L., Wang, L.C., Means, T.K., 2013. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16 (12), 1896
1905. Jin, L., Lohr, C.V., Vanarsdall, A.L., Baker, R.J., Moerdyk-Schauwecker, M., Levine, C., et al., 2008. Characterization of a novel alphaherpesvirus associated with fatal infections of domestic rabbits. Virology 378, 13
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2088. Kapoor, A., Victoria, J., Simmonds, P., Wang, C., Shafer, R.W., Nims, R., et al., 2008. A highly divergent picornavirus in a marine mammal. J. Virol. 82, 311
320. Krishnan, M.N., Ng, A., Sukumaran, B., Gilfoy, F.D., Uchil, P.D., Sultana, H., et al., 2008. RNA interference screen for human genes associated with West Nile virus infection. Nature 455 (7210), 242
245. Liu, D., Graber, J.H., 2006. Quantitative comparison of EST libraries requires compensation for systematic biases in cDNA generation. BMC Bioinform. 7, 77. Lovley, D.R., 2003. Cleaning up with genomics: applying molecular biology to bioremediation. Nat. Rev. Microbiol. 1 (1), 35
44. Mata, M.A., Satterly, N., Versteeg, G.A., Frantz, D., Wei, S., Williams, N., et al., 2011. Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor. Nat. Chem. Biol. 7 (10), 712
719. Mcfadden, B.F., 2006. Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. Cell 124, 767
782. Meliopoulos, V.A., Andersen, L.E., Birrer, K.F., Simpson, K.J., Lowenthal, J.W., Bean, A.G., et al., 2012. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. FASEB J. 26 (4), 1372
1386. Mihindukulasuriya, K.A., Wu, G., St Leger, J., Nordhausen, R.W., Wang, D., 2008. Identification of a novel coronavirus from a beluga whale by using a panviral microarray. J. Virol. 82, 5084
5088. Moss, T.W., 2007. Association of vaccinia virus fusion regulatory proteins with the multicomponent entry/fusion complex. J. Virol. 81, 6286
6293. Ozsolak, F., Milos, P.M., 2011. Single-molecule direct RNA sequencing without cDNA synthesis. Wiley Interdiscip. Rev. RNA 2 (4), 565
570. Ozsolak, F., Platt, A.R., Jones, D.R., Reifenberger, J.G., Sass, L.E., McInerney, P., 2009. Direct RNA sequencing. Nature 461 (7265), 814
818. Panda, D., Das, A., Dinh, P.X., Subramaniam, S., Nayak, D., Barrows, N.J., et al., 2011. RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses. Proc. Natl Acad. Sci. USA 108 (47), 19036
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866. Russell, J.R., Huang, J., Anand, P., Kucera, K., Sandoval, A.G., Dantzler, K.W., 2011. Biodegradation of polyester polyurethane by endophytic fungi. Appl. Environ. Microbiol. 77 (17), 6076
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Schaffer, U., Schlosser, A., Mu¨ller, K.M., Scha¨fer, A., Katava, N., Baumeister, R., et al., 2010. SnAvi
a new tandem tag for high-affinity protein-complex purification. Nucleic Acids Res. 38 (6), e91. Schoenfeld, T., Patterson, M., Richardson, P.M., Wommack, K.E., Young, M., Mead, D., 2008. Assembly of viral metagenomes from yellowstone hot springs. Appl. Environ. Microbiol. 74, 4164
4174. Stertz, S., Shaw, M.L., 2011. Uncovering the global host cell requirements for influenza virus replication via RNAi screening. Microbes Infect. 13 (5), 516
525. Storch, G., 2007. Diagnostic virology. In: David, P.M., Knipe, M. (Eds.), Field’s Virology. Lippincott/Williams & Wilkins, Philadelphia, PA, pp. 565
604. Ternette, N., Yang, H., Patridge, T., Llano, A., Cedeno, S., Fischer, R., et al., 2015. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells. Eur. J. Immunol. 46 (1), 60
69. Watanabe, T., Watanabe, S., Kawaoka, Y., 2010. Cellular networks involved in the influenza virus life cycle. Cell Host Microbe 7 (6), 427
439. Zhang, L., Ding, L., Cheung, T.H., Dong, M.Q., Chen, J., Sewell, A.K., et al., 2007. Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol. Cell 28 (4), 598
613. Zhang, T., Breitbart, M., Lee, W.H., Run, J.Q., Wei, C.L., Soh, S.W., et al., 2006. RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol. 4, e3. Zheng, J., Sugrue, R.J., Tang, K., 2011. Mass spectrometry based proteomic studies on viruses and hosts—a review. Anal. Chim. Acta 702 (2), 149
159. Zheng, J., Tan, B.H., Sugrue, R.J., Tang, K., 2012. Current approaches on viral infection: proteomics and functional validations. Front. Microbiol. 3, 393.

Further Reading Bexfield, N., Kellam, P., 2011. Metagenomics and the molecular identification of novel viruses. Vet. J. 190 (2), 191
198. Zhang, J., Niu, D., Sui, J., Ching, C.B., Chen, W.N., 2009. Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis: insights on liver angiogenesis. Proteomics 9 (10), 2836
2845.

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3 Algal Biotechnology: An Update From Industrial and Medical Point of View Hemaiswarya Shanmugam1, Ramaraj Sathasivam2, Raja Rathinam3, Kulanthaiyesu Arunkumar4 and Isabel S. Carvalho3 1

Anna University, MIT Campus, Chennai, Tamil Nadu, India 2Sangmyung University, Seoul, South Korea 3University of Algarve, Faro, Portugal 4Central University of Kerala, Kasaragod, India

3.1 MICROALGAE—AN INTRODUCTION Algae are a large and diverse group of eukaryotic organisms and represent a vast variety of photosynthetic species dwelling in diverse environments (Mata et al., 2010). They are distributed worldwide in the sea, in freshwater, in marine water, and in most situations on land. Most are microscopic; cell size can vary from 1 μm up to 10 m (Fig. 3.1). Some can produce resting stages called cysts that can survive in sediments for at least 10 to 50 years. The most important classes are green algae, red algae, and diatoms. The total number of algal species is estimated around 72,500 (Guiry, 2012). Algae comprise a variety of unicellular and some simple multinuclear and multicellular eukaryotic organisms such as green algae, diatoms, red algae, brown algae, and dinoflagellates. Cyanobacteria have a prokaryotic cell structure typical of bacteria and conduct photosynthesis directly within the cytoplasm, rather than in specialized organelles.

3.1.1 Biological Importance of Microalgae 3.1.1.1 Foods Microalgae are rich in carbohydrates, proteins, and lipids. However, most of the microalgae are abundantly rich in vitamins and minerals like vitamins A, B1, B2, C, and E, nicotinate, biotin, folic acid, pantothenic acid, niacin, iodine, potassium, iron, magnesium, and

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00003-6

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(A)

10µm

10 µm (C)

(D)

5 µm (E)

FIGURE 3.1 Morphological structure of a few microalgae. (A) Chlamydomonas sp., (B) Chlorella sp., (C) Dunaliella sp., (D) Botryococcus braunii, (E) Chlorella vulgaris stained by Nile red, (F) microalgae culture in a mini raceway pond.

(B)

50µm (F)

calcium. Nowadays, commercially made biomass is promoted as a health food, in the form of pills, capsules, and liquids (Pulz and Gross, 2004). Microalgae Spirulina (Arthrospira) and Chlorella are also leading in the algal market due to the ease of biomass production. Spirulina has high protein content and excellent nutrient value (Spolaore et al., 2006). It has 62% amino acid and is a rich source of vitamin A and B12 of naturally mixed carotenoids and xanthophyll. Chlorella is used in the healthy food market, as well as for feed and aquaculture. Dunaliella tertiolecta and Euglena gracilis are being sold as health foods in Japan. Most of the carotenoids have therapeutic value including antiinflammatory and anticancer activities, which are largely attributed to their strong antioxidant nature. The market value for carotenoids was about US$1.2 billion in the year 2010 and these carotenoids were produced by chemical synthesis. Dunaliella salina is a green halophilic, unicellular alga that produces β-carotene. It has numerous useful properties in food, feed, pharmaceutical, and cosmetic divisions (Sathasivam et al., 2014). Under unfavorable conditions it can accumulate high amounts of β-carotene (10% 14%) of algal dry weight (Raja et al., 2007; Sathasivam et al., 2012; Sathasivam and Juntawong, 2013). Intake of algal β-carotene showed protective effect against atherosclerosis in both mouse and humans. For example, in mouse and humans, administration of β-carotene inhibits low-density lipoprotein oxidation; influences plasma I. MICROBIAL AND PLANT TECHNOLOGIES

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(A)

(B)

PLATE 3.1 (A) A R&D type of photobioreactor. (B) Concrete raceway pond with a paddle wheel. (C) DAF tank. (D) Culture dryer (industrial scale). (E) Flocculated culture.

(C)

(D)

(E)

triglycerides, cholesterol, and high-density lipoprotein levels; and decreases many kinds of cancer and degenerative diseases. Astaxanthin is the second important carotenoid produced by the freshwater green algae Haematococcus pluvialis. It has protective properties against cancer, inflammatory diseases, metabolic syndrome, diabetes, diabetic nephropathy, neurodegenerative diseases, and eyerelated diseases (Cysewski and Lorenz, 2004). Astaxanthin is used in aquaculture as a pigmentation source as well as in nutraceuticals, food, and feed industries. The annual worldwide aquaculture market of astaxanthin is worth US$230 million with a normal cost of US $2,500/kg. Chlorella zofingiensis also produces astaxanthin but it is meagre amount (Pelah et al., 2004). Canthaxanthin is a type of secondary carotenoid which is perfectly suitable as a diet coloring dye; it delivers color to egg yolks. Astaxanthin and canthaxanthin show vitamin E substances of the liver. Coelastrella striolata, Scenedesmus komareckii (Hanagata, 1999), aplanospores of D. salina (Borowitzka and Huisman, 1993), and Ch. zofingiensis are the microalgae that have the ability to produce large amounts of canthaxanthin (Plate 3.1). I. MICROBIAL AND PLANT TECHNOLOGIES

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3.1.1.2 Feed Microalgae play a significant part in nutritional food for animals which extends from aquaculture to farm animals (Shields and Lupatsch, 2012). Currently more than 40 strains are used in the aquaculture field. Among this genus, Spirulina is widely used as a feed because the pigment of this alga is suitable in the aquaculture industries especially as a feed for fishes which live in tropical areas. The carotenoid from Dunaliella and Haematococcus and lutein from Muriellopsis sp. and Scenedesmus almeriensis (Del Campo et al., 2007) play a major role in the growth of fish larvae. Astaxanthin is needed for growth and existence of shrimp, salmon, and trout (Lakeh et al., 2010). When chicken were fed Porphyridium sp. it was found that the cholesterol of egg yolk was lower by about 10% and the color of egg yolk was darker due to higher carotenoid. 3.1.1.3 Fatty Acids Microalgae are also responsible for the production of essential fatty acids (EFAs), particularly the long-chain polyunsaturated fatty acids (PUFAs) like γ-linolenic acid (GLA) (18:3 ω-6), arachidonic acid (AA) (20:4 ω-6), eicosapentaenoic acid (EPA) (20:5 ω-3), and docosahexaenoic acid (DHA) (22:6 ω-3) (Borowitzka, 2013). As humans and animals lack the requisite enzymes to synthesize PUFAs of more than 18 carbon atoms, they have to obtain them from their foods (Certik and Shimizu, 1999). DHA is a most important structural fatty acid for brain and eye development in babies and has been shown to assist in cardiovascular health condition in adults (Ward and Singh, 2005). Also, GLA is an important precursor in the synthesis of prostaglandins. In this respect DHA, EPA, and GLA are extremely effective. DHA oil comes to market from Crypthecodinium cohnii and it holds 40% 50% DHA but not any EPA or PUFAs. The oil rich in both DHA and EPA is produced by Schizochytrium strain and it has also reached the market (Ward and Singh, 2005). EPA is produced in large amounts by Chlorella minutissima (Seto et al., 1984). Nannochloropsis and Nitzschia also have the ability to produce EPA-rich oil (Spolaore et al., 2006). 3.1.1.4 Cosmetics Extracts taken from microalgae are available in the form of face, skin care, and hair products. Spirulina extract is rich in protein and thus repairs skin aging, whereas the extract from Chlorella vulgaris stimulates collagen synthesis in skin and helps in wrinkle reduction. A component from Nannochloropsis oculata has excellent skin-tightening functions and D. salina has the capability to significantly increase cell proliferation and energy metabolism of the skin. Microalgae such as Porphyridium and Rhodella and many cyanobacteria have the ability to produce polysaccharides (De Philippis et al., 2011). Other active compounds like mycosporine are used in cosmetics that have potential application in natural sunscreen. Mycosporine is produced by cyanobacteria and some algae (Llewellyn and Airs, 2010). 3.1.1.5 Biofertilizers Microlgae are in practice in the agri industry as biofertilizer and to maintain the soil in a healthy form. Microalgae can offer more than 20 kg nitrogen/(ha year) when they are

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used as biofertilizers. Cyanobacteria (BGA) establish a key group which are capable of fixing atmospheric nitrogen. Like other countries, India has also commercialized the production of BGA inoculants. The extract of BGA consists of several active compounds which stimulate plant growth and development (Aly et al., 2008). The cyanobacteria, namely Anabaena, Nostoc, Aulosira, Tolypothrix, and Scytonema, are also used in paddy field to get more yield. 3.1.1.6 Anticancer Activity A major part of death in men and women throughout the world happens due to cancer. Nowadays natural products show a significant role in cancer therapy. Anabena and Nostoc sp. play a major role because they can yield over 120 secondary metabolitic products having anti-HIV, anticancer, antifungal, antimalarial, and antimicrobial activities. Also most of the marine cyanobacteria are potential producers of bioactive compounds that are effective in killing cancer cells (Boopathy and Kathiresan, 2010; Shanab et al., 2012). Under nitrogen conditions, the two cyanobacterial species Nostoc muscorum and Oscillatoria sp. showed an increased and comparable antioxidant and anticancer activities. Chlorosulfolipid, a novel compound isolated from Poterioochromonas malhamensis, was shown to inhibit protein tyrosine kinase activity (Gerwick et al., 1994). 3.1.1.7 Antiviral In recent years, a number of infectious diseases have been developed by viruses. Because of this reason, algae have gained more attention as possible providers of antiviral agents (Borowitzka, 1995). The sulfated polysaccharide, calcium spirulan, derived from Spirulina platensis shows antiviral activity by inhibiting the entry of enveloped viruses such as herpes simplex virus, human cytomegaloviruses, and measles virus into the cell (Ayehunie et al., 1998). The sulfated polysaccharide from Porphyridium has also been shown to display antiviral activities against HSV-1, HSV-2, and varicella zoster virus by preventing the adsorption of the virions (Huleihel et al., 2001). The extract from Dunaliella primolecta (Ohta et al., 1998) showed antiviral activity against HSV, and the screening of extract from 600 cultures of cyanobacteria showed a hit rate of 10% in inhibiting cellular infection of HIV-1, HSV-2, and antirespiratory syncytia virus (Patterson et al., 1993). 3.1.1.8 Antibacterial Under unfavorable conditions, microalgae has the ability to accumulate secondary metabolites (Skulberg, 2004). The first antibacterial compound isolated from microalgae is by Pratt et al., 1944. Chlorella can accumulate a combination of fatty acids like chlorellin, which is liable for inhibition against Gram-positive and Gram-negative bacteria. Microalgae Phaeodactylum tricornutum showed cell lysate activity against Gram-positive and Gramnegative bacteria [including multidrug-resistant Staphylococcus aureus (MRSA)], even at micromole levels. Hexadecatrienoic acid isolated from P. tricornutum displays activity against S. aureus (the Gram-positive pathogen).

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3.1.1.9 Antifungal Algae are bioactive natural resources and due to the medical importance they have been investigated for the control of fungal pathogens. In recent years, there have been many reports of microalgae-derived compounds that have a broad range of biological activities, such as antiviral, antibacterial, and antifungal activities against human pathogens (Vallinayagam et al., 2009). The culture filtrates of Anabaena flosaquae, Anabaena oryzae, Ch. vulgaris, No. muscorum, Nostoc humifusu, Oscillatoria sp., Phormidium fragile, Sp. platensis, and Wollea saccata were tested for antifungal activity. Among all the strains, only Sp. platensis, Oscillatoria sp., and No. muscorum showed maximum effect on fungal mycelium. 3.1.1.10 Biofuel The depletion of fossil fuel is a major problem for human beings as it appears imminent. To replace the fossil fuel, the alternate source for biodiesel is only microalgae. The oil content of microalgae may range from 16% to 68% dry weight and the oil yield can reach up to 136,900 L/ha compared to other plant crops, which ranges from 172 to 5950 L/ha (Chisti, 2008). Chlorella appears to be a potential feedstock for biodiesel production (Fig. 1B). For example, Chlorella protothecoides produces a crude lipid content of 55.2% dry weight when grown under heterotrophic condition on glucose. At present, Raphidophyceae, Botryococcus, and some other microalgae are drawing attention, as they produce a large volume of carbon hydride of carbon numbers 30 40. 3.1.1.11 CO2 Sequestration The increasing concentration of CO2 in the atmosphere is considered to be one of the main causes of the global warming problem. Electrical power plants are responsible for over one-third of the US emissions or about 2.2 3 109 tonnes CO2/year (Kada, 2001). The chlorophycean member exhibited capability to fix CO2 while arresting solar energy 10 50 times more than the land plants. E. gracilis is one of the high CO2-tolerant species. This species enhanced its growth at 5% 45% concentration of CO2 (Nakano et al., 1996), whereas Chlorella sp. T-1 and Scenedesmus sp. can withstand up to 100% and 80% of CO2 concentration, respectively (Maeda et al., 1995; Hanagata et al., 1992). 3.1.1.12 Wastewater Treatment The practice of algae in wastewater treatment appears to be gifted for microalgal growth joined with biological washing. Cyanobacteria were reported to be effectively used for treatment of organic pollutants from paper industry wastewater (Pinto et al., 2003). Ch. vulgaris successfully demonstrated the elimination of nitrogen and phosphorus from wastewater with 72% nitrogen and 28% phosphorus (3 8 mg/L NH1 4 and 1.5 3.5 mg/L PO23 ) (Aslan and Kapdan, 2006). Other algal cultures such as Chlorella, Scenedesmus, and 4 Spirulina have eliminated nutrients. 3.1.1.13 Bioremediation/Phycoremediation Industrialization has led to increased emission of pollutants into ecosystems. Metals are taken up by algae through adsorption (Gosavi et al., 2004). The alga Scenedesmus obliquus was found to accumulate more Cd and Zn with higher phosphorus concentrations,

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whereas Selenium (Se) accumulation was found to be inhibited. Shehata and Badr (1980) cultured Scenedesmus in different concentrations of copper, cadmium, nickel, zinc, and lead to evaluate their effects on the growth of algae. The concentration of metal that reduced Scenedesmus growth was 0.5 mg/L for Cu, 0.5 mg/L for Ni, 2 mg/L for Cd, and 2 mg/L for Zn.

3.2 SEAWEEDS (MACROALGAE)—AN INTRODUCTION There is a high demand for arable land in those countries with increasing population, especially in Asia and Africa, because of converting cultivable land to habitation, low rainfall, conversion of forest area for cultivation, and soil pollution. Thus, ocean becomes a suitable alternate to meet the demand of growing population because nearly 71% of the surface and 97% of the water of our planet earth is occupied by ocean. More than 3.5 billion people depend on ocean for their primary source of living. In 20 years, this could be doubled to 7 billion (http://www.savethesea.org/STS%20ocean_facts.htm). It is firmly believed that the first living cell of the planet earth emerged from the ocean which contains several forms of bio lives. Being a primary producer, marine flora is composed of unicellular to multicellular forms that support all other fauna in the ocean as they grow, multiply, and disintegrate. Out of marine flora, 90% are algae, and 50% of the global photosynthesis is algal derived. Thus, every second molecule of oxygen we inhale comes from algae, and algae reuse every second molecule of carbon dioxide we exhale (Melkinian, 1995). There are two types of algae in the oceans: benthos and phytoplanktons. Dead algae drift to the open ocean and form a source of food for detritus and filter feeders in ecosystems further away. Thus, the productivity of the benthic algae in shallow waters directly or indirectly alters the efficiency of the marine ecosystem. Seaweeds like red, green, and brown based on their pigmentation and none are poisonous. So far 1500 species of Chlorophyceae, 6500 species of Rhodophyceae, and 1780 species of Phaeophyceae have been recorded worldwide in the ocean. They form very tiny, quite large and normally grows up to 30 m long. The primary production per square meter of the kelp is among the highest in the world, comparable to the tropical jungles. Seaweed is an alternative and relatively less known source of food and chemicals. Growing seaweed needs no land. It does not require transport (Fig. 3.2). Besides these, seaweeds have various advantages over classical crop plants such as high biomass yield, require neither arable land nor additional H2O, and can be engineered to increase the efficiency of photosynthesis (Jickells and Spokes, 2001). Seaweeds are rich in carbohydrates, which can be converted in to sugars and finally to alcohol and other fuel forms by biotechnology. But the chemical composition of seaweed is significantly different from mainland plants. So, new biotechnological tools need to be developed to convert the unique marine biomass into sugars. Further, the water footprint of biofuel production from conventional agricultural biomass is extremely large, running into the use of large

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FIGURE 3.2 (A) Seaweed as benthic forms; (B) red, Gracilaria edulis (G.) S.; (C) green, Ulva lactuca L.; (D) brown, Turbinaria decurrens; (E) Portieria hornemannii (Lyngbye) P.Silva-Red; (F) coralline alga, Corallina elongata E. & S.; (G) coralline alga, Amphiroa fragilissima (L.) Lam.

amount of freshwater per liter of fuel obtained. All seaweeds at one stage are unicellular as spore or zygote and may be temporarily planktonic.

3.2.1 Seaweed Phycocolloids Phycocolloids are polysaccharides of high molecular weight composed of simple sugars extracted from freshwater and marine algae. Till date only polysaccharides extracted from red and brown algae like agar, carrageenan, and algin had economic and commercial significance. These water-soluble colloids are non-crystalline substances exhibits viscous and sticky properties in the solution which refers to seaweed gum. They give excellent jellying, stabilizing, and emulsifying properties, with many applications in many industries.

3.2.2 Phycocolloids of Red Seaweeds The sulfated polysaccharides are categorized as agar and carrageenans by their stereochemistry. Galactans with 4-linked α-galactose of the L-series are called as agar and D-series are carrageenans (Knutsen et al., 1994).

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3.2.2.1 Agar and Its Structure Agar is a structural carbohydrate in the cell walls of some members of Rhodophyceae which are otherwise called agarophytes. Species of Gelidium, Gracilaria, Gelidiella, Ahnfeltia, Pterocladia, Aconthopeltis, and Annfeltia are common agarophytes. The structure of agar is an alternating sequence of 3-linked β-D-galactopyranose and 4linked 3,6-anhydro-α-L-galactopyranose. In natural state, agar is in the form of calcium salt or a mixture of calcium and magnesium salts. It is a combination of polysaccharides which contains two main fractions: agarose, a neutral polymer; and agaropectin, a charged, sulfated polymer. The gelling fraction agarose is a neutral linear molecule free of sulfates in regular structure. Agaropectin is a sulfated nongelling fraction (3% 10% sulfate), comprising agarose and different percentage of ester sulfate, D-glucuronic acid, and meager amounts of pyruvic acid. Agarose has at least two-thirds of natural agar-agar.

3.2.2.1.1 CARRAGEENAN

Carrageenan is another major sulfated hydrocolloid found in some family members of the class Rhodophyceae such as Solieriaceae, Rhabdoniaceae, Hypneaceae, Phyllophoraceae, Gigartinaceae, Furcellariaceae, and Rhodophyllidaceae (Istinii et al., 1994). Structurally it is a linear polymer formed by alternate units of D-galactose and 3,6-anhydro-galactose (3,6-AG) joined by α-1,3- and β-1,4-glycosidic linkage. It contains a repeated structure of alternating 1,3-linked β-D-galactopyranose and 1,4-linked α-D-galactopyranose units. The yield of carrageenan is 75.6% in Kappaphycus striatum and 71.0% in K. alvarezii recorded on dry weight (Pereira et al., 2009). At least 15 different types of carrageenans are categorized based on their structural characteristics which include sulfate patterns and the presence or absence of AnGal on D-units. However, industrially suitable carrageenans are available in the form of κ, ι, and λ. It is yet another high molecular weight polysaccharide with 15% 40% of estersulfate content. 3.2.2.2 Polysaccharides of Brown Seaweeds The primary carbohydrates in Phaeophyceae members (brown seaweeds) are mannitol, laminarin, alginate, and fucoidan. 3.2.2.2.1 ALGINATE

Quantitatively the major polysaccharide of the brown seaweeds is alginic acid reaching up to 40% of the dry weight depending on species, for example, 22% 30% in Ascophyllum

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nodosum, 25% 44% in Laminaria digitata, 17% 33% in L. hyperborea, 25% 38% in L. hyperborea, and 17% 45% in Sargassum sp. It is a polysaccharide consisting of unbranched chains comprising β-l,4-linked D-mannuronic acid and α-l,4-linked L-guluronic acid. Alginates are used commercially as thickening agents by the food and pharmaceutical industries as binders, gelling agents, and wound absorbents.

3.2.2.2.2 LAMINARIN

Laminarin is a water-soluble storage polysaccharide of low molecular weight found in some members of brown seaweeds constituting up to 35% of the dry weight. Laminarin is a linear polymer of 1,3-β-D-glucan with a side branching comprised of 1,3-β-D-glucopyranose with some 6-O-branching in the main chain and some β-1,6 intrachain links (Dunstan and Goodall, 2007).

3.2.2.2.3 FUCOIDAN

Fucoidan is a sulfated polysaccharide of long branched chains of sugars with high fucose. Fucoidan type, its sulfation, molecular weight, and sugar differ in each and every species. Hence, fucoidan in Fucus vesiculosis has 90% fucose; however, Undaria holds equal amount of fucose and galactose. Fucoidans are structurally different among species of brown seaweeds and classified into two groups: (1) fucoidans with central chains composed of 1,3-linked α-L-fucopyranose residues and (2) fucoidans with central chains composed of 1,3- and 1,4-linked α-L-fucopyranose residues. The fucoidan of Turbinaria conoides revealed 33% 34% terminals, 27% 28% linked, and 21% 22% branched in the 1,3-linked main chain. It should be recorded that the fucoidans are of high molecular weight and also there is some percentage of smaller fucoidan-type molecules with proteins (Jiao et al., 2011).

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3.2.2.3 Polysaccharides of Green Seaweeds 3.2.2.3.1 ULVAN

Ulvan is the water-soluble polysaccharide found in green seaweeds of the order Ulvales. It has xylose, sulfate, rhamnose, and iduronic and glucuronic acids as major elements. Yield from 8% to 29% on dry weight was reported in Caulerpa, Ulva, Enteromorpha, and Monostrom (Lahaye and Robic, 2007). Structurally it shows great complexity and variability. The repeating disaccharide units are ulvanobiouronic acid 3-sulfate types comprising either glucuronic or iduronic acid. Furthermore, there are minor repeat units that have sulfated xylose replaced by the uronic acid or glucuronic acid as a branch on O-2 of rhamnose-3-sulfate (Tabarsa et al., 2012).

So far, these polymers have not been used commercially; however, Lahaye and Robic (2007) proposed that ulvan could be the source of (1) rare sugar precursors for the synthesis of fine chemicals, (2) oligosaccharides that could be used as pharmaceuticals, and (3) a gelling agent for designing gels with precisely controlled textures. 3.2.2.3.2 FOOD

Hawaiians, Chinese, Japanese, Malaysian, and other peoples in many countries of the world are consuming seaweed as fresh food, salad, and various prepared forms in their regular diet. Apart from a rich source of phytochemicals, fibers, and minerals that improve

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the mineral content and reduce the salt level of human body, seaweeds are also recognized as a chief source of glyconutrients. Trace glyconutrients are needed for the body to function properly and to ensure a basic communication between cells that eventually keep the body balanced. The most important glyconutrients are fucose, galactose, glucose, mannose, and xylose. 3.2.2.4 Prebiotic Potential of Polysaccharides Present in Seaweeds Gibson and Roberfroid introduced the concept of prebiotic in 1995. They defined it as a fermented component that allows certain variations in the activity of the gastrointestinal microflora that favors the host health. Prebiotics must satisfy three criteria: (1) resistant to acid and enzymatic hydrolysis in the upper gastrointestinal tract (GIT); (2) act as a substrate for the growth of microflora and alter their profile; and (3) induce luminal or systemic effects to host health. Health can be maintained by promoting the beneficial bacteria and suppressing the pathogens in the GIT of lives.

Harmful bacteria

Beneficial Bacteria

Gastrointestinal tract Bacteroides, Prevotella, Eubacterium, Clostridium and Bifidobacterium,

Lactobacillus, Staphylococcus, Enterococcus, Streptococcus, Enterobacter and Escherichia

The use of prebiotic is a cost-effective one and prebiotics act via modulation of intestinal microbial populations, thereby improving the health (O’Sullivan et al., 2010). 3.2.2.5 Mechanism of Action of Prebiotics Compared with other polysaccharide sources, seaweeds are promising sources for prebiotics (Fig. 3.3), for example, Rhodophyceae, Phaeophyceae, and Chlorophyceae (up to 60% dry weight). Also agar, carrageenan, alginate, laminarin, fucoidan, galactan, ulvan, and so on with varying structures are potential sources of prebiotics (O’Sullivan et al., 2010). 3.2.2.6 Nutraceuticals It is an end product of isolated nutrients, dietary supplements, herbal products, specific diets, and processed foods. Polysaccharides, fatty acids, proteins, amino acids, vitamins, minerals, phlorotannins, and pigments extracted from various seaweeds were proved to have potent antioxidant, anticancer, antiviral, anticoagulant, antidiabetic, antiallergic,

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FIGURE 3.3 Seaweed as a promising source for prebiotics.

antiinflammatory, antihypertensive, antibacterial, and radioprotective properties. Advances in the process technology have helped in the extraction of commercially important nutraceuticals from seaweeds including ω-3 PUFAs, carotenoids, fucoxanthin, phycoerythrin, and β-carotene (Kim, 2013). 3.2.2.7 Cosmetics and Cosmeceuticals Cosmeceuticals resulted from cosmetics and pharmaceuticals where a specific product has some active components. Greeks and Roman used them as medicinal remedies and in cosmetic. Cosmetic applications of seaweeds are well known from long back and they are used in preparation of soap, lotions, cleansers, cream, foam to shave, shampoo,

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FIGURE 3.4 Seaweeds as a source of bioactive molecules with a wide range of biological activities.

and so on. Cosmeceutical improvement became significant due to the presence of bioactive compounds. Compared to land plants and animals, seaweeds have health-endorsing molecules and materials like dietary fiber, ω-3 fatty acids, necessary amino acids, vitamins A, B, C, and E, which are essential for cosmeceuticals. These are found in seaweeds growing in extreme cold at polar regions (Thomas and Kim, 2013; www. bohemia-style.com). 3.2.2.8 Pharmaceuticals Seaweeds are a promising natural source of bioactive molecules with a broad range of biological activities (Fig. 3.4). The bioactive compounds in seaweeds were identified as polysaccharides, fatty acids and hydroxyl unsaturated fatty acids, glycolipids, steroids, phenolics, and terpenoids. Besides these, halogenated compounds with bromine, chlorine, and even iodine metabolites like diterpenes and triterpenes are also reported to possess bioactivities related to ichtyotoxic, antioxidant, antimalarial, insecticidal, and cytotoxic properties. 3.2.2.9 Seaweeds as Biological Control Against Animal and Plant Pathogens Apart from commercially viable phycocolloids (Jiao et al., 2011), seaweeds are also known to be a rich source of bioactive compounds extracted in organic solvents (Fig. 3.5)

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Seaweeds (Arunkumar et al., 2011) Extraction by combination of polar and non-polar solvents

Individual solvent extraction either by polar (water, methanol, ethanol, acetone) or nonpolar (petroleum ether, diethyl ether, chloroform)

Crude extracts contain broad range of antimicrobial potential

Fractionated forms

Semipurified/purified forms

characterized as acrylic acids, fatty acids and hydroxyl unsaturated fatty acids, sulpho/glycolipids, steroids, phenolics and terpenoids, lauric acid, palmitic acid, linolenic acid, oleic acid, stearic acids

Active against animal pathogens under in vitro and in vivo assay

Active against plant pathogens under in vitro and in vivo assay

FIGURE 3.5 Seaweeds contain rich source of bioactive compounds.

(Arunkumar et al., 2010) in crude or purified forms active against pathogenic microbes of animal and plant origin (Jime´nez et al., 2011). 3.2.2.10 Bioremediation Growing living organisms to treat or reduce the pollutants level is known as bioremediation. Integrated seaweed cultivation coupled with aquaculture is to reduce the environment pollution of marine ecosystem. Commercially viable seaweed species, such as Ulva, Enteromorpha, Gracilaria, Kappaphycus, Laminaria, Porphyra, Condrus, and Undaria, have been successfully developed as biofilter in aqua farms (Chung et al., 2002). 3.2.2.11 Pigment Extraction and Production Seaweeds are a rich source of pigments namely chlorophylls, carotenoids, and phycobiliproteins (phycocyanin, allophycocyanin, and phycoerythrin). Carotenoids are organic pigments found in chloroplasts and chromoplasts of seaweeds. Carotenoids show antioxidant properties depending upon the species of seaweeds. These are capable to quench single oxygen and scavenge free radicals. The most important carotenoids are β-carotene, fucoxanthin, and tocopherol. Dry algal biomass consists of β-carotene ranging from 36 to 4500 mg/kg. Fucoxanthin comprises 70% of total carotenoid content (Holdt and Kraan, 2011).

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3.2.2.12 Seaweeds Tissue Culture

Seaweed tissue culture

Genetic engineering of crop plants

Micropropagation using tissue culture methods

Large scale propagation of clones with superior traits

Bioprocess technology

Production of high-value chemicals in pharmaceuticals, neutraceuticals, etc.

Micropropagation

Cultivation, secondary metabolites production, genetic improvement, sustainable development, and utilization of seaweed

Protoplast biotechnology

To get protoplast cell wall treated with lytic enzymes

Enzymatic methods:combination of enzymes-to date B9 species, green: cellulase or in combination with macerozyme, brown and red: alginase and agarase/ carrageenase and cellulase

Genetic transformation

Transient expression of the bacterial betaglucoronidase gene (gus) in the Kappaphycus alvarezii

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Development of techniques to isolate plant organs, tissues, and cells has led to opportunities in plant biotechnology and cell cultures for in vitro genetic manipulation and commercially viable products. This tissue culture technique was initiated in 1978 and has enhanced the prospects of seaweeds (Reddy et al., 2008). Currently, the demand for seaweed has exceeded the supply of seaweed from natural stock. 3.2.2.13 Drug Delivery Presently, drug delivery field is given more attention as it keeps a lengthy therapeutic effect at a lower dose. A dose generally has one or more active values with different substances added in order to enable the preparation and administration which promotes the consistent release and bioavailability of the drug devoid of degradation. It is now known that excipients can possibly influence the drug action. Phycocolloids, especially alginate polymers, have a vital role in drug designing due to the absence of toxicity. Alginates can be customized to fitting in well with a person’s needs in pharmaceutical and biomedicals. 3.2.2.14 Bioenergy Owing to the depletion of fossil fuels, there is a need for sustainable energy. Biomass is stored solar energy if the biomass grown for energy equals the consumption, then there would be no excess CO2 in the atmosphere. Biomass is composed of cellulose/lignin complex substances and is very difficult to degrade. Whereas sulfated polysaccharides are free from lignin and low content of cellulose make them a simpler material for bioconversion than the higher plants (Huesemann et al., 2010; www.oilgae.com) (Fig. 3.6). 3.2.2.15 Biofuel The global yield of cultivated seaweeds was about 8 million metric tons wet weight per year with an approximate cost of US$6 billion in 2003, with 50% growth being spent as human food. In comparison, the contribution of seaweeds as renewable fuels is insignificant and even the likely seaweeds for biofuels have been accepted for several years along with ethanol, butanol, or other fermentations. The annual production is roughly estimated as 80,000 100,000 t wet biomass per hectare. The seaweed is a nonarable and alternate crop to animal feedstock as potential biomass for ethanol production. 3.2.2.16 Biomineralization Some seaweeds that biochemically precipitate carbonates up to 80% in their body weight are called calcareous algae (CA) and the phenomenon is called biomineralization or biocalcification. CA belong to members of Chlorophyceae and Rhodophyceae (also

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Biomass

Thermochemical conversion

Biochemical conversion

Direct combution

Electricity

Pyrolysis

Oil, Syngas, Charcoal

Thermo chemical liquefaction

Oil

Gasification

Syngas

Photobiological hydrogen production

Hydrogen

Alcoholic fermentation

Ethanol

Anaerobic digestion

Methane, Hydrogen

FIGURE 3.6 The flow chart shows the production of energy from seaweed biomass.

called coralline algae), and few Phaeophyceae, and to some extent Cyanophyceae, which was recorded in the past, but not now. CA accumulate biominerals mainly into three forms of crystals, namely, calcite (CaMgCO3), aragonite (CaCO3), and dolomite (CaMg (CO3)2). Calcite and dolomite are rhombohedral crystals whereas aragonite is an orthorhombic one. Besides these crystals, CA crystallize biominerals of unpredicted shape and size which have potential in various industrial and pharmaceutical applications (Fragoso et al., 2010). 3.2.2.17 Bionanocrystallization The impact of ocean acidification on calcifying algae can be predicted based on the solubility of calcite and aragonite as amorphous CaCO3 and other poorly defined minerals as in a precursor phase. Estimating the impact of changing carbonate ion concentration in calcifiers provides an understanding that controls chemical variations on biogenic carbonates. Further, structural identification of amorphous, cryptocrystalline, and nanocrystalline CaCO3 in vivo by nanoscale spectroscopy through synchrotron-based technology helps to understand the mechanisms of incorporation of trace impurities (Mg, Sr, B, and S) in calcifying algae. This will be an index for determining paleoclimatic variations, through proxies. A nanocrystal is a crystalline particle with at least one dimension measuring less than 1000 nm (1 nm 5 1029 m).

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3.3 CONCLUSION

Seaweeds

Exraction of pigments/ bioactive compounds using standard solvents/methods

Antimicrobial activities against animal and plant pathogens

Extraction of phycocolloids/sulfated polysaccharides

Nutraceuticaland pharmaceutical compounds

Gelling and stabilizing agents for food, bakery, confectionaries, textiles etc.

Pharmaceutical, prebiotics etc.

Spent biomass

Anaerobic digestion— methane, hydrogen

Photobiological hydrogen prodiction

Ethanol production by fermentation

To conclude, a vast research on extraction, processing, and cultivation of seaweeds is currently being undertaken to standardize the methods and strategies in place to develop more than one product from these resources as proposed in the scheme. This chapter revealed the recent advances and potential new applications in the form of biotechnological tools for seaweeds. This chapter focuses on the chemical and structural nature of compounds isolated from seaweeds brought the potentially viable bioactive compounds.

Acknowledgments The author Rathinam Raja would like to thank the Foundation for Science and Technology-FCT, Portugal, for funding him.

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Skulberg, O.M., 2004. Bioactive chemicals in microalgae. In: Richmond, A. (Ed.), Microalgal Culture. Blackwell Science, Oxford, pp. 485 512. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambet, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101 (2), 87 96. Tabarsa, M., Lee, S.J., You, S.G., 2012. Structural analysis of immunostimulating sulfated polysaccharides from Ulva pertusa. Carbohydr. Res. 361, 141 147. Thomas, N.V., Kim, S., 2013. Beneficial effects of marine algal compounds in cosmeceuticals. Mar. Drugs 11, 146 164. Vallinayagam, K., Arumugam, R., Kannan, R.R., Thirumaran, G., Anantharaman, P., 2009. Antibacterial activity of some selected seaweeds from Pudumadam Coastal Regions. Global J. Pharmacol. 3 (1), 50 52. Ward, O.P., Singh, A., 2005. Omega-3/6 fatty acids: alternative sources of production. Process Biochem. 40, 3627 3652.

Further Reading Alcalde, M., 2007. Laccase: biological functions, molecular structure and industrial applications. In: Polaina, J., MacCabe, A.P. (Eds.), Industrial Enzymes: Structure, Function and Applications. Springer, New York, pp. 459 474. , ISBN:978-1-4020-5376-4. Henson, J.M., Butler, M.J., Day, A.W., 1999. The dark side of the mycelium: melanins of phytopathogenic fungi. Annu. Rev. Phytopathol. 37, 447 471. Available from: http://www.bohemia-style.com. Available from: http://www.oilgae.com. Available from: http://www.savethesea.org. Kerby, N.W., Stewart, W.D.P., 1988. The biotechnology of microalgae and cyanobacteria. In: Rogers, L.G., Gallon, J.R. (Eds.), Proceedings of the Phytochemical Society of Europe, Biochemistry of the Algae and Cyanobacteria. Clarendon Press, Oxford, pp. 319 334. Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., et al., 1989. Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. J. Biol. Chem. 264 (1), 472 478.

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4 Omics Approaches in Fungal Biotechnology: Industrial and Medical Point of View Natesan Sudhakar1, Hemaiswarya Shanmugam2, Sekar Kumaran1, Ana Coelho3, Ricardo Nunes3 and Isabel S. Carvalho3 1

Muthayammal College of Arts and Science, Rasipuram, Tamil Nadu, India 2Anna University, MIT Campus, Chennai, Tamil Nadu, India 3University of Algarve, Faro, Portugal

4.1 INTRODUCTION Fungi exist in two unique morphologic growth forms, the unicellular yeast and the filamentous fungi. The osmotrophic growth habit of fungi is highly efficient to colonize various habitats and make the fungi the principal degraders of biomass in the terrestrial environments and infect both plants and animals. Omics pertains to the collective technology used to explore the functions, associations, and actions of the biologic molecules which make up the cells of an organism. The current classification of omics incudes genomics intended for DNA, transcriptomics intended for messenger RNA, proteomics meant for peptides and proteins, and metabolomics in support of advanced products of metabolism. Technologic developments allow parallel examination of thousands of genes and proteins by means of high-throughput analysis equipments. Moreover, hypothesismotivated research and breakthrough-driven investigation by means of omics methodologies are complementary and complete. The suffix “ome” as utilized in molecular biology relates to a totality. Omes can offer simple shorthand to encapsulate a subject. Researchers are quickly trying out omes and omics. Fungal omics are now extensively applied to assist comprehend both the fundamental fungal biology and connected applications. The next-generation sequencing techniques have shaped Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00004-8

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a qualitative radical alteration and new questions to be approached (Kabran et al., 2012). Moreover, it is easy and cheap to re-sequence the complete genome to agree on a point mutation as disparate to follow the conventional path of genetic mapping and subsequent gene cloning and sequencing. Furthermore, the ready accessibility to genome and transcriptomes has changed the status of model organisms. In addition, omics technology provides the tools required to look at the variations in DNA, RNA, proteins, along with other cellular substances between species and among species. These kinds of molecular profiles can differ with cell or tissue coverage to molecules or drugs and therefore have potential use in toxicologic assessments. Omics experiments can often be conducted in high-throughput assays that produce tremendous amounts of data on the functional and/or genomic alterations within the cell.

4.2 INSIGHTS INTO FUNGAL GENOMICS Genomics is the study of genes and their functions. Among the eukaryotic genome sequences presently accessible, over 50% come from the kingdom Fungi. Furthermore, 50% of the fungal genes determined thus far are completely new to science, implying they are distinctive to fungi. In addition, genetic analysis shows fungi might contain many distinctive coding sequences. Because of the benefits of the rapid development of next-generation sequencing (NGS) technology, bioinformatics algorithms, and the comparatively smaller sizes of fungal genomes compared with some other eukaryotes, sequencing and evaluation of fungal genomics become much simpler. It is arguable that the most interesting concepts are rising from the accessibility to multiple genomes. With the acquisition of fungal genomic data, other omics data, like proteomics data, have been increasingly reported. Consequently, appropriate data mining of those omic data in depth and the obtained information can benefit our knowledge of the complicated fungal biologic techniques from genotype and structure to phenotype, including cell communications and pathogen host connections and beyond. In the prospect, high-throughput quantitative proteomics, associated with transcriptomic sequencing, is set to show a big deal in revealing the role of every protein in fungi. There are genes that are orphan with no assigned functionality and gene families. Highthroughput genomic and proteomic assays help in understanding the functional role of these orphan genes. (Kabran et al., 2012; Calo et al., 2014). The emerging branches of fungal genomics are as follows: Comparative genomics:

Study of the relationship of genome structure of fungi and function across different biologic species

Functional genomics:

Study of the fungal protein functions and interactions

Epigenomics:

Study of the entire epigenetic modifications of a cell

Metagenomics recovered:

Study of fungal metagenomes, that is, genetic material directly from environmental samples

Microarray is a high-throughput screening consisting of assays designed to generate and analyze several information from one experiment. Genomics and proteomics research has been advanced throughout the advancement of experimental techniques that increase

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throughput like microarrays. The reports usually involve searching for modifications in gene expression patterns by cells or tissue under various conditions. Microarrays provide a platform for analyzing the changes in several genes simultaneously. Furthermore, the fungal communities of the human gut microbiota continue to be overlooked for a long time due to the relatively low quantity of fungi with respect to bacteria, and only lately few reports have investigated its dynamics in health status or disease (Strati et al., 2016). The application of metagenomics to the fungal communities happens to be restricted to the very less representation of fungal species with regard to bacteria. An amplicon-based ITS1 metagenomics evaluation of fecal fungal population from 111 healthy subjects was conducted to study their tolerance towards gastrointestinal (GI) tract challenges and their vulnerability to antifungals (Strati et al., 2016). Furthermore, fungal species isolated were showing many phenotypic characteristics associated with intestinal environment like tolerance to body temperature, to acidic and oxidative stress, and to bile salts exposure as well. Moreover, a high incidence of azole resistance in fungal isolates, having potential and significant clinical impact, was observed. Fungi are the major plant pathogens. Moreover, Fusarium sp. hosts differ from cucumbers to humans. A recent study cites a total of more than 20 lakhs life-threatening fungal infections per year with Aspergillus sp., Candida sp., Cryptococcus sp., and Pneumocystis sp. as the major global opportunistic pathogens and with mortalities attainment changeable between 20% and 90%. Information derived from whole genome sequencing is at this time been almost usually used to deal with phylogenetic problems (Liu et al., 2006). There are three studies which have resolved the phylogeny of the fungal kingdom through whole genome data and resolved primarily associations among the ascomycetes, highlighting the accessibility to entire genomes at the time (Fitzpatrick et al., 2006).

4.3 INSIGHTS INTO FUNGAL TRANSCRIPTOMICS Transcriptomics is the analysis of transcriptomes and their functions. Transcriptome is the set of all RNA molecules, including tRNA, rRNA, mRNA, as well as other noncoding RNA, created in one or a population of cells. The fungal transcriptome is the all-inclusive group of RNAs encoded by the fungi genome. Microbial transcriptome and metatranscriptome information is very important for the comprehension of host pathogen interactions that are forecasting immune resistance to particular antibiotics which are quantifying gene expression changes and monitoring disease progression. Next-generation RNAseq of viruses, bacteria, and fungal transcriptome is now a standard way of assessing metatranscriptome and transcriptome information. Metatranscriptomes include all RNAs encoded with several organisms in a sample that is complex. The catch of gene expression signatures that are pathogens straight from the host assures to fuel our insight into the exceedingly modern nature of microbial harmfulness. Furthermore, we found a high consistency of azoles opposition in fungal isolates, with possible and substantial scientific impact. The recent findings of many host-infecting fungal transcriptome give new opportunities to analyze the shared traits of plant and animal pathogeneses, which may coordinate the sensible outline of more extensive range of antifungal agents. Quality subtelomeric and clustering gene repertoire collections can be of good significance. Pathogenic microorganisms

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becoming immune to traditional drugs are dramatically rising due to the growth of secondary resistance or inherent primary resistance as a consequence of long-term antimicrobial treatments. Because of invading fungal pathogens and host share a significant number of cells which are metabolically and physiologically possible goals thus, significant challenges are encountered by the generation of new antifungal drugs. Thus, new and economical strategies to fight fungal infections are crucial. For this purpose, no side effects for the host that was infected and novel antimycotics with distinctive fungal goals are desperately desired. The prevalence of microbial diseases in people continues to be drastically increasing within the last few decades. This really is largely due to the rising number of patients afflicted by disorders or immunosuppressive illnesses, like leukemia or AIDS, or the immunosuppressive unwanted effects of cancer chemotherapeutics (Zitvogel et al., 2008). Treatments against fungal infections cost around 3 billion dollars/year in the United States alone. Of those, Candida sp. infections cost around 2 billion dollars. Chitosan is a characteristic compound with a shown antimicrobial action (Lopez-Moya et al., 2016). Chitosan hinders development of filamentous fungi and yeast (Kulikov et al., 2014; LopezMoya et al., 2015). Besides, transcriptomics uncovers plasma membrane homeostasis and oxidative metabolism genes as crucial players in the response of fungi to chitosan. Omics technologies advance our understanding about the mode of activity of chitosan and help its enhancement as an antifungal and gene modulator. These studies open new potential outcomes to create chitosan as a promising antifungal treatment (Liu et al., 2011).

4.4 INSIGHTS INTO FUNGAL PROTEOMICS Proteome is the whole complement of proteins, including the modifications made to a certain set of proteins, produced by the organism. Characterizing the identification, function, regulation, and the interaction of cellular proteins of the organism, the proteome, may be a significant accomplishment. Furthermore, sequencing of many fungal proteomes continues to be accomplished. Nevertheless, now a noteworthy challenge in current fungal proteomics is to comprehend the expression, function, and regulation of the entire set of proteins encoded by fungal genomes. Moreover, proteomic studies in fungi like Trichoderma harzianum (Grinyer et al., 2005) and T. atroviride (Grinyer et al., 2004) confer knowledge to comprehend the mechanism required for the biologic control of phytopathogens. The emerging branches of fungal proteomics are as follows: Immunoproteomics: Study of large sets of proteins (proteomics) involved in the immune response Nutriproteomics: Comprehensive information on proteins from three different areas: host organism, food, and resident microorganisms. Proteogenomics: An emerging field of biologic research at the intersection of proteomics and genomics. Proteomics data are used for gene annotations. Structural genomics: Study of three-dimensional structure of every protein encoded by a given genome using a combination of experimental and modeling approaches

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Consolidating MS-based protein identification with the uniqueness of immunoblotting presents a promising procedure for the distinguishing proof of immunoreactive fungal antigens. This examination strategy has discovered particular use inside finding the immunoproteome of fungal diseases. Widespread immunoreactivity in human sera against Aspergillus fumigatus Glit and cryptococcosis (Datta et al., 2009) recommends that immunoaffinity purification of antibody from human or animal sera using recombinant fungal antigens may represent a precious supply of antigen-specific reagent for recognizing local protein in the living being. Explaining how a proteome changes in light of biotic anxiety, for example, fungal attack, is crucial to grasp molecular mechanisms of fundamental host pathogen communications and pathogenesis. In the past years, there has been an upheaval in the change of new methodologies for deciding huge number of proteins expressed in cells and for globally finding the variations in protein expression of the different cell states. The primary precise methodologies for measurement of protein protein interactions depended on high-resolution two-dimensional electrophoresis (2-DE). Measurement by 2-DE gives benefits, not because of general familiarity but also its high determination control segregates protein isoforms with posttranslational adjustments. The most standard methods for quantitative MS-based proteomics are situated in light of specific marking of proteins with stable isotopes, for example, 2H, 13C, 15N, or 18O. Certain innovative procedures, particularly protein separation and assessment, are actually in view of capabilities and stay hard to automate. Separation systems like difference gel electrophoresis (DIGE) can be more amiable to robotization. Be that as it may, reproducibility still remains a test in protein detachment. DIGE additionally enhanced the capability of 2-DE as different test samples may be labeled with particular dye and investigated on a similar gel. In any case, measurement through 2-DE similarly experiences downsides as well. The method keeps on being difficult, of restricted reproducibility and semiquantitative. Besides, 2-DE indicates terrible measurement proficiency for proteins with particular capacities like extraordinary sizes, vast hydrophobicity, low plenitude, or extreme isoelectric point. Moreover there is no protein equivalent of PCR for enhancement of low plenitude proteins, thus an extensive range of recognition in one to numerous million substances for each cell is required. Proteins have properties ascending from their folded structure; in this manner, nonspecific techniques are hard to plan and apply, and the assessment and requirement for PTMs give a significant issue. Interestingly, MS keeps on being adjusted in the previous two decades to empower really quantitative proteomics. Mass spectrometry based proteomics has profited from the few genomes sequenced recently which are beneficial to the kingdom of fungi and can be accessible for some more species also. The ascomycetes, whose genomes have been of late sequenced, have a place with all subphyla of Ascomycota. Apart from the huge group represented by the ascomycetes, basidiomycetous genomes are likewise sequenced, particularly from the white rot fungi and yeast. On the off chance the genomes of different animals were analyzed through disentangling of DNA sequences and comparing putative regulators with learning of fungal strain varieties, which includes normally occurring strains, as well as mutant strains. Numerous reciprocal innovations are being created alone or in mix conspicuous in the arsenals of proteomics and viable or basic genomics in expression of profiling or molecular interaction screening. These include protein arrays, the yeast two hybrid system, phage

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display antibody libraries, surface improved laser desorption and ionization and biologic activity profiling of families of proteins like proteases. The proteome is alterable, highlighting the issues at which a cell is uncovered or, for example, a particular disease process. There’s in this manner conceivably a major number of proteomes for each cell sort. Theory driven research with wary determination of the careful elements of the proteome that offer data pertinent to the specific biomedical question is particularly vital, given that the bottleneck is likely to lie not in deciding the proteins however in their downstream portrayal.

4.5 INSIGHTS INTO FUNGAL METABOLOMICS Metabolomics pertains to the completeness in evaluating the metabolic state of a cell, organ, or organism, so as to determine biochemical alterations which are characteristic of specific disease states or toxicity insults. However, metabonomics pertains to the quantitative description of the dynamic multiparametric metabolism response of living systems to pathophysiologic stimuli or genetic change. Typical metabolomics experiments involve the identification and quantitation of more and more endogenous substances in a biologic sample using chemical techniques like chromatography and MS. The output from all of these techniques is compared to computerized libraries of MS tracings to help the recognition of the substances which are present. There has been some disagreement on the precise differences between metabonomics and metabolomics. The distinction between the two terms is not related to selection of analytic platform: this is just due to utilization amongst distinct groups which have popularized the distinct terms, although metabonomics is more connected with NMR spectroscopy and metabolomics with MS-established techniques. A subset of clusters putatively associated with the virulence of significant pathogens create secondary metabolites. A vital enzyme, most typically a nonribosomal peptide or polyketide synthase, creates a precursor molecule, which can be later changed by other enzymes encoded by the cluster. These genes normally make one product of small molecular weight for nonribosomal peptides, for example, polyketides, terpenes, and indole alkaloids, which possess a limited taxonomic distribution and are dispensable for cellular development. In order to recognize biochemical changes which are characteristic of particular disease states or noxious insults, metabolomics pertains to the complete assessment of the metabolism state of the cell, organ, or organism. The result from all of these techniques is compared to computerized libraries of MS tracings to ease identification of the compounds which are present. Phytotoxic properties of several compounds and the nicely documented cytotoxicity have long recognized them as putative virulence factors. Candidate gene analysis and gene expression profiling of multiple secondary metabolite producing species present us with the primary chance to evaluate their function as a collective molecular characteristic employed by fungi to beat worldwide challenges. Other secondary metabolites have impacts on virulence which are specific to the period of disease as well as the host environment. Furthermore, NMR spectroscopy/chromatographic and electrophoretic separations coupled with MS techniques are employed for metabolomics studies. The secretome is the totality of released molecules that are organic and inorganic components by biologic cells, tissues, organs, and microorganisms. The concept of secretome is advantageous as it might refer to every one of the diagnostic candidates naturally created. Other than the secretome,

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it is coveted to look at the proteomes of movement vesicles and secretory organelles at whatever point the experimental goal is to break down protein secretion. Proteomics of the secretome can be utilized to look for spanking new biopolymer degrading enzymes, such as polysaccharidases and proteases. Lately, the proteome and secretome, related to the utilization of two sugars D-xylose and D-maltose by Aspergillus niger, were looked at by high-determination 2-DE (Lu et al., 2010). Moreover, portion of the intracellular proteome was evaluated by DIGE. The utilization of sugars powerfully influenced the makeup of secretome, yet had just a humble impact on the intracellular proteome. From the adjustments in the proteome found for the different conditions, it was presumed that the alterations were because of the change in the shake flask cultures. Nevertheless, the culture problems as pH control or oxygen control largely affect the structure of the intracellular proteome, highlighting the requirement for those parameters for the metabolism process. Adav et al. (2010) explained that around hundred proteins were perceived from the culture broth of A. niger after 6 days of fermentation. Despite the fact that among the hundreds of proteins most are hydrolytic like polysaccharidases and proteases, it should be understood that these are starvation conditions that do not represent to lignocellulose-prompted conditions. Braaksma et al. (2010) concentrated the secretome of A. niger grown on D-galacturonic acid, under which circumstance the pectinases had been the more transcendent enzymes and D-sorbitol for which the carbohydrases especially α- and β-glucosidases had been the most predominant enzymes. Under issues of starvation, because of carbon source depletion proteases were predominantly present in the secretome, similar to the outcomes portrayed by Adav et al. (2010).

4.6 BIOINFORMATICS The first fungal genome sequence was published during 1996, and as far back as then the quantity of fully sequenced fungi has expanded massively. Fungal genomics likewise prompted a major measure of genome-scale functional data like transcriptomes and proteomes for fungi. The openness to these loads of biologic data gives a chance to systematically analyze and characterize fungi based on their biologic information. Bioinformatics tools mine data from large databases of scientific information. These tools are most often used to analyze large sets of genomic information. Nevertheless, bioinformatic tools are being developed for other types of scientific information like proteomics. The US National Center for Biotechnology Information (NCBI) serves as an integral source of genomics information and bioinformatics tools for researchers. Bioinformatics and databases of biologic information can be used to generate maps of cellular and physiologic pathways. This integrative approach is called computational biology. Bioinformatic tools are being used to extract standards from large-scale to introduce a cell and the organism to foresee the computational frameworks of higher multifaceted nature (for example, the connection arrangements of the cell forms the phenotypic organisms). Virtual evaluation systems will be provided by these in silico models for assessing the toxic reactions of cells, tissues, and organisms. The obligation of natural capacities in silico explained that the qualities are unannotated and remains an imperative test. Along with RNAseq for the assessment of altered gene expression, developing access to online assets

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for viable classification of fungal genes and proteins is essential (Priebe et al., 2011), and robust analytic strategies such as quantitative proteomics will contribute significantly to this challenge. In addition, fungal diseases are an expanding risk for worldwide well-being, and for immunocompromised patients specifically. These diseases are portrayed by the association between the fungal pathogen and host cells. The correct mechanism in host and fungal pathogen cooperation stays to be completely illustrated. A multi-omics approach, for instance, information from genomics, proteomics, and metabolomics together, would permit a more thorough knowledge of both the host and the pathogen. Investigation on diverse-omes of biologic information, notwithstanding rising single-particle representation systems, helps with deciding natural pertinence of multi-omics information. Additionally, complete portrayals of host fungal pathogen frameworks are currently conceivable, and usage of these cutting-edge multi-omics techniques may yield progresses in better comprehension of both host science and fungal pathogens at the system scale.

4.7 SAMPLE PREPARATION CHALLENGES Since fungi have an incredibly tough cell wall, powerful extraction of nucleic acid and proteins are a key stride for molecular studies. Sufficient advancement was made in the field of fungal proteomics in the past many years, because of the enhancements in sample preparation, high-resolution protein separation strategies, tandem MS application for effectively distinguishing and depicting protein, and bioinformatics innovation. Nevertheless, various technologic problems still exist. For proteome extraction, an immaculate procedure would reproducibly grab all types of proteins with low issues of contaminations. The protein extraction system of rice blast fungi, Magnaporthe grisea (Kim et al., 2005), offers reproducible fungal proteins on two-dimensional gels. In this technique, proteins are extricated utilizing Mg/CHAPS extraction buffer containing the following components: 0.5 M Tris HCl pH 8.3, 20 mM MgCl2, 2% CHAPS, 20 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF). A most broadly utilized method for protein precipitation is using 10% trichloroacetic acid (TCA) with acetone, which is typically more powerful than either TCA or acetone alone. In addition, the cell wall is thought to bring about inadequate protein extraction from certain group of fungi, that is, basidiomycetous cells, and accordingly the use of fungal protoplast is of extraordinary intrigue.

4.8 FUNGAL OMICS—A MEDICAL PERSPECTIVE The improvement of extensive and inexpensive omics platforms offers data types for the network medication. While network medication supplies a fundamentally different strategy to understanding disease etiology, it will also lead to key variations in how

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illnesses are treated, with multiple molecular targets that might require adjustment in a coordinated, dynamic style. Much remains to be learned regarding the optimal approaches to integrate various omics data types and also to perform network analyses. Furthermore, massive parallel sequence methods additionally prompted the improvement of RNAseq, and by the by, along with the genome, know the transcriptome, and this in many growth problems or developmental phases.

4.8.1 Role of Fungi on Immunocompromised Patients Fungal infections are believed to happen in over a million people every year, and latest evidence suggests the rate is increasing. Vaccines are not available and despite the enhanced analysis and treatment, the treatment of fungal infections, especially in immunocompromised hosts, is a difficult endeavor. Significantly, the potential infection risk and its clinical outcome vary somewhat even among individuals with comparable predisposing factors. This has prompted study regarding the interaction of the fungi with the host within an endeavor to understand the molecular and cellular causes underlying variable susceptibility to infection. The past decade has seen a remarkable improvement in technology and computational methods. The combination of new omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, together with the use of both established and alternative in vivo models of infection, provides nearly comprehensive views of the dynamics of system-wide host fungus molecular interaction networks during infection. By giving a more profound comprehension of the overall complexity of the biologic, biochemical, and biophysical molecular processes controlling fungus host interaction, this knowledge is likely to allow the design and approval of predictive models of infection initiation, development, and result, ultimately contributing to new clinical methods based on individualized diagnostics and treatment. The integration of individual characteristics into clinically relevant procedures to forecast progression and the chance of fungal infection, and the effectiveness of antifungal prophylaxis and treatment, holds the promise of initiating innovations helping patients affected by or at danger of fungal infections. Latest technologies in omics lead to lay the basis for methodologies incorporating clinical data and omics to support individualized diagnostic and curative interventions. Investigations of fungal communities revealed that the human being intestine mycobiota vary in function in a sex-associated manner of people’s life phase (Strati et al., 2016).

4.8.2 Fungi and the Gut Microflora The intestine of human being is an intricate ecologic niche where fungi, protozoa, archaea, bacteria, and viruses live together with the host in a very complex interaction (Human Microbiome Project Consortium, 2012). However, fungi hold a useful part in the functioning/physiology of the host even though the bacterial population outreaches the number of fungi in the intestinal tract (Huffnagle and Noverr, 2013). Moreover, latest studies revealed that whilst the composition of the bacterial community is comparatively steady with time, the fungal community living in the mammalian intestine gets major changes throughout the life (Dollive et al., 2013). This fetch to the final outcome that environmental

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fungi may influence their makeup and that gut fungal community tends to be more changeable than bacterial ones (Underhill and Iliev, 2014). Regardless of signs that fungi live in the mammalian GI tract with the host immune system (Underhill and Iliev, 2014), features and the makeup of the mycobiota in healthful hosts happen to be poorly investigated. The widespread fascination of phenotypes and pathogenic fungi as well as the method through which they confirm the disease is among the significant base that fetch to fail the beginning part of the commensal fungal community. A latest research study demonstrated the organization of inflammatory bowel disease (IBD) to modification of the intestine mycobiota. Particularly Sokol et al. (2017) demonstrated that IBD patients put up with an inferior percentage of Saccharomyces cerevisiae and higher percentage of Candida albicans compared to healthful subjects. Moreover, Sokol et al. (2017) emphasized the lifestyle, in Crohn’s illness, of connected modifications between fungal and bacterial communities. On the other hand, function of intestine mycobiota is very important, the reports revealed that harmful groups are causing disorder in the intestinal part. However, a number of yeasts happen to be clinically prescribed to get an extended time due to their possible probiotic properties, indicating a favorable function of some mushrooms for the host wellbeing. An excellent illustration of valuable fungus is represented by S. cerevisiae var. boulardii, used for the aid of gastroenteritis (Hatoum et al., 2012). In order to decrease the gap of host intestinal information with its own interaction of a cohort on healthful subjects through fungal cultivations, metagenomics and phenotypic assays should be analysed. For the better improvement of the intestinal fungal community by means of amplicon to cohort the healthful subjects established ITSOne metagenomics that were targeted, looking at age and sex groups differences. Dimensions of fungal abundance in each sample showed no variations which is based on the culture established that we found an augmented number of intestinal fungal community in women compared to men (Sokol et al., 2017). The mix of fungal cultivation and metagenomics enabled the commensalism connected characteristics of isolated fungi as well as a detailed knowledge of the fungal intestine community structure linked to the healthy status. We discussed relatively the outcomes of growing and sequencing to significantly assess the use of metagenomics-based strategies to fungal gut population.

4.9 FUNGAL OMICS—AN INDUSTRIAL PERSPECTIVE Human beings utilize fungi since early history as direct food resources, as antibiotics, and for organic acids production. Fungal enzymes happen to be by and large used in modern applications for a considerable measure of decades, which changeover plant biomass into fermentable sugars. In the earlier decade, total genome sequencing of filamentous fungi enhanced the potential to foresee the encoded proteins altogether, particularly hydrolytic enzymes required for the biosynthesis of metabolites of significance. The incorporation of genome sequence data with phenotypes necessitate, in any case, the understanding of every proteins in the cell in a biologic system, given by proteomics. Moreover, fungi have been used for quite numerous years as resources of extracellular enzymes. The accessibility to entire genomes led the hunt for enzymes with improved qualities.

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However, in most cases enzymes were associated with chitin, cellulose and lignin degradation. In addition, fungi act as possible assisting agent in softwood pulping. Furthermore, lignocellulose oxidation is carrying out by low molecular weight metal binding substances which are separated by the wood degrading fungus. To recognize enzymes with new, encouraging potential, accessibility to its genome sequence, transcriptome, and proteome information is vital. Comparative to bacteria, lack of protein information in fungi is a restrictive factor. There are more than lakhs of protein sequences that were publicly accessible, as contrasted a quarter century ago. In any case, the strategies to get them, while enhancing consistently, with obvious improvement, have not experienced a comparative progressive change to that embodied by NGS techniques. Numerous secondary metabolites are nonribosomal proteins, and furthermore the genes required with their synthesis are clustered. Thus it is moderately easy to perceive in genomes these groups required with their action, as they frequently incorporate more than one enzyme. Numerous bioinformatics techniques have been contrived to identify secondary metabolite gene clusters. Only one representation of those conceivable outcomes is the recognition of the gene clusters accountable for the activity of the first-line therapeutic agent pneumocandin in the genome of Glarea lozoyensis. A comparable circumstance is surviving in the Fusarium sp. and Cochliobolus species (Wiemann et al., 2013): among the previous, F. fujikuroi may possibly orchestrate secondary metabolites having a place with 45 distinct families. Of these 13 17 clusters include polyketide synthases, yet just 3 are common to all the Fusaria analyzed (Nesic et al., 2014).

4.10 CONCLUSION Some fungi infects and causes disease in plants and animals, while others have obvious possibility of the management of insect pests. Besides, fungi may also be a rich reservoir of industrially useful enzymes and healing metabolites. Multiple technologies including progress in bioinformatics and protein MS and genome and transcriptome sequencing now enable comprehensive investigation of fungal biochemistry. The fungal proteins function encoded either by in silico or qualities that are unannotated, stays troublesome. An assortment of methodologies including genomics, proteomics, and metabolomics are summarized, which, when used with different strategies together, seem to give authoritative and complete data on fungal protein function. Unquestionably, there is much to be acquired from the relative assessment of fungal transcriptomes from the start of infection. The advancement of post-genomic fungal examinations has incited interest in supporting the bioinformatic tools, and one-stop comparative genome database which connects the gene function that homologues in other organisms by their genome database on microarrays that are rendering in different information which exists on any fungal pathogens. This needs databases and reasonably arranged datasets that interconnect data of varied species sources, an objective that made exploratory information and need to know form into a need if assets are to be maximally utilized (Tables 4.1 4.5).

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TABLE 4.1 Some Medicinal and Nutritional Properties of Commonly Used Edible Macrofungi Fungi

Appearance

Nutritional Properties

Medicinal Properties

Lentinula edodes

Dark brown cap with white stalk

Contains high protein with all essential amino acids; well-known natural source of vitamin D; adenine and choline content effective in preventing the occurrence of cirrhosis of the liver as well as vascular sclerosis

Tyrosinase contained in Lentinula edodes tends to lower blood pressure. Lentinam, an active polysaccharide a (1 3) b-D-glucan reduces cancer and cholesterol and enhances TH1 response

Volvariella volvacea

Pink gills and spore prints; lack a ring; have an Amanita-like volva at the stem base. The gills of young Volvariella are white

A natural source of antioxidant due to high β-carotene content

Contains a fungal immunomodulatory protein FIP-Vvo that induces TH1-specific cytokines (IL-2, IFN-c, LT), TH2-specific cytokine (IL-4)

Flammulina velutipes

Convex shaped cap; moist and sticky when fresh; color variable—dark orange brown to yellowish brown; gills attached to the stem and whitish to pale yellow, becomes dark rusty brown on maturity

Mannofucogalactan, a heterogalactan derived from Flammulina, is known to possess nutritional values

Induces antibody production by modulation of TH-cell. Differentiation and function

Pleurotus ostreatus

White, gray-brown or ivory colored and resembles oyster shell like shape. The white gills run down its short, off-centered white stalk

Unique flavor and aromatic properties; considered to be rich in protein, fiber, carbohydrates, vitamins, and minerals. Among the volatile compounds that constitute edible mushroom flavor, 1-octen-3-ol is considered to be the major contributor

Promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic, and immunomodulation activities

Tuber melanosporum

Fruiting body or truffle is round, pitted, and white when young but darkens as it matures

Truffle has tantalizing taste and aroma and is most sought after delicacy with great economic value

Regarded as therapeutic food having anticarcinogenic, anticholesterolemic, and antiviral properties and also prophylactic properties with regard to coronary heart disease and hypertension

Ganoderma lucidum

Large, hard, and leathery fungus with sessile or stalked basidiocarps having tiny pores undersurface

Used in dietary preparation and to make tea or soup. Protein comprises only 7.3% of dry weight. Glucose accounted for 11% and metals 10.2% of dry mass (K, Mg, Ge, and Ca being the major trace components)

GLIS, a proteoglycan isolated from the fruiting body, is a B-cell stimulating factor. This compound stimulates B lymphocyte activation, proliferation, differentiation, and production of immunoglobulins

Auricularia polytricha

Ear-shaped structure of fruiting body

Rich in P, Mg, K, and Se; high dietary fiber content more than 50% of net weight. Helps in relieving constipation

The fruiting body produces a new immunomodulatory protein (APP) enhancing the production of both nitric oxide (NO) and tumor necrosis factor-a (TNF-a), suggesting that APP is an immune stimulant and can increase the immune response of its host. APP activates murine splenocytes, markedly increasing their proliferation and gammainterferon (IFN-c) secretion

Tremella fuciformis

Gelatinous, jelly-like basidiocarps having leaf-like folds

Widely eaten in the east; high fiber content makes it popular among dieters and cholesterol-affected people

Very high dietary fiber content, have potential hypocholesterolemic effect, similar to other high-fiber foods

Morchella esculenta

White ridges and dark brown pits; with age both ridges and pits turn yellow

Morels are a feature of many cuisines including Provencal.

Methanolic extracts from Morchella esculenta include antioxidant activity, reducing power, scavenging effects on radicals, and chelating effects on ferrous ions; contains galactomannan that induces macrophage activity

Morchella elata

Ridges are gray or tan; pits are brown and elongated when young; turn black with age

Rich in vitamin D2

Chinese believe it can cure tuberculosis, high blood pressure, and common cold

Morchella semilibera

Small caps and long bulbous stems. The bottom of the cap is attached directly to the stem

Spongy texture of young morels makes delicious dishes

The ethanolic extract of Morchella has 85% of antioxidant property

Agaricus bisporus

The original wild form bear a brownish cap and dark brown gills but more familiar ones are with a white cap, stalk and flesh, and brown gills

Fairly rich in vitamins like vitamin B and minerals like sodium, potassium, phosphorus, and selenium. Raw mushrooms are naturally cholesterol and fat free

Effective in reducing blood serum cholesterol; good dietary source of B-complex vitamins especially riboflavin

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TABLE 4.2 Enzymes Associated with Food and Feed Bio-processing Industries

Enzymes

Butter and butter oils

Catalase, glucose oxidase, lipase

Cheese rennet

Lipase, proteinases

Animal feed

Amylase, glucoamylases, glucanase, cellulases, pentosanases, xylanases, proteinases, phytases

Alcohol

Amylase, amyloglucosidase, β-glucanases, cellulases, cellobiase, pectinase, proteinases

Biscuits

Amylases, cellulases, hemicellulases, proteinases, pentosanases

Breads

Amylases, amyloglucosidases, cellulases, glucanases, glucose oxidase, hemicellulases, lipases, pentosanases, proteinases

Brewing

Acetolactase, decarboxylase, amylases, amyloglucosidase, cellulase, glucanase, lipase, pentosanase, proteinase, xylanase

Coffee

Cellulase, hemicellulases, galactomannanase, pectinase

Confectionery

Amylase, invertase, pectinase, proteinase

Egg processing

Proteinase, lipase phospholipase, Catalase, glucose oxidase

Fats

Esterase, glucose oxidase, lipases

Fish

Proteinase

Dairy products

Lactase, proteinase, sulfhydryl oxidase, lactoperoxidase, lysozyme, peroxidase, catalase

Dibittering

Peptidase, naringinase

Flavors

Glucanase, peptidase, proteinase, esterase, lipases, amylase

Fructose

Glucose isomerase, inulinase, amylase, amyloglucosidase, cellulase, glucanases, hemicellulases, isomerase, lipase, phospholipase, pectinases, proteases

Fruit, cloudy juices

Amylases, pectinases, cellulases, proteinase

Fruit extracts

Anthocyanase

Tea

Cellulase, glucanase, pectinase, tannase

Wine

Amylase, amyloglucosidase, cellulase, glucanase, hemicellulase, pectinases, proteases, glucose oxidase, catalase, pentosanase, anthocyanase

Malt extract

Amylase, amyloglucosidase, cellulase, glucanase, proteinase, xylanase

Animal oil/fats

Esterases, lipases, proteinase

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TABLE 4.3 Some Common Enzymes Used as Additives in Food and Feed Enzyme

Applications

Pullulanase

Antistaling agent in baked goods

Cellulase

Animal feed

Naringinase

Debitter citrus peel

β-Amylase

Brewing, maltose syrup

β-Glucosidase

Transforms isoflavone phytoestrogens in soymilk

α-Amylase

Starch syrups, fermentation, ethanol, animal feed

Lactase

Eliminates lactose from dairy foods

Xylanase

Aspergillus niger (and var. awamori), Aspergillus oryzae, Trichoderma reesei (longibrachiatum)

Pectinase

Fruit processing

Proteases

Brewing, baking goods, protein processing, distilled spirits

TABLE 4.4 Recombinant Enzymes from Fungi Enzyme

Host

Donor

Catalase

Aspergillus niger

Aspergillus sp.

Cellulase

Aspergillus oryzae

Humicola sp.

β-Galactosidase

A. oryzae

Aspergillus sp.

β-Glucanase

Trichoderma reesei (longibrachiatum)

Trichoderma sp.

Glucose oxidase

A. niger

Aspergillus sp.

Lipase

A. oryzae

Candida sp., Rhizomucor sp., Thermomyces sp.

Phytase

A. niger, A. oryzae

Aspergillus sp.

Xylanase

A. niger (and var. awamori), A. oryzae, T. reesei (longibrachiatum)

Aspergillus sp., Thermomyces sp., Trichoderma sp.

Chymosin

A. niger var. awamori

Calf

Protease

A. oryzae

Rhizomucor sp.

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TABLE 4.5 Enzyme Applications in Different Sectors FOOD INDUSTRY Laccase

Phenolic remotion from the food and beverage Ascorbic acid determination Sugar beet pectin gelation

Lignin peroxidase

Source of natural aromatics Production of vanillin

Manganese peroxidase

Production of natural aromatic flavors PULP AND PAPER INDUSTRY

Laccase

Depolymerization of lignin Delignify wood pulps Bleaching of kraft pulps

Lignin peroxidase

Decolouriment of kraft pulp Mill effluents

Manganese peroxidase

Kraft pulp bleaching TEXTILE INDUSTRY

Laccase

Textile dye degradation and bleaching

Lignin peroxidase

Textile dye degradation and bleaching

Manganese peroxidase BIOREMEDIATION Laccase

Biodegradation of xenobiotics Polycyclic aromatic hydrocarbons (PAHs) degradation

Lignin peroxidase

Degradation of azo, heterocyclic, reactive, and polymeric dyes Mineralization of environmental contaminants Xenobiotic and pesticides degradation

Manganese peroxidase

PAHs degradation Synthetic dyes Bleach from paper-producing plants DDT, PCB, TNT

ORGANIC SYNTHESIS, MEDICAL, PHARMACEUTICAL, COSMETICS AND NANOTECHNOLOGY APPLICATIONS Laccase

Polymers production Coupling of phenols and steroids Medical agents (Continued)

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TABLE 4.5 (Continued) Carbon nitrogen bonds construction Complex natural products synthesis Personal hygienic products Biosensors and bioreporters Lignin peroxidase

Functional compounds synthesis Cosmetics and dermatological products Bioelectro-catalytic activity at atomic resolution

Manganese peroxidase

Acrylamide polymerization Polymer styrene degradation Direct electron transfer (DET)

References Adav, S.S., Li, A.A., Manavalan, A., Punt, P., Sze, S.K., 2010. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes. J. Proteome Res. 9, 3932 3940. Braaksma, M., Martens-Uzunova, E.M., Punt, P.J., Schaap, P.J., 2010. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data. BMC Genomics 19 (11), 584. Calo, S., Shertz-Wall, C., Lee, S.C., Bastidas, R.J., Nicola´s, F.E., Granek, J.A., et al., 2014. Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature 513 (7519), 555 558. Datta, K., Bartlett, K.H., Baer, R., et al., 2009. Spread of Cryptococcus gattii into Pacific Northwest region of the United States--a comprehensive review of the emerging outbreak, produced by a multidisciplinary team of investigators in Canada and the USA. Emerg Infect Dis. 15 (8), 1185 1191. Dollive, S., Chen, Y.Y., Grunberg, S., Bittinger, K., Hoffmann, C., Vandivier, L., et al., 2013. Fungi of the murine gut: episodic variation and proliferation during antibiotic treatment. PLoS One 8 (8), e71806. Fitzpatrick, D.A., Logue, M.E., Stajich, J.E., Butler, G., 2006. A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis. BMC Evol. Biol. 6, 99. Grinyer, J., Mckay, M., Nevalainen, H., Herbert, B.R., 2004. Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum. Curr. Genet. 45, 163 169. Grinyer, J., Hunt, S., Mckay, M., Herbert, B.R., Nevalainen, H., 2005. proteomic response of the biological control fungus Trichoderma atroviride to growth on the cell walls of Rhizoctonia solani. Curr. Genet. 47, 381 388. Hatoum, R., Labrie, S., Fliss, I., 2012. Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications. Front. Microbiol. 3, 421. Huffnagle, G., Noverr, M., 2013. The emerging world of the fungal microbiome. Trends Microbiol. 21, 334 341. Human Microbiome Project Consortium, 2012. A framework for human microbiome research. Nature 486 (7402), 215 221. Kabran, P., Rossignol, T., Gaillardin, C., Nicaud, J.M., Neuve´glise, C., 2012. Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica. DNA Res. 19 (3), 231 244. Kim, K.M., Cho, S.K., Shin, S.H., Kim, G.T., Lee, J.H., Oh, B.J., et al., 2005. Analysis of differentially expressed transcripts of fungal elicitor- and wound-treated wild rice (Oryza grandiglumis). J. Plant Res. 118, 347 354. Kulikov, S.N., Lisovskaya, S.A., Zelenikhin, P.V., Bezrodnykh, E.A., Shakirova, D.R., Blagodatskikh, I.V., et al., 2014. Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida albicans: molecular weight-activity relationship. Eur. J. Med. Chem. 74, 169 178.

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Liu, X., Yang, F., Song, T., Zeng, A., Wang, Q., Sun, Z., et al., 2011. Effects of chitosan, O-carboxymethyl chitosan and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl] chitosan chloride on lipid metabolism enzymes and low-density-lipoprotein receptor in a murine diet-induced obesity. Carbohydr. Polym. 85, 334 340. Liu, Y.J., Hodson, M.C., Hall, B.D., 2006. Loss of the flagellum happened only once in the fungal lineage: phylogenetic structure of kingdom Fungi inferred from RNA polymerase II subunit genes. BMC Evol. Biol. 6, 74. Lopez-Moya, F., Colom-Valiente, M.F., Martinez-Peinado, P., Martinez-Lopez, J.E., Puelles, E., Sempere-Ortells, J.M., et al., 2015. Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens. Fungal Biol. 119, 154 169. Lopez-Moya, F., Kowbel, D., Nueda, M.J., Palma-Guerrero, J., Glass, N.L., Lopez-Llorca, L.V., 2016. Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan. Mol. BioSyst 12 (2), 391 403. Lu, X., Sun, J., Nimtz, M., Wissing, J., Zeng, A.P., Rinas, U., 2010. The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate. Microb. Cell Fact. 9, 23. Nesic, K., Ivanovic, S., Nesic, V., 2014. Fusarial toxins: secondary metabolites of Fusarium fungi. Rev. Environ. Contam. Toxicol. 228, 101 120. Priebe, S., Linde, J., Albrecht, D., Guthke, R., Brakhage, A.A., 2011. FungiFun: a web-based application for functional categorization of fungal genes and proteins. Fungal Genet. Biol. 48, 353 358. Sokol, H., Leducq, V., Aschard, H., Pham, H.P., Jegou, S., Landman, C., et al., 2017. Fungal microbiota dysbiosis in IBD. Gut 66, 1039 1048. Strati, F., Di Paola, M., Stefanini, I., Albanese, D., Rizzetto, L., Lionetti, P., et al., 2016. Age and gender affect the composition of fungal population of the human gastrointestinal tract. Front. Microbiol. 7, 1227. Underhill, D.M., Iliev, I.D., 2014. The mycobiota: interactions between commensal fungi and the host immune system. Nat. Rev. Immunol. 14, 405 416. Wiemann, P., Sieber, C.M., von Bargen, K.W., Studt, L., Niehaus, E.M., Espino, J.J., et al., 2013. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog 9 (6), e1003475. Zitvogel, L., Apetoh, L., Ghiringhelli, F., Kroemer, G., 2008. Immunological aspects of cancer chemotherapy. Nat. Rev. Immunol. 8, 59 73.

Further Reading Freitag, J., Ast, J., Bo¨lker, M., 2012. Cryptic peroxisomal targeting via alternative splicing and stop codon readthrough in fungi. Nature 485 (7399), 522 525.

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C H A P T E R

5 Genetic Engineering for Plant Transgenesis: Focus to Pharmaceuticals Surender Khatodia and S.M. Paul Khurana Amity University Haryana, Gurgaon, India

5.1 INTRODUCTION Plants have been used as source for many medicinally important drugs. Advancement in molecular biology and genetic engineering for manipulation of plant’s genome expanded the use of plants for medical products (Twyman et al., 2012). Plant Molecular Farming (PMF) is described as genetically modifying plants for recombinant proteins to produce medicinal and industrial compounds of human value. Molecular farming of plants has been used for the production of pharmaceutical products, such as monoclonal antibodies, vaccines, cytokines, growth factors, and enzymes (Twyman et al., 2012). This chapter highlights the procedure of using plants as bioreactor for recombinant protein production through plants, increasing recombinant protein accumulation in plants, plantibodies, edible vaccines, commercial status, and chloroplast genome engineering for plant-made pharmaceuticals (PMPs). PMF consists of three broad subdivisions including PMPs, plant-made vaccines, and plant-made industrials (Davies, 2005). The PMF production platform comprises three components: (1) the plant of interest such as different crops, vegetables, and fruits; (2) expression system such as stable transformation, plant viruses, transient expression, and chloroplast transformation; and (3) production in open fields and greenhouses. There is a straightforward basic combination of developments in molecular genetics and agricultural biotechnology for pharmaceutical production (Davies, 2005). Plant transgenesis comprises the insertion of any gene of interest into any crop species for a desired trait to bring together useful genes from unrelated sources (Agarwal et al., 2016). So, the

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gene for any high-value protein could be expressed in crops and grown on a large scale for extraction and purification of the recombinant protein after characterization (Davies, 2005). There are many vectors available now, which are designed for very high and stable transgene expression, in particular crop species and novel tissue culture techniques also developed for the generation of transgenic plants from recalcitrant species.

5.2 PLANTS AS BIOREACTORS Using plants as bioreactor for recombinant protein production has several advantages including the low cost of establishment, and the scalability of plants and products generally regarded as safe in comparison to the mammalian cells, Escherichia coli, yeast, and insect cells, which have limitations like high production cost and low yield (Twyman et al., 2012). Plants as a bioreactor fulfill the requirement for the production of large quantities of economic and safe therapeutic recombinant proteins. For the commercial production of pharmaceutical proteins, plants as a bioreactor have a number of advantages over conventional expression systems as follows: 1. The eukaryotic posttranslational modification such as glycosylation and disulfide bond required for biological activity of therapeutic proteins. 2. There is no risk of contamination with human pathogens using plants as bioreactor, which is a major concern. 3. There are simple and inexpensive plant growth requirements, compared to traditional cell culture systems, allowing unrestricted scalability. 4. The vigorous and static nature of plant system makes handling, purification, and their ability to be used raw as oral vaccines easy. The concept of using Plants as Bioreactor needs to focus on the objective of purifying the product from transgenic plants instead of altering the phenotype of the plant (Fischer et al., 2014). Various products that are produced in plants include mainly vaccine antigens, antibodies, nutritional supplements, enzymes, and biodegradable plastics (Sharma and Sharma, 2009; Fig. 5.1).

5.2.1 Recombinant Protein Production From Plants The technology of plant genetic engineering has been applied to plants for producing a recombinant protein to use plants as bioreactor. This has developed rapidly in the last decade and comprises different production platforms and many target pharmaceuticals (Fig. 5.2). The whole process of production of recombinant proteins from plants includes the following constitutional steps. 1. 2. 3. 4.

Choice of the host plant species Optimization of coding sequence of the target gene in relation to the host Selection of expression cassette and creation of the expression vector Approaches for increasing heterologous protein accumulation in plants

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FIGURE 5.1 Plants as bioreactors for biotechnological advances. Source: Adapted from Sharma, A.K., Sharma, M.K., 2009. Plants as bioreactors: recent developments and emerging opportunities. Biotechnol. Adv. 27, 811
832.

LB

RB T

P GOI

Cloning and expression vector construction

T

Transformation Co-cultivation into agrobacterium with agrobacterium

Nuclear transformation

Edible

on

ap Top pl ica ica l tio n

Purificati

Selection

Transgenic plant

P Marker

Regeneration

FIGURE 5.2 Different constitutional steps in the process of PMF. Source: Adapted from Chan, H., Daniell, H., 2015. Plant-made oral vaccines against human infectious diseases—are we there yet? Plant Biotechnol. J. 13, 1056
1070 and Yao, J., Weng, Y., Dickey, A., Wang, K.Y., 2015. Plants as factories for human pharmaceuticals: applications and challenges. Int. J. Mol. Sci. 16, 28549
28565.

5. 6. 7. 8.

Genetic transformation of the gene construct into the plant genome Regeneration and selection of plants expressing the desired protein Identification and characterization of the plant line for commercial production Purification and characterization of the recombinant protein

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5.2.1.1 Plants for Open-Field and Greenhouse Production of Pharmaceuticals The model plant tobacco was the species of choice for most of the recombinant protein production experiments, but now a number of other plant species are also being used including tomato, banana, rice, maize, wheat, carrot, soybean, pea, potato, lettuce, and alfalfa (Sharma and Sharma, 2009). The selection of the plant species initially depends on the form of the recombinant protein to be finally used, then the life cycle of the host, biomass yield, containment, as well as scale-up costs. A broad range of plant species have been used for molecular farming, based on the types of plants as follows: 1. Dry seed cereals and grain legumes (e.g., maize and rice): The recombinant protein could be directed to the seeds for long-term storage of the target protein. 2. Leafy crops (e.g., tobacco and alfalfa leaves): The recombinant protein could be synthesized in an aqueous environment. 3. Fruit and vegetable crops (e.g., tomatoes and bananas): These could be used as oral vaccines, because the raw plant material is edible. 4. Oil crops (e.g., safflower): The recombinant protein could be directed into oil bodies in the seeds, which facilitates downstream processing. 5.2.1.2 Plant-Based Expression Systems Expression system choice depends on many criteria including the form of protein, the posttranslational modifications, scale of production, downstream processing, costs, environmental issues, confinement and containment, and regulatory requirements for drug authorization. There are two broad choices of expression systems for PMPs, i.e., Stable and Transient expression system. Transgenic plants have been the most commonly used expression platform for the production of proteins of pharmaceutical importance because of the flexibility and the efficiency in scaling up (Twyman et al., 2012). Chloroplast transformation in plants is an alternative stable expression system to nuclear transformation for the production of therapeutic proteins in plants because of the high-level accumulation of protein products and transgene containment in chloroplasts (Twyman et al., 2012; Jin and Daniell, 2015). The plastid genome is highly polyploid; therefore, the copy number of any introduced transgene can be amplified by as many as 10,000 per cell, and the expression levels in plant chloroplasts could be ranging from 5% to 20% of the total soluble proteins. Another important plant-based platform for the production of recombinant pharmaceutical proteins in plants is transient gene expression system. Plant viruses have been used for high level of expression of transgenes for vaccine delivery and therapeutic protein production using transient gene expression system (Ko et al., 2010). The transient gene expression system results in rapid and high protein production, which is not achievable via stable gene expression. 5.2.1.3 Bioreactor-Based Plant Systems Plant production platforms are also developed using hairy roots, plant cell culture, moss, and algae, usually in fully contained bioreactors. 1. Hairy roots: The hairy root cultures in liquid medium can be induced in plant species by transformation with Agrobacterium rhizogenes for the production of variety of plant

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metabolites, which could be excreted into the liquid medium and purified easily (Larsen and Curtis, 2012). 2. Plant Cell Suspension Cultures: Plant cell suspension culture could also be used for the whole-plant system
based production of pharmaceuticals. Plant cells such as bacteria could grow using conventional bioreactors and allows the production of correctly folded and assembled proteins with rapid doubling times (Twyman et al., 2012; Larsen and Curtis, 2012). 3. Moss: Physcomitrella patens, a moss variety that could be modified by homologous recombination for the production of pharmaceutical proteins. Physcomitrella can easily be cultured in photobioreactors with a high level of purity and rapid growth rates for protein production in the medium (Ko et al., 2010). 4. Algae: Chlamydomonas reinhardtii is a single-cell species that could be used for the production of pharmaceutical proteins, which can be grown under high density and large volumes for easy and cost-effective downstream processing (Gregory et al., 2012). 5.2.1.4 Increasing Heterologous Protein Accumulation in Plants The main challenge in using plants as protein production system is the initial low yield. Now various molecular approaches have been developed to increase the product yields in plant-based production systems. The main approach for the increased heterologous protein production in plants includes modulations in gene expression at transcription and translation levels along with the subcellular targeting of the proteins. Transcription and Translation Boosting: Promoter elements can have a dramatic effect. The most commonly used constitutively expressed promoters for recombinant protein expression in plants are 35S promoter of cauliflower mosaic virus, rice actin-1 gene, and corn ubiquitin-1 gene. The level of mRNA of a transgene and protein product could be boosted by using novel synthetic promoters by combining the most active sequences of multiple well-characterized natural promoters, e.g., addition of the poly(A) signal from nopaline synthase (nos) gene and inclusion of one or more introns in a gene construct (Rose, 2002). Furthermore, the efficiency of mRNA translation could be boosted by addition of a 50 nontranslated sequence of the natural genes to the transgene construct like leader sequence of tobacco mosaic virus and rice seed storage protein genes. Codon usage optimization of transgenes with the plant species preferred codon usage could also be used for enhancing foreign protein expression in plants (Twyman et al., 2012). Subcellular targeting of recombinant proteins to the suitable compartment of the plant cell is also critical for obtaining high levels of accumulation of foreign proteins by addition of a signal for the specific organelle (Jha et al., 2012). 5.2.1.5 Purification of Recombinant Proteins From Plants After successful optimization of expression of the recombinant protein in plants, the recovery and purification is the next step to the commercialization of plant-made recombinant proteins. There are two alternatives for recombinant protein purification: one is affinity fusion
based and another is non
affinity-based purification. Affinity fusion
based protein purification is done with a partner protein, which is fused with the target recombinant protein in the transgene construct. The fusion tag could prevent proteolysis, increase solubility and stability, and facilitate isolation and

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purification of target proteins in downstream processing. Some of the examples of fusion partner are like Zera, a proline-rich N-terminal domain derived from the maize storage protein zein and polyhistidine tag, usually comprising six consecutive histidines (Twyman et al., 2012). The gene fusion results in the expression of an affinity-tagged fusion protein that can be easily purified via an affinity matrix column for the purification of recombinant proteins from various expression systems. The elastin-like polypeptides (ELPs) are also used as a fusion partner to increase recombinant protein accumulation in plants and for non
affinity-based purification. ELPs are artificial biopolymers containing repeats of the pentapeptide sequence. An ELPs-based non
affinity-based method for protein purification has been depicted in Fig. 5.3 (Wiktorek-Smagur et al., 2012; Chan and Daniell, 2015). A nonchromatographic purification was done by using the inverse temperature transition of the pentapeptide sequence for separation of the ELP-fused polypeptides from the insoluble aggregates by inverse transition cycling). Another method for recovering plant-made proteins via the nonaffinity method is Oleosin fusion, which is hydrophobic proteins of the oil bodies of plant seeds (Moussavou et al., 2015). The recombinant protein and Oleosin fusion products can be easily separated as oil bodies by flotation centrifugation.

5.3 PLANT-MADE PHARMACEUTICALS PMPs have been in Phase II and Phase III clinical trials for many disease models for either injections or oral delivery, and some are commercialized also (Sack et al., 2015). There are two major classes of product in development using genetic engineering of plants for pharmaceuticals—plant-derived antibodies (Plantibodies) and plant-derived subunit

FIGURE 5.3 Purification of ELPylated target proteins from plants using inverse transition cycling (WiktorekSmagur et al., 2012).

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First expression of vaccine-related protein (antibodies, MAbs) in transgenic tobacco plant.

First ever vaccine produced in plant: virus surface antigen for hepatitis B (HBsAg) was expressed in transgenic tobacco plant.

Plant-derived recombinant protein successfully immunized mice against rabbies.

Hiatt et al. (1989)

Mason et al. (1992)

Modelska et al. (1998)

First ever modified tobacco plant cells-derived vaccine was approved for use in chicken against newcastle disease virus (NDV) developed by dow agro sciences.

Miller et al. (2004)

Magnifection: A novel approach was introduced for expressing recombinant vaccines in plants.

Gleba et al. (2005)

Plant-based epitope presentation of CRPV-LI, CPRV-L2 immunized rabbits and proved efficacy of plant-derived HPV vaccine.

First Phase I human clinical trials of anti-idio type vaccine produced in tobacco against non-hodgkin’s iymphoma

First preclinical and phase I clinical trials of virus like particles (VLPs) expressed in tobacco against H5NI influenza virus.

Recombinant glucocerebrosidase: a bio therapeutic expressed in carrot suspension cell culture approved by FDA to treat gaucher's disease.

Completed clinical trials of autologous vaccine for follicular lymphoma

Kohl et al. (2007)

MeCormick et al. (2008)

Laanger (2011)

Rader (2013)

Clinical trials.gov (2014)

FIGURE 5.4 The timeline of development events in PMPs. Source: Modified from Fahad et al., 2015.

vaccines (Edible Vaccines). The production of pharmaceuticals protein in plants started in 1980s, initially in transgenic tobacco. The first antibody was expressed in tobacco, and the structural authenticity of plant-made recombinant proteins was confirmed for the first time in 1992 to produce the hepatitis B virus surface antigen as a vaccine (Pniewski, 2013). The timeline of development events for PMPs has been presented in Fig. 5.4 (Fahad et al., 2015).

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5.3.1 Plantibodies Antibodies are glycoproteins with specific binding activity, which could be used for the diagnosis, prevention, and treatment of disease. When the antibodies are produced in plants, they are called Plantibodies (Hiatt et al., 1989). The complex immunoglobulins, Fab fragments, single variable domains, antibody-fusion proteins, large single-chain antibodies, and heavy-chain antibodies have been expressed in correct folding and assembly successfully in plants (Fischer et al., 2014; Liao et al., 2015). To express the antibodies in plants, the complementary DNAs of heavy and light chains of monoclonal antibody (mAb) are cloned and transferred into the plant genome (Yao et al., 2015). Alteration of the mAb antigen-binding site could be done by mutagenesis to redesign the antigen specificity and increase binding affinity with high antigen specificity (Ko et al., 2010). The heavy and light chain genes can be introduced to plant cells either at the same time by using a single transformation event or the genes are inserted separately in plant lines and co-expressed by crossing these individual lines.

5.3.2 Edible Vaccines Charles J. Arntzen, a plant biotechnologist, first devised the way to induce the transgenic plants to produce the encoded proteins in form of vaccines in the edible parts. The plant parts producing the vaccine proteins could be eaten as Edible vaccines (Langridge, 2006; Fig. 5.5). The transgenic plants producing subunit vaccine proteins could be used for the largescale production and delivery of vaccines to induce protective immune responses (Daniell et al., 2009; Chan and Daniell, 2015). Transgenic vegetable and fruit plants are ideal for FIGURE 5.5 Dr Charles J. Arntzen, the scientist behind edible vaccines (The Biodesign Institute at Arizona State University, United States).

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FIGURE 5.6 An outline for the process of oral delivery of plant-derived vaccine antigens. The antigen genes are introduced and expressed in lettuce chloroplasts via particle bombardment. The confirmation of stable integration into all of the chloroplast genomes in each plant cell and characterization of dosage and functionality were done and transplastomic lines are transferred to the greenhouse to increase biomass. Harvested leaves are lyophilized below 20 C, powdered, and stored in moisture-free environment. Orally administered Cholera Toxin subunit B (CTB)-fused antigens are proposed to be taken up by mucosal cells. Antigens are then captured by antigen-presenting cells, inducing antigen-specific T and B cells to elicit cell-mediated immunity and humoral immunity (Chan and Daniell, 2015).

producing oral vaccines, as they could enable antigens to reach the gut-associated lymphoid tissue by protecting from the acidic environment in the stomach (Chan and Daniell, 2015). An outline for the process of oral delivery of plant-derived vaccine antigens in lettuce chloroplasts have been depicted by Chan and Daniell (2015; Fig. 5.6).

5.3.3 PMPs: Commercial Status The first PMP approved and commercialized for human use is ELELYSO (taliglucerase alfa) in carrot cell suspension for the treatment of Gaucher’s disease manufactured by Protalix BioTherapeutics in 2012. Protalix has taken a normal version of the human gene affected in Gaucher’s disease and introduce it into carrot cells. The lower production costs will allow the company to sell Elelyso for just 75% of the price of Cerezyme, the most popular drug for Gaucher’s disease in the market. Mapp biopharmaceutical has produced a drug in tobacco leaves called ZMapp, which has been used to combat the 2014 Ebola virus

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outbreak in Africa (Arntzen, 2015). Several examples of PMP products commercially available and those in clinical trials are listed in Tables 5.1 and 5.2 (Sack et al., 2014).

5.4 CHLOROPLAST GENOME ENGINEERING FOR PHARMACEUTICALS Chloroplast genome engineering has led to stable integration and expression of transgenes to express pharmaceutical proteins, antibiotics, vaccine antigens, and industrial enzymes. There are several advantages of transforming the chloroplast over the nuclear TABLE 5.1 Examples of Plant-Derived Pharmaceutical Products Assessed in Clinical Studies (Sack et al., 2014) Company

Products

Main application

Current status

Protalix

Elelyso

Gaucher’s ERT

FDA-approved for the United States

PRX-102 (alpha galactosidase) Fabry ERT

Phase I/II

PRX-12 (oral glucocerebrosidase)

Gaucher’s ERT

Phase II

VEN100 (lactoferrin)

Antibiotic-associated diarrhea; antiinflammatory

Phase II

VEN 120

Inflammatory bowel disease

Phase I

VEN130

Osteoporosis

Phase I

Biolex (now Synthon)

Locteron

HCV

Phase II/IIb

Icon Genetics

NHL vaccine MAPP66

HSV/HIV

Phase I/II

Medicago

H5

Pandemic influenza vaccine

Phase II/III, approved for emergency use

Ventria

H5 intradermal seasonal influenza vaccine

Phase I

Planet Biotechnology

CaroRX

Anti-caries antibody

Approved as medical device

Fraunhofer IME

HIV antibody

Microbicide

Phase I

Fraunhofer CMB

HA vaccine

Vaccine

Phase I

VAXX/Arizona State University

NoroVAXX

Vaccine

Phase I

MAPP

ZMapp

Ebola antibody cocktail

Emergency use Phase I

Fabry ERT

Scheduled for Phase I

Greenovation ERT, enzyme replacement therapy.

I. MICROBIAL AND PLANT TECHNOLOGIES

TABLE 5.2 Commercially Available Plant-Produced Recombinant Proteins (Sack et al., 2014) Product

Company

Plant system

Application

Advantage

References

Elelyso

Protalix

Carrot suspension

Injectable pharmaceutical

Plant-specific glycosylation

www.protalix.com/Pastores et al. (2014)

Growth factors, cytokines

ORF

Barley seeds

Cell culture supplement

Endotoxin-free

Magnusdottir et al. (2013)

Growth factors

ORF/SifCosmetics

Barley seeds

Cosmetic ingredient

Animal-free

www.orfgenetics.com

Growth factors, cytokines, antibodies

AgrenVec

Tobacco leaves transient

Research reagent

Animal-free, costs

www.agrenvec.com

Albumin. transferrin, lactoferrin, lysozyme

Ventria/InVitria

Rice seeds

Cell culture supplement

Animal-free

www.ventria.com/www.invitria. com/Alfano et al. (2014)/ Youngblood et al. (2014)

Aprotinin (nonclinical grade)

KBP

Tobacco leaves transient

Research reagent

Cost

http://www. kentuckybioprocessing.com/

Laccase, trypsin, avidin

ProdiGene/Sigma

Corn seeds

Technical reagent

Cost

www.sigmaaldrich.com

Canine interferon alpha

NAIST

Strawberry fruits

Veterinary pharmaceutical/oral

Cost, minimal processing

www.infiniteenzymes.com

Cellobiohydrolase I

Infinite enzymes

Corn seeds

Technical enzyme

Cost

Antibody

CIGB

Transgenic tobacco

Used for the purification of a hepatitis B virus vaccine

Cost, animal-free

Collagen

CollPlant

Transgenic tobacco

Tissue culture. Animal free. pharmaceutical “virgin” collagen applications are envisaged

Growth factors, cytokines, antibodies

NBM

Rice cell suspension Bioreagents, cosmetic ingredients

Animal-free, endotoxin-free

www.collplant.com

http://www.nbms.co.kr/? page_id5244&lang5en

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Cp DNA

Cp DNA P Marker

Cp DNA

T

RBS P Marker

GOI

P GOI RBS GOI

Helium

T

gas

RBS GOI

Cloning and expression vector construction

Cp DNA T

Particle bombardment

Chloroplast transformation

Selection

Regeneration

Maternal inheritance

FIGURE 5.7 Chloroplast transformation using particle gun bombardment of chloroplast vectors is followed by two to three rounds of antibiotic selection and subsequent regeneration of homoplasmic transformants (Chan and Daniell, 2015).

genome like high levels of expression, multigene engineering, gene containment via maternal inheritance, and subcellular compartmentalization. Chloroplast genome-engineering for PMF making up to 70% of total leaf protein and the ability to express in edible leaves permits oral delivery and significantly reduces the production costs (Jin and Daniell, 2015). In the chloroplast engineering technology, transgenes are inserted into the chloroplast genome by site-specific homologous recombination that eliminates the gene silencing, positional effects, and pleiotropic effects in the transgenic lines (Verma et al., 2008). Expression of antigens by chloroplast genome in leaves also allows complete transgene containment, high gene expression levels, and facilitates several important posttranslational modifications (Jin and Daniell, 2015). Extensive optimization was undertaken to develop a reproducible expression system utilizing species-specific chloroplast vectors, endogenous regulatory sequences, and optimal organogenesis/hormone concentrations to directly generate transplastomic lines without callus induction to make Chloroplast Bioreactors for pharmaceuticals (Chan and Daniell, 2015; Fig. 5.7). Lettuce serves as the only reproducible transplastomic system for oral delivery of vaccines and pharmaceuticals. More than 40 pharmaceuticals and vaccine antigens have been expressed via the chloroplast genome (Jin and Daniell, 2015; Table 5.3).

5.5 FUTURE DIRECTIONS The concept of PMF is well established now for the production of pharmaceuticals with the recent approval from FDA to the latest research and development of plant-based production systems using molecular biotechnological approaches. The FDA-approved Elelyso developed by Protalix have become as success story of this field, which fueled the interest of scientists to produce Ebola vaccines in plants. The PMP is a promising field for costeffective prevention of infectious disease and epidemics around the world. The therapeutic compounds could be produced in plants using cell or root cultures, greenhouses, and the fields. The recent developments for increased heterologous protein expression in plants have made the commercialization of the PMP products possible. In conclusion, the PMF could be used to produce affordable healthcare solutions for the developing countries to reduce the disease burden.

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TABLE 5.3 Chloroplast Bioreactors for Functional Pharmaceuticals and Vaccine Antigens (Jin and Daniell, 2015) Pharmaceutical/vaccine antigen

Expression system

Expression level

CTB
AMA1 (malarial vaccine antigen apical membrane antigen-1)

Lettuce Tobacco

CTB
MSP1 (malarial vaccine antigen merozoite surface protein-1)

Functional evaluation

References

7.3% TSP 13.2% TSP

Long-term dual immunity against two major infectious diseases: cholera and malaria

Davoodi-Semiromi et al. (2010)

Lettuce Tobacco

6.1% TSP 10.1 TSP

Long-term dual immunity against two major infectious diseases: cholera and malaria

Davoodi-Semiromi et al. (2010)

EDA (extra domain A-fibronectin)

Tobacco

2.0% TSP

Retains the proinflammatory properties of the EDA produced in Escherichia coli

Farran et al. (2010)

Parvovirus immunogenic peptide 2L21 fused to a tetramerization domain

Tobacco

6% TSP

Immunogenic response in mice

Ortigosa et al. (2010)

Immunogenic fusion Tobacco protein F1-V from Yersinia pestis

14.8% TSP

Oral delivery of F1-V protected 88% of mice against aerosolized Y. pestis; F1-V injections protected only 33% and all control challenged mice died. Oral boosters conferred protective immunity against plague

Arlen et al. (2008)

Coagulation factor IX

Tobacco

3.8% TSP

Prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice

Verma et al. (2010)

BACE (human β-site APP cleaving enzyme)

Tobacco

2.0% TSP

Immunogenic response against the BACE antigen in mice

Youm et al. (2010)

Human papillomavirus L1 protein

Tobacco

21.5% TSP

Confirmed the formation of capsomeres

Waheed et al. (2011a,b)

Proinsulin

Tobacco

47% TSP

Oral delivery of proinsulin in plant cells or injectable delivery into mice showed reduced blood glucose levels

Boyhan and Daniell (2011)

PA (dIV) (domain IV of Bacillus anthracis protective antigen)

Tobacco

5.3% TSP

Demonstrates protective immunity in mice against anthrax

Gorantala et al. (2011)

Human thioredoxin 1 protein

Lettuce

1% TSP

Protected mouse insulinoma line 6 cells from hydrogen peroxide

Lim et al. (2011)

Thioredoxins
human serum albumin fusions

Tobacco

26% TSP

The in vitro chaperone activity of Trx m and f was demonstrated

Sanz-Barrio et al. (2011)

HPV16 L1 antigen fused with LTB

Tobacco

2% TSP

Proper folding and display of conformational epitopes

Waheed et al. (2011a,b)

Exendin 4 (EX4) fused to CTB

Tobacco

14.3% TSP

CTB
EX4 showed increased insulin secretion similar to the commercial EX4 in β-TC6

Kwon et al. (2013)

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TABLE 5.3 (Continued) Pharmaceutical/vaccine antigen

Expression system

Expression level

Functional evaluation

References

CTB
ESAT-6 (6 kDa early Tobacco secretory antigenic target) Lettuce

Up to 7.5% 0.75%

Hemolysis assay and GM1-binding assay confirmed functionality and structure of the ESAT-6 antigen

Lakshmi et al. (2013)

CTB
Mtb72F (a fusion polyprotein from two tuberculosis antigens, Mtb32 and 39)

Tobacco

Up to 1.2%

Not reported

Lakshmi et al. (2013)

CTB fused to MBP (myelin basic protein)

Tobacco

2% TSP

Amyloid loads were reduced Kohli et al. (2014) in vivo in brain regions of 3 3 TgAD mice fed with bioencapsulated CTB
MBP. Aβ(1
42) accumulation was reduced in retinae and loss of retinal ganglion cells was prevented in 3 3 TgAD mice treated with CTB
MBP

Coagulation factor VIII (FVIII) antigens: heavy chain (HC) and C2 fused with CTB

Tobacco

80 or Feeding of the HC/C2 mixture 370 μg/g in substantially suppressed T helper fresh leaves cell responses and inhibitor formation against FVIII in hemophilia A mice

Sherman et al. (2014)

TSP, total soluble protein.

References Agarwal, S., Grover, A., Khurana, S.M.P., 2016. Plant molecular biotechnology: tools to develop transgenics. In: Khan, M.S., et al., (Eds.), Applied Molecular Biotechnology: The Next Generation of Genetic Engineering. CRC Press, Boca Raton, FL. Alfano, R., Youngblood, B.A., Zhang, D., Huang, N., MacDonald, C.C., 2014. Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells. Bioengineered 5, 180
185. Arlen, P.A., et al., 2008. Effective plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts. Infect. Immun. 76, 3640
3650. Arntzen, C., 2015. Plant-made pharmaceuticals: from ‘Edible Vaccines’ to Ebola therapeutics. Plant Biotechnol. J 13, 1013
1016. Boyhan, D., Daniell, H., 2011. Low-cost production of proin- sulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol. J. 9, 585
598. Chan, H., Daniell, H., 2015. Plant-made oral vaccines against human infectious diseases—Are we there yet? Plant Biotechnol. J. 13, 1056
1070. Daniell, H., Singh, N.D., Mason, H., Streatfield, S.J., 2009. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant. Sci. 14, 669
679. Davies, H., 2005. Plant-made pharmaceuticals: an overview and update. In: Eaglesham, A., Bessin, R., Trigiano, R., Hardy, R.W.F. (Eds.), Agricultural Biotechnology: Beyond Food and Energy to Health and the Environment. NABC Report 17. National Agricultural Biotechnology Council, Ithaca, pp. 59
70. Davoodi-Semiromi, et al., 2010. Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol. J. 8, 223
242.

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990. Fischer, R., Buyel, J., Schillberg, S., Twyman, R., 2014. Molecular farming in plants: the long road to the market. In: Howard, J.A., Hood, E.E. (Eds.), Commercial Plant-Produced Recombinant Protein Products SE-3, Biotechnology in Agriculture and Forestry. Springer Verlag, Berlin, pp. 27
41. Gorantala, J., et al., 2011. A plant based protective antigen [PA (dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax. Vaccine 29, 4521
4533. Gregory, J.A., Li, F., Tomosada, L.M., Cox, C.J., Topol, A.B., Vinetz, J.M., et al., 2012. Algae-produced pfs25 elicits antibodies that inhibit malaria transmission. PLoS One 7, e37179. Hiatt, A., Caffferkey, R., Bowdish, K., 1989. Production of antibodies in transgenic plants. Nature 342, 76
78. Jha, S., Agarwal, S., Sanyal, I., Jain, G.K., Amla, D.V., 2012. Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants. Plant Sci. 196, 53
66. Jin, S., Daniell, H., 2015. The engineered chloroplast genome just got smarter. Trends Plant. Sci. 20, 622
640. Ko, K., Brodzik, R., Steplewski, Z., 2010. Production of antibodies in plants: approaches and perspectives. Curr. Top. Microbiol. Immunol. 332, 55
78. Kohli, N., et al., 2014. Oral delivery of bioencapsulated proteins across blood
brain and blood
retinal barriers. Mol. Ther. 22, 535
546. Kwon, K.C., et al., 2013. Oral delivery of bioencapsulated exen- din-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol. J. 11, 77
86. Lakshmi, P.S., et al., 2013. Low cost tuberculosis vaccine anti- gens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro. PLoS One 8, e54708. Langridge, W.H.R., 2006. Edible vaccines. Scientific American. 16, 46
53. Larsen, J.S., Curtis, W.R., 2012. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots. BMC Biotechnol. 12, 21. Liao, Y., Pingli, H., Senzhao, C., Yao, M., Zhang, J., 2015. Plantibodies: a novel strategy to create pathogenresistant plants. Biotechnol. Genet. Eng. Rev. 23, 253
272. Lim, S., et al., 2011. Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts. Plant Mol. Biol. 76, 335
344. Magnusdottir, A., Vidarsson, H., Bjornsson, J.M., Orvar, B.L., 2013. Barley grains for the production of endotoxinfree growth factors. Trends Biotechnol. 31, 572
580. Moussavou, G., Ko, K., Lee, J., Choo, Y., 2015. Production of monoclonal antibodies in plants for cancer immunotherapyBioMed. Res. Int. 2015 (2015), Article ID 306164, 9 pages . Available from: http://dx.doi.org/ 10.1155/2015/306164. Ortigosa, S.M., et al., 2010. Stable production of peptide antigens in transgenic tobacco chloroplasts by fusion to the p53 tetra- merisation domain. Transgenic Res. 19, 703
709. Pastores, G.M., et al., 2014. A Phase 3, multicenter, open-label, switchover trial to assess the safety and efficacy of taliglucerase alfa, a plant cell- expressed recombinant human glucocerebrosidase, in adult and pediatric patients with Gaucher disease previously treated with imiglucerase. Blood Cells Mol. Dis. 53, 253
260. Pniewski, T., 2013. The twenty-year story of a plant-based vaccine against hepatitis B: stagnation or promising prospects?. Int. J. Mol. Sci. 14, 1978
1998. Rose, A.B., 2002. Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA 8, 1444
1453. Sack, M., et al., 2015. From gene to harvest: Insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants. Plant Biotechnol. J. 13, 1094
1105. Sack, M., Hofbauer, A., Fischer, R., Stoger, E., 2014. The increasing value of plant-made proteins. Curr. Opin. Biotechnol. 32, 163
170. Sanz-Barrio, R., et al., 2011. Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts. Plant Biotechnol. J. 9, 639
650. Sharma, A.K., Sharma, M.K., 2009. Plants as bioreactors: recent developments and emerging opportunities. Biotechnol. Adv. 27, 811
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Sherman, A., et al., 2014. Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells. Blood 124, 1659
1668. Twyman, R.M., Schillberg, S., Fischer, R., 2012. Molecular Farming in Plants: Recent Advances and Future Prospects. Springer Science 1 Business Media. Available from: http://dx.doi.org/10.1007/978-94-007-2217-0. Verma, D., Samson, N.P., Koya, V., Daniell, H., 2008. A protocol for expression of foreign genes in chloroplasts. Nat. Protoc. 3, 739
758. Verma, D., Moghimi, B., LoDuca, P.A., Singh, H.D., Hoffman, B.E., Herzog, R.W., et al., 2010. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemo- philia B mice. Proc. Natl. Acad. Sci. USA 107, 7101
7106. Waheed, M.T., et al., 2011a. Plastid expression of a double-pen- tameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes. Plant Biotechnol. J. 9, 651
660. Waheed, M.T., et al., 2011b. Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines. Transgenic Res. 20, 271
282. Wiktorek-Smagur, A., Hnatuszko-Konka, K., Gerszberg, A., Kowalczyk, T., Luchniak, P., Kononowicz, A.K., 2012. Green way of biomedicine
how to force plants to produce new important proteins. In: C ¸ iftc¸i, Y.O. (Ed.), Transgenic Plants - Advances and Limitations. InTech. Available from: https://doi.org/10.5772/31145. Yao, J., Weng, Y., Dickey, A., Wang, K.Y., 2015. Plants as factories for human pharmaceuticals: applications and challenges. Int. J. Mol. Sci. 16, 28549
28565. Youm, J.W., et al., 2010. High-level expression of a human beta- site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice. Transgenic Res. 19, 1099
1108. Youngblood, B.A., Alfano, R., Pettit, S.C., Zhang, D., Dallmann, H.G., Huang, N., et al., 2014. Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells. J. Biotechnol. 172, 67
72.

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C H A P T E R

6 Agricultural Biotechnology: Engineering Plants for Improved Productivity and Quality Indra A. Padikasan, Karthik Chinnannan, Sathiya Kumar and Govindaraju Subramaniyan Periyar University, Salem, Tamil Nadu, India

6.1 INTRODUCTION OF AGRICULTURAL BIOTECHNOLOGY 6.1.1 Origin and Definition of Agricultural Biotechnology The word agriculture (agri 5 field; culture 5 tillage) means tillage of the soil leading to production of crops. The origin of agriculture could be traced to the early human attempts at settling down in congenial environments and gradually exploiting the animal and plant resources, which were found easily accessible. Undoubtedly, wild plants, their roots, fruits, and seeds were among the principal plant resources belonging to the vegetable kingdom used as food for ancient man. In perspective, 90% of the evolutionary span has emerged about 9000 7000 BC and that industrialization on a large scale is a product of the 19th and 20th century (Fig. 6.1). Agricultural biotechnology has been a long time practice, which was initiated by the farmers to improve the agriculturally important plants using traditional breeding techniques. A good example of traditional agricultural biotechnology is the development of disease-resistant wheat varieties by crossbreeding different wheat types until the desired disease resistance was present in a resulting new variety. In recent years, this skill has reached a stage where scientists can choose one or more specific useful genes from the plants, animals, bacteria, and viruses for developing the plants with desirable characters.

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00006-1

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Copyright © 2018 Elsevier Inc. All rights reserved.

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2000

1950

Genome research (1990) smart breeding Genetic engineering (1970) Green revolution and tissue culture (1960)

FIGURE 6.1 Historical development of agriculture biotechnology.

Hybrid crop 1900 Mendel’s laws (1865) 1850 AD BC 5000

Pasteur (1860) fermentation Key agricultural developments wine and beer landmarks in biotechnology Plant and animal domestication

10,000

6.1.2 Plant Breeding Program All living organisms have the ability to improve themselves through natural way to adapt the different environmental conditions. However, detectable evaluation was taken more than hundred years. The man then learned how to domesticate and breed plants to develop crops to his own wish and needs using various means including biotechnology. Agricultural biotechnology is the term used in crop and livestock improvement through biotechnological tools, which encompasses elements of the conventional breeding, plant physiology, biochemistry, molecular biology, molecular genetics, bioinformatics, and microbiology. 6.1.2.1 Conventional Plant Breeding Plant breeding is one of the earliest and foremost attempts of the earnest man, which had led him towards civilization. Ever since the beginning of agriculture, consciously or unconsciously, human created genotypes that are more efficient in the production in high quality and quantity of food, fiber, and fuel plants. The early farmers were aware of selecting better seeds for getting better harvest. The selection of features such as faster growth, higher yields, pest and disease resistance, larger seeds, or sweeter fruits has dramatically changed domesticated plant species than wild relatives. Different workers defined plant breeding as follows: according to Smith (1966), “plant breeding is the art and science of changing the genetic patterns of plants in relation to their economic uses.” In general, three main steps are involved in plant breeding process such as (1) plant selection, (2) hybridization, and (3) hybrid vigor (Fig. 6.2). When the science of plant breeding was further developed in the 20th century, plant breeders better understood how to select superior plants and breed them to create new and improved varieties of different crops. This has dramatically increased the productivity and quality of the plants.

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Elite variety

89

FIGURE 6.2 Overview flowchart of the conventional plant breeding.

Screening and selection (Based on the visible, behavioral, quality, and quantity traits)

Hybridization

Hybrid vigor

Field trial

Modified elite variety

6.1.2.2 Modern Plant Breeding The process of developing new crop varieties requires many steps, and it will take almost 25 years. However, applications of modern plant breeding and agricultural biotechnology have considerably shortened the time. It currently takes 7 10 years for new crop varieties to be developed. One of the tools, which make it easier and faster for scientists to select the plant traits, is called modern plant breeding. The major modern plant breeding techniques are 1. tissue culture, 2. genetic engineering, and 3. marker-assisted breeding (MAB).

6.1.3 Application of Modern Agriculture 6.1.3.1 Yield Increase Modern plant breeding techniques increased productivity of major agriculture crops such as corn, rice, sorghum, wheat, and soybean over the years. For example, the productivity of corn rose from about 2000 kg/ha in the 1940s to about 7000 kg/ha in the 1990s. In England, it took only 40 years for wheat yields to rise from 2000 to 6000 kg/ha by using various modern breeding techniques. 6.1.3.2 Enhancement of Compositional Traits Breeding for plant compositional traits to enhance nutritional quality or to meet an industrial need is the major plant breeding goal. Previously, high-nutrient quality crop varieties (e.g., high lysine or quality protein maize) have been developed for use in various parts of the world by modern plant breeding techniques.

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6.1.3.3 Crop Adaptation Crop plants are being produced in regions to which they are not native, because breeders have developed cultivars with modified physiology to cope with variations, for example, in the duration of day length (photoperiod). Photoperiod-insensitive cultivars will flower and produce seed under any day length conditions. The duration of the growing period varies from one region of the world to another.

6.2 GENETIC ENGINEERING STRATEGIES FOR CROP IMPROVEMENT 6.2.1 Introduction of Plant Genetic Modification Technical and biological discoveries in the 1970s led to a new era of DNA analysis and manipulation. The term “genetic engineering” refers to “Alteration in the genome of a cell or organism brought about by the uptake and incorporation of introduced, alien DNA.” It will not replace conventional breeding but facilitate the efficiency of crop improvement. It is possible because plant cells are totipotent, enabling regeneration of a new plant from an isolated cell. Cloning, in this sense, refers to the isolation and amplification of defined pieces of DNA. Key among these was the discovery of two types of enzymes that made DNA cloning possible. The restriction enzymes cut the DNA from any organism at specific sequences of a few nucleotides, generating a reproducible set of fragments. The other enzyme type, called DNA ligases, can covalently join DNA fragments at their termini that have been produced by restriction enzymes. Thus, ligases can insert DNA restriction fragments into replicating DNA molecules such as plasmids, resulting in recombinant DNA molecules. The recombinant DNA molecules can then be introduced into appropriate host cells, most often bacterial cells. All descendants from such a single cell, called a clone, carry the same recombinant DNA molecule.

6.2.2 Plant Transformation Techniques Numerous methods have been developed and used to introduce “foreign” DNA into plant cells, leading to transformed plant phenotypes. In plants, widely used techniques are the Agrobacterium-mediated transfer and bombardment methods. 6.2.2.1 Physicochemical Methods A number of valuable physical and chemical methods such as particle bombardment, macro and micro injection, electroporation, vacuum infiltration mediated transformation, ultrasound and shock wave mediated transformation, electrophoresis and polyethylene glycol, calcium phosphate-DNA coprecipitation, and liposome mediated transformation are used to transfer genes in plant cells. These methods are widely cost-effective and show an unpredictable pattern of foreign DNA interaction.

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6.2.2.2 Biological Methods 6.2.2.2.1 AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION

Agrobacterium tumefaciens is a soil bacterium that has the ability to infect plant cells and transfer a defined sequence of their DNA to the plant cell by infection and a causative agent of crown gall disease. Agrobacterium tumefaciens cell contains a plasmid known as the Ti (tumor-inducing) plasmid (140 235 kb). Most of the Ti plasmids have following regions: (1) T-DNA region: responsible for tumor induction. Sequences homologous to this region are always transferred to plant nuclear genome. This region encodes the enzymes responsible for the phytohormone synthesis, so that the incorporation of these genes in plant nuclear genome leads to the synthesis of phytohormones in the host plant. The phytohormones in their turn alter the developmental program, leading to the crown gall formation. (2) An opine synthesis (OS) region responsible for the synthesis of unusual amino acid or sugar derivatives, which are collectively given the name opines. (3) On either side of the T-DNA, there is a short sequence of 24 bp called border sequence. Both the left and right border sequences are essential for tumor induction. (4) Origin of replication: responsible for replication. (5) Tra region: responsible for conjugation. (6) Inc region: responsible for incompatibility among plasmids in a bacterium. (7) Vir region: responsible for virulence region and plays a crucial role in the transfer of T-DNA into the plant nuclear genome. 6.2.2.2.2 VIRUS-MEDIATED PLANT TRANSFORMATION

Virus-based vectors have been an efficient tool for the transient, high-level expression of foreign proteins in plants. These vectors are derived from plant viruses, e.g., Tobacco Mosaic Virus, and are manipulated to encode the protein of interest. Within a plant cell, the virus-based vectors are autonomously replicated, can spread from cell to cell, and direct the synthesis of the encoded protein of interest. The advantages of this method are the applicability to whole plants and thus a much faster and the high-level expression of the desired protein within a short time. 6.2.2.2.3 IN PLANTA TRANSFORMATION

Most of the transformation procedures discussed so far have one major disadvantage that they rely on tissue culture for the regeneration of whole plants. In this typical experiment, inflorescence shoots are cut at their bases and wound sites are inoculated with Agrobacterium with concurrent vacuum infiltration. Selection of transformants is usually achieved by germinating the seeds on a medium containing antibiotics (Bechtold et al., 1993).

6.3 APPLICATIONS OF GENETICALLY MODIFIED CROPS Genetic engineering technology has helped to increase crop productivity by introducing new qualities into the target crop. This technique has enabled the production of transgenic plants in food, fiber, vegetable, fruit, and forest crops. In recent times, agriculture biotechnology innovation provides new solution to the age-old problems. Genetic engineering helps to introduce specifically desirable genes without transfer of any undesirable genes

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FIGURE 6.3 Biotic and abiotic stresses that affect plant growth, development, and yield.

Biotic and abiotic stresses Biotic stresses

Abiotic stresses Herbicides

Insect

Disease caused by bacteria, fungi, and virus

Temperature stress (heat, freezing, chilling) Drought salinity

Water – deficit stresses

Intense light

Flood Heavy metal

from donor organisms, which normally occurs in the conventional breeding methods. The major objective of plant biotechnology is to develop plants that are resistant to biotic and abiotic stresses (Fig. 6.3).

6.3.1 Resistance to Biotic Stress Genetic transformation has led to the possibility of transforming crops for enhanced resistance to insects and pathogens; it is rapidly moving towards commercialization. These advances are from the basis of chemical-free and economically viable approach for pest and disease control. 6.3.1.1 Insect Resistance It is estimated that about 15% of the world’s crop yield is lost due to the insect attacks. The damage to crops is mainly caused by insect larvae and to some extent adult insects. The majority of the insects that damaged crops belong to the following order: Lepidoptera, Coleoptera, Orthoptera, and Homoptera. In the past few decades, only the chemical pesticides were used to control the pests. Scientists have been looking for alternate methods of pest control for the following reasons: • Environmental pollution • Toxic to nontarget organisms • Difficult to deliver target part (root, stems, and fruits). It is fortunate that scientists have been able to discover new biotechnological alternatives to chemicals pesticides, thereby providing insect resistance to crop plants. Transgenic plants with insect-resistant transgenes have been developed. About 40 genes obtained from microorganisms, plants, and animals have been used to provide insect resistance in plants.

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6.3.1.1.1 RESISTANCE GENE FROM MICROORGANISMS

Microorganisms contribute a major part in insect resistance. Bacillus thuringiensis is an entomocidal bacterium that produces an insecticidal cry protein. Cry genes code for insecticidal cry protein, which differ in their spectrum of insecticidal activity. Most Bt toxins are active against lepidopteran larvae, but some are specific for dipteran and coleopteran insects. Similarly, some other insecticidal toxins were obtained from other microbial source such as Ipt gene (isopentyl transferase) from A. tumefaciens, cholesterol oxidase gene from Streptomyces, and Pht gene from Photorhabdus luminescens. 6.3.1.1.2 RESISTANCE GENES FROM HIGHER PLANTS AND ANIMALS

Currently there are two major groups of plant-derived genes that are used to confer insect resistance on crop plants by retarding insect growth. The groups are (1) proteinase and amylase inhibitors and (2) lectins (pea lectin, rice lectin, jacalin, etc.). Resistance genes of animal origin are serine protease inhibitors from mammals and tobacco hornworm. 6.3.1.2 Disease Resistance A large number of plant defense response genes encoding antimicrobial proteins have been cloned. Most of these are transcriptionally activated in response to infection or exposure to microbial elicitor macromolecules. The products of defense response genes may include (1) hydrolytic enzymes (chitinase, 1-3 β-D glucanase, and other pathogenesisrelated proteins), (2) ribosome inactivation proteins, (3) antifungal proteins, (4) biosynthetic enzymes for production of antimicrobial phytoalexins, (5) wall-bound phenolics, osmotins, thionins, lectins, etc., and (6) hydrogen peroxide. 6.3.1.3 Virus Resistance Virus infection is retarded cell division, excessive cell division, and cell death, which leads to the lowered product yield and sometimes complete crop failure. The chemical methods used to control various plant pathogens will be ineffective with respect to plant viruses because the viruses are intracellular obligate parasites. It is possible to immunize plants against viral damage by expressing viral proteins in the plant cells. With the advances made in genetic engineering, it has become a reality to develop transgenic plants with virus resistance. This is mostly done by employing virus-encoded genes, virus-coated proteins, satellite RNA, antisense RNAs, and ribozymes. In recent years, some attempts are also made to provide virus resistance to plants by using animal genes.

6.3.2 Resistance to Abiotic Stresses Plants are constantly being subjected to environmental stresses, which may result in the deterioration of crops and are the responsible factors for the low yield. Plants are depended on the subtle internal mechanisms for tolerance of various stresses. The in situ tolerance of crop plants, whenever present, is inadequate and therefore cannot give protection against the stresses. A wide range of strategies are required to engineer plants against a particular type of stress tolerance. Some of the abiotic stresses and the recombinant strategies developed to overcome them are described.

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6.3.2.1 Herbicide Resistance The use of herbicides to control weeds plays a pivotal role in modern agriculture. A major effort has been devoted in several laboratories to engineer herbicide-resistant plants. Recently, various improvements have been accomplished in herbicide resistance as single genes govern the resistance, in which three approaches have been followed: (1) overproduction of a herbicide-sensitive biochemical target, (2) structure alteration of biochemical target resulting in reduced herbicide affinity, and (3) detoxification degradation of the herbicide before it reaches the biochemical target inside the plant cells. 6.3.2.2 Tolerance to Water-Deficit Stresses The environmental conditions such as temperature (heat, freezing, and chilling), water availability (shortage due to drought), and salinity influence the plant growth, development, and yield. The abiotic stresses due to temperature, drought, and salinity are collectively regarded as water-deficit stresses. Plant cells are subjected to several osmotic stresses due to water deficit. However, it produces certain compounds, collectively referred to as osmoprotectants or osmolytes, to overcome the osmotic stress. Osmoprotectants are nontoxic compatible solutes. The production of osmoprotectants is species-dependent. The formations of mannitol, proline, and glycine betaine are more closely linked to osmotic tolerance. Some progress has been made in this direction. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding key enzymes isolated. In fact, some progress has been made in the development of transgenic plants with high production of osmoprotectants.

6.4 GENETIC MANIPULATION FOR CROP QUALITY With the advances made in plant genetic engineering, improvement in crop yield and quality has become a reality. The crop yield is primarily dependent on the photosynthetic efficiency and the harvest index. There are a wide range of crops that have been manipulated by scientists for improved yield and quality. Some examples of transgenic crops with improved quality have been summarized in Table 6.1. The quality of the crop is dependent on a wide range of desirable characters nutritional composition of edible parts, flavor, processing quality, self-life, etc. In this chapter, only selected examples are briefly described below.

6.4.1 Transgenic for Improved Fruit Storage The first transgenic food product was developed by Calgene, USA, in 1994, with the properties of delayed ripening flavr savr tomato. The improved fruit ripening or long shelf life of tomato is suitable for food processing developed by two approaches: (1) antisense RNA technology and (2) degradation of 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene by ACC deaminase genes. Based on the PG sequence, a complimentary PG gene was constructed and the tomato plant was transformed. The resulted transgenic plant could produce RNA RNA pairing by binding of both sense and antisense mRNA of PG

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TABLE 6.1 Lists of Transgenic Crops for Commercial Use Trait

Crop (Trait Detail)

Gene

Transformation method

References

Protein and amino acids

Bahiagrass (protein)

vspB

Biolistic

Luciani (2007)

Soybean (amino acid balance)

Zein

Somatic embryogenesis

Dinkins et al. (2001); Rapp (2002)

Maize (amino acid composition; protein)

Opaque-2

Self-pollination

Cromwell et al. (1967); O’Quinn et al. (2000)

Maize (Lys and Met)

dzs10

Agrobacteriummediated

Lai and Messing (2002)

Potato (Met)

ts

Agrobacteriummediated

Zeh et al. (2001)

Sorghum (Lys)

Bar and HT12

Agrobacteriummediated

Zhao et al. (2003)

Rice (α-linolenic acid)

Omega-3 fatty acid desaturase gene

Agrobacteriummediated

Anai et al. (2003)

Maize (fructan)

Ubi1-P-int and GmFAD3

Agrobacteriummediated

Caimi et al. (1996)

Potato (fructan)

SacB

Particle bombardment

Hellwege et al. (2000)

Wheat (phytase)

Phytase-encoding gene

Particle bombardment

Brinch-Pedersen et al. (2000)

Lettuce (iron)

Exogenous ferritin gene

Agrobacterium tumefaciens

Goto et al. (2000)

Maize (phytase and ferritin)

Aspergillus phytase

Particle bombardment

Drakakaki et al. (2005)

Delayed ripening

Tomato

accD gene

A. tumefaciens

Reed et al. (1995)

Golden rice

Indica rice

Lycopene β cyclase

Particle bombardment

Datta et al. (2003)

Carbohydrate

Minerals

gene. This would inhibit the expression of PG gene product, thus preventing the attack of gene upon pectin in the cell wall of the ripening fruit and thereby preventing the softening fruit (Fig. 6.4).

6.4.2 Golden Rice About one-third world’s population depends on the rice stable food. The milled rice is usually consumed is almost deficit in β-carotene the (provitamin A) as such vitamin A deficiency is a nutritional disorder world over. To overcome this problem, it was proposed

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FIGURE 6.4 Illustration showing the steps of antisense RNA technology.

FIGURE 6.5 An outline of pathway for the biosynthesis of β-carotene

Isopreoid units Sequential addition

(provitamin A).

Geranyl geranyl diphosphate Phytoene synthase Phytoene Carotene desaturase Lycopene Lycopene β-cyclase 13 β-carotene (provitamin A)

to genetically manipulated rice to produce β-carotene in the rice endosperm. The genetic manipulation to produce golden rice requires introduction of three genes encoding the enzymes namely phytoene synthase, carotene desaturase, and lycopene β-cyclase (Fig. 6.5).

6.4.3 Eco-Social Impact of Genetically Modified Crops Genetic engineering is exemplified as a revolution to hunger and environmental problems in agriculture, which is best developed and allocated by private firms, market

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mechanisms, and intellectual property rights (IPR). These resources inspire towards the molecular disciplines, global standardization of property to knowledge, and germplasm (Kathleen, 2003). The positive prognosis includes differences in the role of genetic and crop diversity in various farming system, cultural and genetic diversity, and economic vulnerability. The commodity relation determines the level of risk in the region and the degree of access. Difference in them leads to incompatible paradigms for valuation, access and use rights, exchange and conservation of crop genetic resources. Deliberately, many difficulties are evaded to address the elemental questions of execution and societal change. It deals with the commercialization of food consumption, disposal, politics, and all institutions of food in need that recompense for seasonal food and climatic variation for the food system is strictly defensible (Chappell and Liliana, 2011). Obstacles commonly proclaimed were new agricultural biotechnologies that can prevent an approaching crisis of productivity, lack of control over food-producing resources by farmers, inadequate storage, transport and marketing infrastructure, depressed farm product prices, lack of incomes and entitlements to food, and lack of political power among small- and medium-scale food producers and poor consumers (Kathleen, 2003). In spite of the widespread international use of genetically modified (GM) crops, the portfolio of accessible crop trait combinations is quiet very narrow. At present, only a few first-generation technologies have been commercialized. The dominant technology is herbicide tolerance in soybeans, which accounts for 70% of the worldwide production. GM maize is the second-most dominant crop of 24% of total maize production. GM crops with weighty area shares include cotton and canola. Bt cotton with resistance to bollworms and budworms is chiefly significant in developing countries. In 2008, India had the largest Bt cotton area with 7.6 million hectors, followed by China with 3.8 million hectors. Firstgeneration GM technologies that are being industrialized include fungal, bacterial, and virus resistance in major cereal along with root and tuber crops (Halford, 2006). Plant tolerance to abiotic stress (drought, heat, and salt) is also being driven on intensively. Yet, because of the complex genetic mechanisms, the effort is at a rudimentary level. Secondgeneration GM technologies in the pipeline comprise product quality improvements for nutrition and industrial purposes. Examples are oilseeds with improved fatty acid profiles; high-amylose maize; and staple foods with enhanced essential amino acids, minerals, and vitamins. The utmost risk is raising GM crop production with the same heritable traits (Jefferson-Moore and Traxler, 2005).

6.4.4 Current Status of GM Plants 1. In 2013, hectarage of biotech crops grew by 5 million hectares, at an annual growth rate of 3%. 2. Notably, developing countries grew more, 54% (94 million hectares) of global biotech crops in 2013. 3. In 2013, a record of 18 million farmers, up by 0.7 million from 2012, grew biotech crops. 4. In 2013, a record of 7.5 million small farmers in China and another 7.3 million in India elected to plant more than 15 million hectares of Bt cotton.

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TABLE 6.2 Top Transgenic Crops Distributed Around the Countries Rank

Country

1

United States

70.1

Maize, soybean, cotton, canola, sugar beet

2

Brazil

40.3

Soybean, maize, cotton

3

Argentina

24.4

Soybean, maize, cotton

4

India

11

Cotton

5

Canada

10.8

Maize, soybean, canola, sugar beet

6

China

4.2

Cotton, papaya, poplar, tomato

7

Paraguay

3.6

Soybean, maize, cotton

8

South Africa

2.9

Soybean, maize, cotton

9

Pakistan

2.8

Cotton

10

Uruguay

1.5

Soybean, maize

11

Bolivia

1.0

Soybean

12

Philippines

0.8

Maize

13

Australia

0.6

Cotton, canola

14

Burkina Faso

0.5

Cotton

15

Myanmar

0.3

Cotton

16

Mexico

0.1

Maize

17

Spain

0.1

Cotton, soybean

18

Colombia

0.1

Cotton, maize

19

Sudan

0.1

Cotton

Total

Area (million hectares)

Biotech crops

175.2

Clive James, ISAAA report (2008).

5. Bangladesh, Indonesia, and Panama approved biotech crop planting in 2013 with plans for commercialization in 2014 and distribution of transgenic crops have been described in Table 6.2.

6.4.5 Goals of Genetic Engineering in Crop Improvement There are about 30 40 crops that have been GM and many more are being added. However, very few of them have got the clearance for commercial use. The ultimate goals of GM crop plants are listed below: 1. Higher yielding and nutritional capacity 2. Resistance to biotic and abiotic stress 3. Improved nitrogen fixing ability

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4. Production of pharmaceutically important compounds 5. Modified sensory attributes, e.g., increased sweetness as in thaumatin 6. Absence of allergens

6.4.6 Concerns About Transgenic Plants The fear about thee harmful environmental and hazardous health effects of transgenic plants still exists, despite the fact that there have been no reports so far in this regard. The transfer of almost all the transgenic plants from the laboratory to the crop field is invariably associated with legal and regulatory hurdles, besides the social and economic concerns. The major concern expressed by public and biotechnologists is the development of resistance genes in insect, generation of super weeds, etc. The farmers in developing countries are much worried about the seed terminator technology which forces them to buy seeds for every new crop. These farmers are traditionally habituated to use the seeds from the previous crop which is now not possible due to seed terminator technology.

6.5 GENETIC ASSISTED PLANT BREEDING 6.5.1 Introduction to Molecular Markers In agriculture, molecular markers are used to improve the existing cultivars, which are lacking in one or more characters. Crossing such cultivars with lines that possess the targeted desired trait could produce the hybrid plant. During the conventional breeding program, the plants have to be selected from the superior recombinants among the several segregated products. Indeed, such a procedure is difficult and time-consuming; it may consist of several crosses, several generations, and the phenotypic selections should be monitored carefully. The linkage drag may create further difficulty to achieve the desired objectives. DNA marker technology and its developments in plant breeding strategies offered more possibilities to plant breeders and geneticists to overcome many of the problems faced during conventional breeding. DNA marker techniques are highly reliable selection tools as they are abundant and stable. Also the molecular markers are not influenced by the environment as well as it is relatively easy to score in an experienced laboratory conditions. While comparing to phenotypic assays, DNA markers are providing great advantages to accelerate the variety development. The important steps in plant breeding are analysis of genetic distance, variety identification and seed purity analysis, and marker-assisted backcrossing (MABC). These applications are collectively called as genome-wide polymorphism, and this can be detected using two defined genotypes (lines) by molecular markers. Hence, the genome-wide polymorphism is used to identify the relative value in the relation to score of the marker in other individual. The usage of multiple markers have proven to be applicable for achieving number of objectives aimed at controlling the lines of plant breeding. Application of DNA markers in the plant breeding are tightly linked to the identification of specific gene,

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monogenic traits, and quantitative trait locus. Table 6.3 summarizes the few advantage and disadvantages of molecular marker. 6.5.1.1 Prerequisites and General Activities of MAB While comparing to the conventional breeding approaches, molecular breeding, mostly referred to as DNA MAB, needs more facilities and complicated equipments. In general, some of the prerequisites listed below are important factors for MAB in plants, and the ideal DNA markers should meet the MAB criteria. • • • • • • • •

Co-dominance in expression Low cost to use Genome-specific in nature Clear distinct allelic features High levels of polymorphism No detrimental effect on phenotype Single copy and no pleiotropic effect Occurrence and even genome-wide distribution

TABLE 6.3 Marker and Its Advantages and Disadvantages S. No Types of markers

Advantages

Disadvantages

1

Restriction fragment length polymorphism (RFLP)

Co-dominance markers, high genomic abundance, good genomic coverage, and can be used across species

No probe or primer information, not reproducible, completely not validated, and dominant markers

2

Randomly amplified polymorphic DNA (RAPD)

Map-based cloning is needed, high genomic abundance, radioactive labeling is not needed, and easy to automate

Need high quantity of DNA, laborious, need radioactive labeling, and difficult to automate

3

Simple sequence repeat (SSR)

Highly reproducible, highly genomic abundance, high polymorphism, and radioactive labeling is not needed

Cannot be used across species, not well tested, and sequence information is needed

4

Amplified length polymorphism

High genomic abundance, high polymorphism, useful for preparing counting maps and can be used across the species

Very difficult due to the changes of patterns with respect to materials used, not reproducible, and good quality of primers are needed

5

Sequence tagged site (STS)

Fairly good genomic coverage, highly reproducible, and useful in counting maps

Sequence information is needed, cloning and characterization information needed, and laborious

6

Isozymes

Useful for evolutionary studies, can be used across the species, and no need for sequence information

Limited polymorphism, laborious, expensive technique, and not easily automated

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An important desirable attribute for the markers to be used is close association with the target gene(s). If the markers are located near to the close proximity of the target gene or present within the gene, selection of the markers will ensure the success in selection of the gene and could be used in the plant breeding program. But some other classical markers have some limitations. The polymorphisms are available throughout the genome, and their presence and absence are not affected by the environment and generally it is not affecting the phenotype. The DNA markers usually detect at all stages of the plant growth, and also the detection using the classical markers has certain limitations for the plant growth stages. Each type of DNA markers has both advantage and disadvantages.

6.5.2 Variety Identification and Seed Purity Analysis Genotyping using DNA is considered as the most reliable method for the identification of fixed lines and varieties. DNA fingerprinting methods have been applied in many breeding programs to analyze the purity of seed lots of inbred lines as well as hybrid seed lots (Rolda´n-Ruiz et al., 2000). The usage of molecular markers in the seed purity analysis is cheaper than the visual inspection of the seedlings. Also this has the opportunity for the fact commercial valorization and saving cost of the expensively produced seeds. Multiplex DNA finger printing methods—RAPD, AFLP, and SSR—are mostly used to detect the polymorphism at low cost for identifying varieties or to determine the hybrid purity. 6.5.2.1 Genetic Distance Analysis All individuals of the germplasm samples are fingerprinted using multiplex fingerprinting technique. A pairwise distance matrix is calculated for each pair of individuals, followed by a clustering algorithm or a Principal Component Analysis. The result of this grouping individuals within the germplasm may vary according to the interrelatedness. Various methods have been validated for calculating the interpreting similarity matrices (Reif et al., 2005). Genetic distance analysis is a powerful tool for assorting the unknown genotypes to the groups within the germplasm. (e.g., heterotic groups in maize) (Lu¨bberstedt et al., 2000). The pairwise similarities are used for designing crosses aimed at maximizing the genetic segregation. It may be either for optimizing the genetic mapping population or expected segregation of the traits. The usage of genetic distances analysis based on molecular marker scores for predicting heterosis has been investigated by Syed and Chen (2004) and Vuylsteke et al. (2000).

6.5.3 MABC Breeding MABC breeding is a standard application in modern plant breeding, and the optimization of MABC strategies was reviewed (Frisch et al., 1999; Reyes Valdes, 2000). In this technique the linkages are sought between the DNA markers and the agronomically important traits such as pathogen resistance, insects, nematodes, abiotic stress tolerance, quantitative traits, and quality parameters. Instead of selecting the characters, the breeders can select the markers that can be detected very easily in the selection scheme.

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Important requirements for the plant breeding programs are follows: • • • •

The markers should be co-segregated or be closely linked with the desired trait. The efficient mean for the screening of large populations should be available. It should be economically cheaper and user friendly. The screening techniques should have high reproducibility across laboratories.

Various strategies have been developed for screening the large number of random, unmapped molecular markers in the relatively short time and for selecting those few markers that reside in the vicinity of the target gene. The efficient molecular markers rely on two principles. 1. To generate hundreds or even thousands of potentially polymorphic DNA segments and rapidly visualize from the single preparation of DNA. 2. To use genetic stocks to identify the thousand DNA fragments those that are derived from the region adjacent to the targeted gene. In the past few decades, the high-volume molecular marker technologies and thousands of loci scattered throughout the genome have been assayed in a matter of weeks or months. The next problem is to determine which of the amplified loci lie near the targeted gene. These strategies have been proved effective. 6.5.3.1 Nearly Isogenic Strategies The breeders have developed the nearly isogenic line (NIL) genetic stocks and have been maintaining these inbred line that are differ at the targeted locus. The NIL isogenic lines are produced when donor line (P1) is crossed to the recipient line (P2). This resulting F1 hybrid is then backcrossed to the P2 recipient to produce the backcross generation (BC1). From this BC1, the single individual containing the dominant allele of the targeted gene from the P1 is selected. This selection is usually based on the basis of phenotype. Again the BC1 is backcrossed with P2, and this procedure is repeated for the number of generations. Generally, in the BC7 generations, genomes mostly derived from the p2, except small chromosomal segment containing allele derived from the P1. The homologous lines for the targeted gene can be selected from the BC7, F2 generations, and these are nearly isogenic with the recipient parent, P2.

6.5.4 Molecular Markers for Hybrid Vigor Hybridization between two unrelated or distantly related species leads to the production of F1 hybrids with increased vigor and fertility, e.g., F1 hybrids are taller, sturdier, and more productive than their parents. This superiority of the progeny over the parents is called as hybrid vigor. Hybrids in crops such as maize, sorghum, rice, and pearl millet have contributed greatly towards increasing the yield potential of the crops. Using the molecular markers on a set of diallel crosses among the eight elite parental lines widely used in the Chinese hybrid rice production program, and the high correlation was found between the specific heterozygosity and midparent heterosis.

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6.6 FUTURE PROSPECTS The feasibility of transgenic approaches for the improvement of crop productivity and quality has been widely accepted. However, many of the transgenic plants have been produced in the past several decades using single-gene transfer methodology. Hence, the use of multigene approaches with simultaneous transfer of several genes deals with considerable promise because of the probable inheritance of all the transgenes in the same locus. In addition, implementation of advances in structural and functional analysis of plant genomes will provide substantial knowledge on biochemical pathways that are responsible in the regulation of more complex traits. This could even enable scientists to recognize and transfer entire biochemical pathways from one plant species to another and to incorporate them into new hosts for the benefit of agriculture. Currently, some of the new proposed strategies for producing transgenic plants with high productivity were emerging by using the inducible specific promotors. Moreover, techniques such as RNA interference and transposon insertional knockouts for the signaling pathways and candidate genes are expected to produce more beneficial characteristics for crop productivity and quality. In addition, the knowledge expanded from the genomic and post-genomic projects assurances a great deal for the transgenic research and for the future agriculture.

References Anai, T., Koga, M., Tanaka, H., Kinoshita, T., Rahman, S.M., Takagi, Y., 2003. Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene. Plant Cell. Rep. 21, 988 992. Bechtold, N., Ellis, J., Pelletier, G., 1993. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris Life Sci. 316, 1194 1199. Brinch-Pedersen, H., Olesen, A., Rasmussen, S.K., Holm, P.B., 2000. Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Mol. Breed. 6, 195 206. Caimi, P.G., McCole, L.M., Klein, T.M., Kerr, P.S., 1996. Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB gene. Plant Physiol. 110, 355 363. Chappell, M.J., Liliana, A.L., 2011. Food security and biodiversity: can we have both? An agroecological analysis. Agric. Hum. Values 28, 3 26. Cromwell, G.L., Pickett, R.A., Beeson, W.M., 1967. Nutritional value of opaque-2 corn for swine. J. Anim. Sci. 26, 1325 1331. Datta, K., Baisakh, N., Oliva, N., Torrizo, L., Abrigo, E., et al., 2003. Bioengineered ‘golden’ indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnol. J. 1, 81 90. Dinkins, R.D., Reddy, M.S.S., Meurer, C.A., Yan, B., Trick, H., Thibaud-Nissen, F., et al., 2001. Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein. In Vitro Cell Dev. Biol. Plant 37, 742 747. Drakakaki, G., Marcel, S., Glahn, R.P., Lund, E.K., Pariagh, S., Fischer, R., et al., 2005. Endosperm-specific coexpression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol. Biol. 59, 869 880. Frisch, M., Bohn, M., Melchinger, A.E., 1999. Comparison of selection strategies for marker assisted backcrossing of a gene. Crop Sci. 39, 1295 1301. Goto, F., Yoshihara, T., Saiki, H., 2000. Iron accumulation and enhanced growth in transgenic lettuce plants expressing the iron-binding protein ferritin. Theor. Appl. Genet. 100, 658 664. Halford, N.G. (Ed.), 2006. Plant Biotechnology: Current and Future Uses of Genetically Modified Crops. John Wiley & Sons, Chichester, UK.

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Hellwege, E.M., Czapla, S., Jahnke, A., Willmitzer, L., Heyer, A.G., 2000. Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots. Proc. Natl. Acad. Sci. USA 97, 8699 8704. James, C., 2008. Global status of commercialized biotech/GM crops: ISAAA Briefs. Jefferson-Moore K.Y. and Traxler G., Second-generation GMOs: Where to from here?, AgBioForum 8, 2005, 143 150. Kathleen McAfee, 2003. Geographies of risk and difference in crop genetic engineering and agrobiodiversity conservation. Geographical Review. Paper prepared for: CAPRI-IPGRI International Workshop on Property Rights, Collective Action and Local Conservation of Genetic Resource, Rome. Lai, J.S., Messing, 2002. Increasing maize seed methionine by mRNA stability. Plant J. 30, 395 402. Lu¨bberstedt, T., Melchinger, A.E., Dussle, C., Vuylsteke, M., Kuiper, M., 2000. Relationship among early European maize inbreds: IV genetic diversity revealed with AFLP markers and comparison with RFLP, RAPD, and pedigree data. Crop Sci. 40, 783 791. Luciani, G., 2007. Genetic engineering to improve nutritional quality and pest resistance in bahiagrass (Paspalum notatum var. Flugge). Online thesis. http://ufdcimages.uflib.ufl.edu/uf/e0/01/98/20/00001/luciani_g.pdf (accessed 15.04.08). O’Quinn, P.R., Nelssen, J.L., Goodband, R.D., Knabe, D.A., Woodworth, J.C., Tokach, M.D., et al., 2000. Nutritional value of a genetically improved high-lysine, high-oil corn for young pigs. J. Anim. Sci. 78, 2144 2149. Rapp, W., 2002. Development of soybeans with improved amino acid composition. In: 93rd AOCS Annual Meeting and Expo, Montreal, American Oil Chemists’ Society Press, Champaign, IL, pp. 79 86. Reed, A.J., Magin, K.M., Anderson, J.S., Austin, G.D., Rangwala, T., Linde, D.C., et al., 1995. Delayed ripening tomato plants expressing the enzyme 1-aminocyclopropane-1-carboxylic acid deaminase: 1. Molecular characterization, enzyme expression, and fruit ripening traits. J. Agric. Food Chem. 43, 1954 1962. Reif, J.C., Melchinger, A.E., Frisch, M., 2005. Genetical and mathematical properties of similarity and dissimilarity coefficients applied in plant breeding and seed bank management. Crop Sci. 45, 1 7. Reyes Valdes, M.H., 2000. A model for marker-based selection in gene introgression breeding programs. Crop Sci. 40, 91 98. Rolda´n-Ruiz, I., Calsyn, E., Gilliland, T.-J., Coll, R., van Eijk, M.-J.-T., De Loose, M., 2000. Estimating genetic Conformity between related ryegrass (Lolium) varieties. 2. AFLP characterization. Mol. Breed. 6 (6), 503 602. Smith, D.C., 1966. Plant breeding: development and success. In: Frey, K.J. (Ed.), Plant Breeding. Iowa State University Press, Ames, pp. 3 54. Syed, N.H., Chen, Z.J., 2004. Molecular marker genotypes, heterozygosity and genetic interaction explain heterosis in Arabidopsis thaliana. Heredity 94, 295 304. Vuylsteke, M., Kuiper, M., Stam, P., 2000. Chromosomal regions involved in hybrid performance and heterosis: their AFLP based identification and practical use in prediction models. Heredity 85, 208 218. Zeh, M., Casazza, A.P., Kreft, O., Roessner, U., Bieberich, K., Willmitzer, L., et al., 2001. Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants. Plant Physiol. 127, 792 802. Zhao, Z.Y., Glassman, K., Sewalt, V., Wang, N., Miller, M., Chang, S., et al., 2003. Nutritionally improved transgenic sorghum. In: Vasil, I.K. (Ed.), Plant Biotechnology 2002 and Beyond. Kluwer Academic Publishers, Dordrecht, the Netherlands, pp. 413 416.

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C H A P T E R

7 Functional Food Biotechnology: The Use of Native and Genetically Engineered Lactic Acid Bacteria Alejandra de Moreno de LeBlanc1, Tessalia D. Luerce2, Anderson Miyoshi2, Vasco Azevedo2 and Jean Guy LeBlanc1 1

Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucuma´n, Argentina 2Universidade Federal de Minas Gerais (ICB/UFMG), Belo Horizonte, Brazil

7.1 INTRODUCTION In March 2014, if one performed a MEDLINE search using the keyword “functional food,” the total hits would be over 2038 articles. From these, more than 70% (1482) were published in 5 years (between 2009 and the beginning of 2014) showing that this field of health promoting foods is currently trending. When the keyword “biotechnology” is crossed with “functional foods” in the same time period, only 83 articles are found, showing that although some research is being conducted in this field, it is only just the beginning. The objective of this chapter is to give an overview of the most recent studies that have shown that native and genetically engineered lactic acid bacteria (LAB) can be used as novel biotechnological tools to produce foods that are beneficial to the health of the consumers.

7.2 DEFINITIONS Before going any further, it is important to clearly define the terms that are going to be described in this chapter, such as functional foods, nutraceuticals, and probiotics.

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A functional food is a food that can be incorporated as a part of a normal diet and provides the consumer with a beneficial effect often related to health promotion or disease prevention in addition to the intrinsic nutritional value of the food. Functional foods were defined by the Institute of Medicine of the US National Academy of Sciences as “foods that encompass potentially healthful products, including any modified foods or food ingredients that may provide a health benefit beyond the nutrients it contains” (Martirosyan, 2009). A more specific definition has been adopted by the Functional Foods Center (Dallas, TX) that states that a functional food is a “natural or processed food that contains known biologically-active compounds which when in defined quantitative and qualitative amounts provides a clinically proven and documented health benefit, and thus, an important source in the prevention, management and treatment of chronic diseases of the modern age.” In this last definition, it is clear that the foods must contain beneficial biologically active compounds, which for the scope of this chapter will include nutraceuticals and/or probiotics. The specific ingredients present in functional foods that can produce a beneficial effect on the host above and beyond their basic nutritional value are denominated nutraceuticals. The term “nutraceutical” was coined from joining the words “nutrition” and “pharmaceutical” in 1989 by Stephen DeFelice, MD, founder and chairman of the Foundation for Innovation in Medicine (FIM), Cranford, NJ (Brower, 1998). According to DeFelice, a nutraceutical can be defined as “a food (or part of a food) that provides medical or health benefits, including the prevention and/or treatment of a disease.” However, the term nutraceutical as commonly used in marketing has no regulatory definition (Brower, 1998). When a functional food aids in the prevention and/or treatment of disease(s) and/or disorder(s) (except anemia), it is called a nutraceutical (Kalra, 2003). The most commonly accepted definition of probiotics was published by the World Health Organization/Food and Agricultural Organization in 2001 that stated that probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit to the host” (FAO/WHO, 2001). However, according to the International Scientific Association for Probiotics and Prebiotics (ISAPP), a nonprofit scientific organization dedicated to advancing the science of probiotics and prebiotics, the term probiotic is commonly misused both commercially, when the term is featured on products with no substantiation of human health benefits, and scientifically, where the term has been used to describe bacterial components, dead bacteria, or bacteria with uncharacterized health effects in humans (http://www.isapp.net/Portals/0/docs/ ProbioticDefinitionClarification.pdf). The ISAPP does not provide a new definition for probiotics; it simply points out the important elements that are contained in the FAO/ WHO definition. This being said, they clarify that a probiotic must (1) be alive when administered; (2) have undergone controlled evaluation to document health benefits in the target host; (3) be a taxonomically defined microbe or combination of microbes (genus, species, and strain level); and (4) be safe for its intended use.

7.3 LACTIC ACID BACTERIA LAB are a heterologous group of microorganisms that have a long history of use as starter cultures for the elaboration of fermented foods because these industrially important bacteria can improve the safety, shelf life, nutritional value, flavor, and overall quality of I. MICROBIAL AND PLANT TECHNOLOGIES

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fermented products. In addition, LAB have been shown to exert a large range of beneficial properties, the reason for which they are frequently used as probiotic microorganisms in a variety of novel products. In addition to their intrinsic beneficial properties, certain strains of LAB have the capability of producing/releasing and/or increasing specific beneficial compounds in foods. These ingredients can be macronutrients (such as unsaturated fatty acids present in some oils), micronutrients (such as vitamins), or nonnutritive compounds (such as hydrolytic enzymes and flavonoids), and can be naturally present in foods (such as omega-3 fatty acids in fish or vitamin C in citrus fruits, etc.) (Hugenholtz and Smid, 2002).

7.4 NUTRACEUTICAL PRODUCTION BY LAB Because LAB are involved in the preparation of a wide range of fermented foods and because of their GRAS (Generally Recognized as Safe) status, the selection of strains delivering nutraceuticals is now the main objective of several research groups. In the following sections, a brief overview of the most recent and relevant works on the use of native and genetically modified LAB for the production of nutraceuticals will be discussed.

7.4.1 Vitamins Among these studies, vitamin production by LAB has recently gained attention of the scientific community (LeBlanc et al., 2014). It has been shown that certain foods fermented with LAB contain elevated levels of B-group vitamins as a result of microbial biosynthesis (LeBlanc et al., 2011b). For this reason, LAB (considered food-grade microorganisms) are the ideal candidates to deliver specific compounds such as vitamins into foods. A detailed description of microbial synthesis of the three most relevant water-soluble Bgroup vitamins (riboflavin, folate, and cobalamin) by LAB has been discussed in detail previously (LeBlanc et al., 2010b, 2011b; Lain˜o et al., 2011) and will not be the focus of this chapter. However, a few recent applications of beneficial microorganisms to increase the concentration of vitamins through microbial biosynthesis will be discussed as an economically attractive alternative to mandatory fortification of foods to produce functional foods. Recently, the riboflavin-producing capabilities of 179 strains of LAB, isolated from a variety of fermented dairy products, were evaluated (Juarez del Valle et al., 2014). Only 42 strains were able to grow in a commercial riboflavin-free medium after which the concentration of this vitamin was determined by high-performance liquid chromatography. Five of these strains (two strains of Lactobacillus (L.) fermentum, one Lactobacillus plantarum, one Streptococcus thermophilus, and one Lactobacillus paracasei subsp. paracasei) were preselected for their capacity to produce elevated concentrations of riboflavin in this commercial B2free medium. These were then inoculated in soymilk to evaluate their capacity to grow in this food matrix to increase its low riboflavin concentrations (309 ng/mL). Only L. plantarum CRL 725 was able to significantly enhance the riboflavin levels in soymilk, doubling (700 ng/ml) more than the initial concentration. In addition to the search for native riboflavin-producing strains, it has been shown that mutants isolated on the basis of their resistance to the toxic riboflavin analog roseoflavin also exhibit I. MICROBIAL AND PLANT TECHNOLOGIES

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a riboflavin-overproduction phenotype (Burgess et al., 2006). Some of these have been shown to provide beneficial effects in vitamin-depleted animals and could be inserted as novel starter cultures (LeBlanc et al., 2005a, 2006). Roseoflavin-resistant variants of L. plantarum CRL 725 were also obtained and evaluated in soymilk. One of the obtained variants was able to increase six times (1860 6 20 ng B2/mL) the initial riboflavin levels of soymilk (Juarez del Valle et al., 2014). Roseoflavin-resistant strains capable of synthesizing riboflavin in soymilk constitute an interesting and economically feasible biotechnology strategy that could be easily adapted by the food industry to develop novel vitamin-bioenriched functional foods with enhanced consumer appeal. In another study, Lactobacillus delbrueckii subsp. bulgaricus and St. thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in a folate-free culture medium and milk after which intracellular and extracellular folate production were evaluated (Lain˜o et al., 2012). From the initial 92 isolated LAB strains tested, 4 of L. delbrueckii subsp. bulgaricus and 32 of St. thermophilus strains were able to grow in the absence of folate. From these, L. bulgaricus CRL 863 and St. thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (between 22 and 135 μg/L) in the folate-free medium. When used to inoculate milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folates. In a follow-up study, the previously identified folate producing L. delbrueckii subsp. bulgaricus (three strains) and St. thermophilus (two strains) were combined and used to elaborate 15 different yogurts, each of which contained different folate concentrations (Lain˜o et al., 2013). The yogurt elaborated with L. bulgaricus CRL 871 and two strains of St. thermophilus (CRL 803 and CRL 415) had a significantly higher folate concentration (180 μg/L) compared to milk and conventional yogurts. These results show that the rational selection of strains was useful in obtaining a yogurt naturally bioenriched in folate with a fourfold increase in vitamin content compared to unfermented milk and a twofold increase compared to conventional yogurts. After these very positive results, this same group demonstrated that the addition of a nonconventional starter culture (Lactobacillus amylovorus CRL 887) to the selected L. bulgaricus CRL 871 and St. thermophilus strains was efficient in producing a new yogurt with even higher folate concentrations (260 μg/L) making this new bio-enriched product a very interesting alternative to fortification with the chemical folic acid (Lain˜o et al., 2015). Recently it has also been reported that LAB and other beneficial microorganisms have the potential to produce vitamins in situ in the gastrointestinal tract (GIT) (LeBlanc et al., 2013b). So far, fragmentary information is available on the de novo synthesis of vitamins by enteric bacteria. In the case of bifidobacteria, the enzymes needed for the biosynthesis of riboflavin seem to be partially or completely absent from the majority of currently available bifidobacterial genomes (Ventura et al., 2007). However, one cannot exclude the possibility that multiple, coexisting microbial species are capable of de novo synthesis (LeBlanc et al., 2013b). With the expanding availability of genome sequences, it is not only possible to identify potential vitamin-producing strains but also to understand the intertwined mechanisms for their biosynthesis, all of which should be exploited to increase the vitamin-producing capacities in the GIT of humans. The association of the human gut microbiota and human health has been reviewed elsewhere (Bakhtiar et al., 2013; de Moreno de LeBlanc and LeBlanc, 2014).

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It has been shown that metabolic engineering can also be used to increase vitamin production by LAB. In one study, a riboflavin consuming strain of Lactococcus (Lc.) lactis subsp. cremoris was converted to a riboflavin “factory” by overexpressing four of its biosynthesis genes (Burgess et al., 2004). This strain was used to produce a novel fermented milk that was able to eliminate most physiological manifestations of riboflavin deficiency (ariboflavinosis), such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values, and hepatomegaly, that were observed using a riboflavin depletion repletion model, whereas a product fermented with a non riboflavin-producing strain did not show similar results (LeBlanc et al., 2005b). Genetically engineered folate-overproducing strains of Lc. lactis were shown to be beneficial of folate-depleted animals in a proof-of-concept study because these were able to revert partial megaloblastic anemia caused by folate deficiency and to increase vitamin levels in the blood (LeBlanc et al., 2010a). To our knowledge, these are the first in vivo studies that have demonstrated that vitamins produced by genetically engineered LAB represent a bioavailable source of these essential micronutrients. In a complementary study, it was shown that these genetically engineered LAB were just as safe as the native strains from which they were derived and thus merit further studies to include them into the food chain (LeBlanc et al., 2010c). Although interesting as to understand the mechanisms involved in vitamin production, these genetically engineered strains cannot yet be included in food products destined for human consumption.

7.4.2 Bioactive Peptides During the fermentation of foods, the hydrolytic enzymes of LAB can release substances with biological activities that can provide benefits to the host. It has long been suggested that latent bioactive peptides in milk proteins could be activated by the proteolytic action of LAB during dairy processing (Gobbetti et al., 2002). These peptides can exert a wide range of effects, such as antimicrobial, antihypertensive, antithrombotic, immunomodulatory, and opioid properties, in addition to aiding in the mineral absorption of calcium. Although many in vitro experiments have shown that potentially bioactive peptides can be produced by the action of LAB on milk proteins, the first proof that such peptides could in fact exert a beneficial effect was shown using murine models where it was demonstrated that peptides released by the proteolytic strain of Lactobacillus helveticus R389 was able to modulate the immune system (LeBlanc et al., 2002) and induce a humoral immune response following an Escherichia coli O157:H7 infection (LeBlanc et al., 2004a). Since these pioneer studies, it has been shown that other LAB are able to produce bioactive peptides from milk proteins. It is important to state that the adequate selection of strains is essential as demonstrated by the variability of hydrolysis of β-, αs1-, and αs2-caseins by 10 strains of St. thermophilus and resulting in the liberation of different bioactive peptides (Miclo et al., 2012). Different Enterococcus faecalis strains from food, environmental, and clinical origins produced angiotensin-converting enzyme (ACE) inhibitory peptides and other bioactive peptides during growth in bovine skimmed milk (Gutiez et al., 2013).

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In another study, it was demonstrated that β-casein hydrolyzate generated by the cell envelope associated proteinase of L. delbrueckii subsp. lactis CRL 581 protects against trinitrobenzene sulfonic acid (TNBS) induced colitis in mice (Espeche Turbay et al., 2012). Bovine casein hydrolysis by Bifidobacterium longum KACC91563 was efficient to produce bioactive peptides with antioxidant activities (Chang et al., 2013). One group produced a novel probiotic-fermented milk with ACE inhibitory peptides produced by Bifidobacterium bifidum MF 20/5 after demonstrating that this strain possessed stronger ACE inhibitory activity than other LAB, including L. helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro and Val-Pro-Pro (Gonzalez-Gonzalez et al., 2013). Not only are bioactive peptides produced from the hydrolysis of milk proteins, recent studies have shown that other food matrices could be used for the production of bioactive peptides by LAB. Peptides with ACE inhibitory activity were generated from porcine skeletal muscle proteins by the action of meat-borne Lactobacillus curvatus CRL 705 and Lactobacillus sakei CRL 1862 (Castellano et al., 2013). Recently, it was shown that enzymatic and microbial conversion of flour components during bread making could determine the accumulation of bioactive peptides (Ganzle, 2014). It was also suggested that LAB could be genetically engineered to synthesize and secrete therapeutic peptides and proteins in the GIT. An interesting review focused on the genetic engineering of Lc. lactis to secrete high-quality, correctly processed, bioactive molecules derived from a eukaryotic background that have a great potential for therapeutic applications once sound environmental containment strategies are put into place (Van Huynegem et al., 2009). In this manuscript, a detailed review is presented on Lc. lactis strains engineered to produce specific antigens, antibodies, cytokines, and trefoil factors, with special regard to immunomodulation, all of which could eventually be added to develop novel functional foods.

7.4.3 Exopolysaccharides Many LAB possess the ability to synthesize extracellular polysaccharides (exopolysaccharides, EPS), which can remain attached to the outer cell wall forming a capsule or are released into the environment contributing to the consistency and rheology of fermented milk products (Notararigo et al., 2013). Antitumor, antiulcer, immunomodulatory, antiviral, and cholesterol-lowering activities are some of the health benefits adduced to these EPS (Nwodo et al., 2012). Increasing attention is being paid to these biomolecules because of their bioactive role and their applicability within the biotechnology, food, and biopharmaceutical industries (Zajsek et al., 2013). One example of industrially important EPS is kefiran which is produced by LAB present in kefir grains that consist of a complex population of LAB and yeasts firmly embedded together. This EPS can be used as a food-grade additive for fermented products because it enhances the rheological properties of chemically acidified skimmed milk gels by increasing their apparent viscosity, and the storage and loss modulus of these gels (Zajsek et al., 2013). It has also been shown that some EPS can act as prebiotics that are compounds that enhance the growth of specific microbial populations such as beneficial bifidobacteria. It was shown that Leuconostoc citreum KACC 91035 is able to produce EPS (isomaltooligosaccharides)

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during kimchi fermentation because of is high glycosyltransferase activity and has been proposed to be used for the synthesis of beneficial oligosaccharides in various fermented foods (Cho et al., 2014).

7.4.4 Antioxidant Enzymes LAB could also be in the treatment of inflammatory bowel disease (IBD) as will be discussed further in Section 7.5. IBD can involve an inflammation associated with oxidative stress, which is characterized by an uncontrolled increase in reactive oxygen species (ROS) concentrations in the GIT. Probiotic LAB strains expressing high levels of antioxidant enzymes could increase these enzymatic activities in specific locations of the GIT and could thus contribute to prevent oxidative epithelial damages. Because few microorganisms produce antioxidant enzymes at the required concentrations to exert biological effects, genetic engineering strategies have been employed to produce antioxidantproducing LAB (LeBlanc et al., 2013a). Superoxide dismutase (SOD) producing Lactobacillus casei BL23 was able to significantly attenuate the TNBS-induced damages in mice as shown by higher survival rates, decreased animal weight loss, lower bacterial translocation to the liver, and the prevention of damage to the large intestines (LeBlanc et al., 2011a). This was in agreement with previous results that have shown that the same SOD-expressing strain of L. casei was able to slightly attenuate the colonic histological damage score of a DSS-induced colitis model (Watterlot et al., 2010). A catalase-producing strain of L. casei BL23 was also evaluated, and it has been shown to significantly decrease the physiological damages caused by TNBS administration (LeBlanc et al., 2011a). Genetically modified LAB to produce antioxidant enzymes could also be included in functional foods because it was recently reported that a St. thermophilus strain selected for its intrinsic antiinflammatory capacity was genetically modified to produce catalase or SOD and administered to mice as bacterial suspension or in fermented milk and their antiinflammatory properties were demonstrated using a TNBS-induced colitis model (del Carmen et al., 2014).

7.4.5 Other Beneficial Enzymes LAB can not only increase or produce beneficial compounds in foods but can also improve the nutritional properties by degrading antinutritional factors. Soy products have an excellent status for their high protein content, and soy proteins contain enough of all the essential amino acids to meet the biological requirements when consumed at the recommended level of protein intake. However, soybeans, as well as other legumes, characteristically contain high concentrations of antinutritional factors such as α-galactooligosaccharides (α-GOS) and phytates that can inhibit the absorption of many essential nutrients and cause serious physiological problems. Hydrolytic digestion of α-GOS (such as raffinose and stachyose) is relatively weak in mammals because they do not possess α-galactosidase (α-Gal) in the upper GIT. The indigestibility of these soluble carbohydrates results in their delivery into the colon where they are rapidly fermented by the resident microbiota resulting in the production

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of large amounts of gas. This induced flatulence greatly hampers the acceptability of soy products as a major food source for humans and animals. It has been reported that some LAB, including L. plantarum, L. fermentum, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus reuteri are able to hydrolyze α-GOS into digestible carbohydrates during vegetable fermentations (LeBlanc et al., 2012). Lactobacillus fermentum CRL 722 and CRL 251 were selected for their high α-Gal activity, which allowed them to degrade raffinose and stachyose in vitro in culture medium and in soymilk (LeBlanc et al., 2004b, 2004c; Garro et al., 1996). It was shown that α-Gal activity was detected in the small intestinal chyme of conventional rats that were administered L. fermentum CRL 722 (LeBlanc et al., 2005c). This activity, even though short-lived, significantly reduced some physiological effects associated with the consumption of nondigestible sugars found in legumes such as soy (LeBlanc et al., 2005c). In a subsequent study, it was shown that when this LAB strain was coadministered with soymilk, a significant reduction in hydrogen emission was observed, showing that α-Gal from L. fermentum CRL 722 remained active in situ, in the GIT of rats monoassociated with Clostridium butyricum, a bacterial species known to produce large amounts of gas from carbohydrate fermentation (LeBlanc et al., 2008). In human microbiota associated rats, L. fermentum CRL 722 also induced a significant reduction of H2 emission (LeBlanc et al., 2008). These findings strongly suggest that L. fermentum is able to partially alleviate the constitutive deficiency of α-Gal in rats and opens the door to various applications in which LAB could be used as a vector for delivery of digestive enzymes in humans and animals. Also, α-Gal genes were inserted in the different Lc. lactis vectors (LeBlanc et al., 2004d), and supplementation with these genetically modified LAB alleviated the undesired physiological effects associated to soy consumption in rats such as increased gas emissions (flatulence) (LeBlanc et al., 2008; LeBlanc et al., 2005c). Phytates, a common component of soybeans and grains can hinder the ability of humans (and other animals) to absorb crucial minerals such as calcium, magnesium, iron, and zinc. Certain LAB produce the enzyme phytase that degrades phytates and therefore may potentially improve mineral bioavailability and absorption. Many studies with phytase-producing strains have been performed and have been reviewed elsewhere (LeBlanc et al., 2012). One of the most studied phytases of LAB is the enzyme produced by Lactobacillus sanfranciscensis CB1, a strain selected among isolates of Italian sourdough because of its high phytase activity (De Angelis et al., 2003). Although most phytase-producing strains have been isolated from sourdough, their application is not limited to this specific fermented cereal. Also, strains that produce elevated levels of phytase have been isolated form soy-based products, such as L. plantarum JK-01 isolated from kimchi (Cho et al., 2007). Leuconostoc mesenteroides KC51, also isolated from kimchi was able to degrade phytate when used to cultivate soymilk (Oh and In, 2009). Although it is clear that microbial phytases are suitable for food fermentations, other enzymes, such as nonspecific acid phosphatase, can also degrade phytate as shown in the low phytase-producing strain of L. plantarum (Zamudio et al., 2001). Following this line of though, it has been reported that LAB can also provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value during fermentation (Reale et al., 2007).

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7.5 PROBIOTIC EFFECTS OF LAB Probiotic microorganisms and fermented foods containing probiotic LAB have grown in popularity due to the increasing number of studies proving that they provide healthpromoting properties. Some of the health benefits which have been claimed for probiotics include improvement of the normal microbiota, prevention of infectious diseases and food allergies, reduction of serum cholesterol, anticarcinogenic activity, stabilization of the gut mucosal barrier, immune adjuvant properties, alleviation of intestinal bowel disease symptoms, and improvement of the digestion of lactose in intolerant hosts. Although many double-blind controlled human clinical trials have been performed, some proposed beneficial effects of probiotics still need to be studied to obtain more information about their mechanisms of action and to confirm their effectiveness.

7.5.1 Probiotics in Intestinal Inflammation The effects of probiotics and fermented products on intestinal disorders have been the most extensively studied because these enter the organism orally and can positively modulate the GIT microbiota. It has been shown that LAB and other probiotic microorganisms can counteract IBDs by stabilizing the gut microbiota and the permeability of the intestinal barrier, and by enhancing the degradation of enteral antigens and altering their immunogenicity (Isolauri et al., 2004). Some of these disease-preventing effects have been recently reviewed (del Carmen et al., 2011a, 2013a); however, some recent examples of different mechanisms by which LAB contained in functional foods exert anti-IBD effects will be given in the following paragraphs. Orally administered Lactobacillus salivarius UCC118 in milk reduced the prevalence of colon cancer and mucosal inflammatory activity in IL-10 knockout mice by modifying the intestinal microbiota in these animals with reduction in Clostridium perfringens, coliforms, and enterococcus levels in the probiotic-fed group (O’Mahony et al., 2001). The administration of yoghurt, with potential probiotic strains, decreased the inflammation by modulation of the host immune response in an acute TNBS-induced mouse model of IBD (de Moreno de LeBlanc et al., 2009). The same yoghurt administered during the remission period in chronic TNBS-induced model of IBD prevented the recurrence of the inflammation in these animals maintaining an anti-inflammatory profile of cytokines in the intestine (Chaves et al., 2011). Both of these effects were related to beneficial changes in the large intestine microbiota of the mice, with increases of bifidobacteria population. The crucial role of interleukin-10 (IL-10) in the development of IBD has been demonstrated by experiments in IL-10-deficient mice. These animals develop a chronic bowel disease resembling Crohn’s disease (CD) in humans, which is in part caused by the loss of suppression of the mucosal immune response toward the normal intestinal microbiota. One of the ways by which probiotics can exert immunomodulatory activities in IBD models is by modulating IL-10 production by the host (de Moreno de Leblanc et al., 2011). Antiinflammatory effects, such as stimulation of IL-10-producing cells, are strain-dependent traits, and their effectiveness also depends on the concentrations used and the method of administration. By causing an increase in the IL-10 levels and in

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consequence decreasing inflammatory cytokines, such as TNF-α, IFN-γ, and IL-17, some LAB can prevent the appearance of local inflammatory diseases and could be used as an adjunct therapy with conventional treatments. Genetically modified LAB have been used to deliver IL-10 in the GIT in animal models (Steidler et al., 2000; del Carmen et al., 2013b) and also in human trials (Braat et al., 2006). These could be included in functional foods because it was previously mentioned that mice that received milk fermented by Lc. lactis strains that produces IL-10 under the control of the xylose-inducible expression system decreased the severity of the inflammation induced by TNBS (del Carmen et al., 2011b). Enhancement of the intestinal barrier function is another mechanism by which probiotic bacteria may benefit the host. This effect may be related to alterations in mucus or chloride secretion or changes in mucosal cell cell interactions and cellular stability through modulation of the cytoskeleton and tight junction protein phosphorylation. Recently, it was reported that milk fermented by Lactobacillus rhamnosus GG regulated intestinal epithelial homeostasis and prevented intestinal inflammation in a DSS-induced mice model with the activation of epidermal growth factor receptor (Yoda et al., 2014). Recently, several randomized controlled trials have confirmed the beneficial effects of probiotics in humans with IBD. Lactobacillus rhamnosus GG is a probiotic bacterium included in many functional foods in the entire world. In a pilot study, it was suggested that L. rhamnosus GG may improve gut barrier function and clinical status in children suffering from mildly to moderately active stable CD (Gupta et al., 2000). In other study, the oral intake of probiotic yoghurt containing Bifidobacterium and Lactobacillus was evaluated in IBD patients in remission phase, which produced an antiinflammatory profile in the serum that may contribute to the health benefices in these individuals (Shadnoush et al., 2013). A randomized, double-blind, placebo-controlled trial demonstrated that a probiotic mixture containing Lactobacillus acidophilus, L. plantarum, L. rhamnosus, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, and St. thermophilus was therapeutical in patients with irritable bowel syndrome due to the stabilization of their GIT microbiota (Ki Cha et al., 2012). Another study showed that probiotic supplementation to patients with ulcerative colitis and severe pouchitis restored the mucosal barrier, which correlated with the bacterial diversity of mucosal pouch microbiota (Persborn et al., 2013). 7.5.1.1 Probiotics and Their Effects on Host’s Immunity and the Prevention of Infections The modulation of the host’s immune response is related to many beneficial effects attributes to probiotic products, such as the decrease of allergies’ prevalence in susceptible individuals, the regulation of inflammatory responses in the gut (discussed above), and the antagonistic effects against intestinal and food-borne pathogens, among others. The intestinal mucosa is the body’s first line of defense against pathogenic and toxic invasions from food. After ingestion, orally administered antigens encounter the gut-associated lymphoid tissue (GALT), which is a well-organized immune network that protects the host from pathogens and prevents ingested proteins from hyperstimulating the immune response through a mechanism called oral tolerance. The main mechanism of protection given by the GALT is humoral immune response mediated by secretory IgA (s-IgA) that

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prevents the entry of potentially harmful antigens while also interacting with mucosal pathogens without potentiating damage. The stimulation of this immune response could thus be used to prevent certain infectious diseases that enter the host through the oral route. Probiotic strains have shown to highly increase s-IgA; therefore, the stimulation of IgA-producing cells is often considered a desirable characteristic in probiotic screening trials (de Moreno de LeBlanc et al., 2005a). LABs are the most common microorganisms evaluated as probiotics by their capacity to modulate the host mucosal immunity. However, the regulation of the intestinal epithelial innate response by probiotic yeast contained in a fermented food, such as kefir, was also reported (Romanin et al., 2010). Lactobacillus casei CRL 431 is a probiotic strain contained in probiotic products that are available in the markets of different countries, including Argentina. The use of animal models demonstrated the immunomodulatory effects on the host exerted by the oral administration of L. casei CRL 431 (Galdeano et al., 2007). These effects were also associated with the protection of against Salmonella enteritidis serovar Typhimurium infection (de Moreno de LeBlanc et al., 2010; Castillo et al., 2011, 2013). Lactobacillus casei DN 114001 is another probiotic strain consumed in many countries, and the effect of the oral administration of milk containing this probiotic bacterium was evaluated in different animal models (de Moreno de LeBlanc et al., 2008a; Galdeano et al., 2009). Milk fermented by L. casei DN 114001 was evaluated as a supplement for the renutrition diet in malnourished mice (Maldonado Galdeano et al., 2011). Probiotic fermented milk improved the gut and systemic immune response in the mice and exerted protection against Salmonella infection. This beneficial effect on the host immunity was also observed in the thymus of the mice (Nunez et al., 2013). The administration of probiotic fermented milk containing L. casei DN 114001 to the mothers during the suckling period and also to their offspring after weaning and until adulthood was evaluated in mice (de Moreno de LeBlanc et al., 2008b). Groups of mice that received this probiotic fermented milk (the mother or the offspring) showed improvement of the intestinal microbiota, an modulation of the gut immune response, which was demonstrated with the stimulation of the IgA-positive cells, macrophages and dendritic cells, and was related to protection against Sa. enteritidis serovar Typhimurim infection in these animals (de Moreno de LeBlanc et al., 2010). A multicenter, double-blind controlled study reported that the administration of fermented dairy product containing the probiotic strain L. casei DN 114001 to free-living elderly individuals was associated with a decreased duration of infection diseases, especially for respiratory infections (Guillemard et al., 2010). The immunomodulatory capacity of L. casei strain Shirota was also reported. The oral administration of this probiotic strain to rats infected with Listeria monocytogenes enhanced host resistance against the infection with increase in cell-mediated immunity (de Waard et al., 2002). The oral administration of L. casei strain Shirota to mice activated not only systemic cellular immunity but also local cellular immunity and reduced influenza virus titer in infected mice (Hori et al., 2002). The oral administration of probiotic microorganisms can influence mucosal sites different to the intestine due to the existence of the common mucosal immune system. Thus, probiotic microorganisms and fermented products orally administered can exert

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beneficial effects against nonintestinal infections such as in pharyngeal mucosa. In vitro studies showed the immunomodulatory capacity of two promising pharyngeal probiotics, L. helveticus MIMLh5 and Streptococcus salivarius ST3 (Taverniti et al., 2012). These strains grow efficiently when co-cultured in milk, suggesting the possibility to prepare a milk-based fermented product containing them for the administration of these bacteria. It was also reported that the surface-layer protein of L. helveticus MIMLh5 plays an important role in mediating bacterial immune-stimulating activity (Taverniti et al., 2013). 7.5.1.2 Probiotics for Obese Hosts The use of probiotics was also evaluated in nonintestinal diseases such as obesity. Recent studies suggest that the GIT microbiota might play a critical role in the development of obesity and LAB were pointed as a candidate for an antiobesity effect (Tsai et al., 2014). A review from 61 original articles showed that the main effect observed at the microbiota level (usually accompanied by weight loss) after probiotic or prebiotic administration in obese hosts was associated with increases in bifidobacteria populations (da Silva et al., 2013). Studies in diet-induced obese mice showed that the supplementation of L. curvatus HY7601 and L. plantarum KY1032 reduced the obesity and modulated proinflammatory and fatty acid oxidation related genes in the liver and adipose tissue; this effect was associated with modulation of the gut microbiota (Park et al., 2013). Recently, the beneficial effect of Lactobacillus coryniformis CECT5711 was demonstrated in a high-fat diet induced mouse model (Toral et al., 2014). Probiotic administration to obese mice induced marked changes in microbiota composition and reduced the metabolic endotoxemia by decrease of the lipopolysaccharide plasma level. Probiotic Lactobacillus gasseri SBT2055 was reported as a strain with antiobesity effect. The administration of milk fermented by this probiotic to rats inhibited dietary fat absorption (Hamad et al., 2009). This probiotic was also administered to high-fat diet induced obese mice and prevented body weight gain, fat accumulation, and proinflammatory gene expression in the adipose tissue (Miyoshi et al., 2014). Nonalcoholic fatty liver disease (NAFLD) is a disease linked to obesity, and the beneficial role of probiotics was also reported (Kelishadi et al., 2013). Recently, it was shown that L. rhamnosus GG protected against NAFLD in a mice model (Ritze et al., 2014). The effect was associated with the increase in total bacterial numbers including the phyla Firmicutes and Bacteroidetes in the distal small intestine. This result was in concordance with the previous one that reported modulation of the microbiota in the small intestine with a concomitant antiobesity effect in mice that received L. rhamnosus GG and L. sakei NR28 (Ji et al., 2012). The effect of probiotics in humans was also observed. A clinical trial with the probiotic bacterium L. salivarius Ls-33 was conducted in obese adolescents to investigate the impact on fecal microbiota (Larsen et al., 2013). Ratios of the Bacteroides Prevotella Porphyromonas group to Firmicutes belonging bacteria were significantly increased after administration of Ls-33; however, these changes were not related to effects on their metabolic syndrome. The probiotic L. gasseri SBT2055 showed beneficial effects in human trials, lowering abdominal adiposity and body weight (Kadooka et al., 2010). A multicenter, double-blind, parallel-group randomized controlled trial conducted in healthy Japanese adults with

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large visceral fat areas showed the reduction in abdominal adiposity when they consumed fermented milk containing a probiotic L. gasseri SBT2055 (Kadooka et al., 2013). Probiotic and conventional yogurt were compared on the lipid profile of type 2 diabetic patients (Ejtahed et al., 2011). A randomized double-blind controlled trial demonstrated that the consumption of probiotic yogurt containing L. acidophilus La5 and B. lactis Bb12 improved total cholesterol and LDL cholesterol concentrations in the patients, contributing to decrease cardiovascular disease risk factors. The authors also reported that the consumption of this probiotic yoghurt improved blood glucose levels and antioxidant status in type 2 diabetic patients (Ejtahed et al., 2012). A randomized doubled-blind controlled clinical trial was performed recruiting obese and overweight individuals who received yogurt containing L. acidophilus La5, Bifidobacterium Bb12, and L. casei DN001 accompanied or not by a low calorie diet (Zarrati et al., 2013). It has been shown that yoghurt consumption had a synergic effect with the low calorie diet on modulation of gene expression in peripheral blood mononuclear cells. 7.5.1.3 Probiotics and Reduction of Cardiovascular Risk Probiotics were reported among dietary strategies to modulate the gastrointestinal microbiota or their metabolic activities and decrease the risk of cardiovascular diseases (Ebel et al., 2014; Taga and Walker, 2008; Tuohy et al., 2014). The improvement of disease biomarkers, especially plasma cholesterol levels, appears to be possible after probiotic administration to lower cardiovascular risk (DiRienzo, 2014). In this sense, it was shown that the administration of a probiotic soy product containing Enterococcus faecium CRL 183 and L. helveticus 416 supplemented or not with isoflavones was associated with an improved cholesterol profile and inhibition of atherosclerotic lesion development in a rabbit model (Cavallini et al., 2011). The authors reported that Enterococcus spp., Lactobacillus spp., and Bifidobacterium spp. were negatively correlated with total cholesterol, non-HDL cholesterol, and lesion size. The intake of the probiotic soy product increased these bacterial species significantly in the fecal microbiota. The influence of diets supplemented with yogurt or probiotic yogurt was examined in spontaneously hypertensive rats, and it was observed that both yoghurt supplements exhibited antihypertensive and hypocholesterolemic effects (Ramchandran and Shah, 2011). A probiotic cheddar cheese was made with L. plantarum K25 and administered to mice to evaluate the effects on serum cholesterol levels (Zhang et al., 2013a). It has been shown that this probiotic cheese can reduce the risk of cardiovascular diseases by decreasing the levels of serum total cholesterol, LDL cholesterol, and triglycerides, and increasing the level of serum HDL cholesterol in mice. 7.5.1.4 Probiotics in Cancer Prevention Colorectal cancer (CRC) is one of the most common cancers worldwide, with incidence rates higher in the Western world. A beneficial approach of probiotics on the gut immune system in the prevention of cancer has also been described (de Moreno de LeBlanc et al., 2007b). There are different mechanisms by which probiotics and fermented products containing viable LAB may lower the risk of colon cancer; some among them are the modulation of the intestinal microbiota (Arthur et al., 2013; Marchesi et al., 2011; Verma and Shukla, 2013), the inactivation of carcinogenic compound (Sreekumar and Hosono, 1998a;

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Sreekumar and Hosono, 1998b; Orrhage et al., 2002), antioxidant effects (described above), and the modulations of the host’s immune response (Matsuzaki et al., 2004; de Moreno de LeBlanc and Perdigon, 2004; Appleyard et al., 2011). Recently, the administration of probiotic Dahi containing L. acidophilus LaVK2 and B. bifidum BbVK3 alone or in combination of piroxicam showed antineoplastic and antiproliferative activities in a model of DMHinduced CRC in rats (Mohania et al., 2014). Probiotics can modify the intestinal microbiota and as result of this action reduce the level of certain enzymes, such as β-glucuronidase and nitroreductase, among others, which convert procarcinogens to carcinogens in the intestine. It was demonstrated using a DMH-induced colon cancer model in mice that animals injected with DMH and fed cyclically with yogurt presented lower enzyme activity levels in the intestinal content than the tumor control group, which increased the activity of these microbial enzymes contributing in this way to the cancer development (de Moreno de LeBlanc and Perdigon, 2005). Probiotics L. rhamnosus GG and L. acidophilus suppresses DMH-induced procarcinogenic fecal enzymes and preneoplastic aberrant crypt foci in early colon carcinogenesis in Sprague Dawley rats (Verma and Shukla, 2013). Modifications in the gut microbiota can also be related with change in the presence of short-chain fatty acids (SCFA) produced by bacterial fermentation of undigested carbohydrates. Using a rat model, it was demonstrated that the symbiotic combination of resistant starch (RS) and B. lactis significantly protected against the development of CRC, which was in part due to an increase in the SCFA by RS intake (Le Leu et al., 2010). There are several studies using animal models that suggest that probiotics have the potential to prevent CRC by modulation of the host’s immune system, especially the cellular immune response. Lactobacillus casei strain Shirota has been demonstrated to have beneficial effects in carcinogenesis animal models, via host immune modulation (Matsuzaki et al., 2004). A possible mechanism of carcinogenesis prevention is the proliferation and activation of NK and T cells (Matsuzaki et al., 2007). Lee et al. (2004) reported that the administration of L. acidophilus SNUL, L. casei YIT9029, and B. longum HY8001 increased the survival rate of mice injected with tumor cells, which was correlated with an increase in cellular immunity (total T cells, NK cells and MHC class II1 cells, and CD42CD81 T cells) (Lee et al., 2004). Yoghurt feeding inhibited tumor growth in a CRC model in mice by modulating of the host immune response (Perdigon et al., 2002). The analysis of cytokine suggested that yogurt feeding stimulated cytokine production when this was required, or inducing downregulation of the immune cells to avoid an exacerbated immune response. This effect would occur mainly through IL-10, which was increased in the tissue of the mice given yoghurt during all the assayed periods (de Moreno de LeBlanc and Perdigon, 2004; de Moreno de LeBlanc et al., 2004). Oxidative stress and epithelial damage are normally linked to pathologies of the GIT so another mechanism by which LAB could prevent certain types of cancer is through the production of antioxidant enzymes that can degrade ROS or impair their formation. A genetically modified strain of L. lactis that produce catalase was evaluated in an experimental DMH-induced colon cancer model, and its capacity to prevent tumor appearance was demonstrated (de Moreno de LeBlanc et al., 2008c).

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As was explained for other pathologies, the effects of probiotic and probiotic foods were also reported for nonintestinal tumors. The antitumor activity of the probiotic L. casei CRL 431 was studied against a fibrosarcoma induced by methylcholanthrene in mice. The administration of the probiotic strain inhibited tumor growth in a dose-dependent form (Perdigo´n et al., 1993, 1995) and stimulated the immune system with high levels of macrophage activation (main infiltrative cells in the tumor), high levels of TNFα, and with a remarkable decrease in the tumor volume. A pilot study suggested that the consumption of a daily probiotic drink by women with a human papillomavirus (HPV)-positive lowgrade squamous intraepithelial lesion diagnosis in their PAP smear had a twice as high chance of clearance of HPV-related cytological abnormalities (Verhoeven et al., 2013). Lactibacillus casei displaying E7 antigen at its surface protected mice against HPV type 16 induced tumors (Ribelles et al., 2013). The administration of probiotic fermented milk containing L. rhamnosus GG and L. casei strain Shirota with chlorophyllin reduced liver precarcinogenic events in rat AFB1induced liver carcinogenesis. This effect was attributed to an increased antioxidant status and decreased expression of oncogenes (Kumar et al., 2011). The beneficial effects of LAB were also reported in animal models of oral cancer (Zhang et al., 2013b) and skin carcinogenesis (Lee et al., 2013). In this last work, Maesil, a member of the genus Rosaceae, was fermented with probiotics and administered to mice in a chemically induced model of skin carcinogenesis. The results showed that Maesil fermented with probiotics inhibited the carcinogenesis through alleviation of oxidative stress. Breast cancer is another type of tumor in which there are reports about the beneficial effects of probiotic administration. Many reports analyzed the association of soy-based products and especially soy isoflavones with breast cancer risk. In this context, soy isoflavone ingestion was studied accompanied with the coadministration of probiotic bacteria, and it was observed that high concentrations of probiotics may alter the metabolism of isoflavones (Cohen et al., 2007). Recently, the consumption of beverages containing L. casei strain Shirota and soy isoflavone was inversely associated with the incidence of breast cancer in Japanese women when they were consumed regularly since adolescence (Toi et al., 2013). The cooperative prevention mechanism of soymilk and L. casei strain Shirota was evaluated using a rat carcinogenic model. It was observed that soymilk prevents the development of mammary tumors and that L. casei strain Shirota suppresses tumor growth (Kaga et al., 2013). Studies performed in humans, by Le et al. (1986), showed a negative association between yogurt consumption and breast cancer development. Similar results were observed in the Netherlands and suggested that these effects would be related to changes in the intestinal microbiota (which could alter the metabolism of estrogen) and to the modulation on the immune system (van’t Veer et al., 1989). In addition to containing LAB, fermented milk can possess nonbacterial components produced during fermentation that may contribute to their antitumor activities (LeBlanc et al., 2002). Thus, cultured dairy products can be proposed to inhibit the growth of many types of cancers, including breast tumors. In this context, milk fermented by L. helveticus R389 (a strain with high proteolytic activity) was studied comparatively with the milk fermented by a proteolytic deficient mutant, and both were able to delay tumor growth in an experimental breast cancer model using BALB/c mice (de Moreno de LeBlanc et al., 2005b, 2005c). This effect was related to the immunoregulatory capacity of the fermented

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milk that decreased IL-6 levels, modulating the relationship between immune and endocrine systems. However, the best modulation of the host immune response was observed in mice fed with milk fermented by L. helveticus R389. Kefir was another fermented product also evaluated in a breast cancer model in mice. Kefir and its cell-free fraction (KF) possess several substances that can exert beneficial effects on the immune system and prevent certain types of cancer (Vinderola et al., 2005). It was observed that mice receiving 2 days cyclical feeding with whole kefir diminished tumor growth, and the same cyclical feeding with KF showed the most significant delay of the tumor growth (de Moreno de LeBlanc et al., 2006). This effect was related principally to a decrease in IL-6. KF caused not only a decrease of this cytokine but also a regulatory response with increased levels of IL-10 in all the samples studied. The results also demonstrated that the most important effect in this tumor model was due to substances released during milk fermentation (and not the microorganisms themselves) (de Moreno de LeBlanc et al., 2007a). Recently, the effect of milk fermented by the probiotic bacterium L. casei CRL 431 was examined on a murine breast cancer model. It was observed that the administration of this probiotic fermented milk stimulated the immune response against this breast tumor, avoiding or delaying its growth when it was preventively administrated and also when the administration started after tumor cells injection (Aragon et al., 2014). 7.5.1.5 Probiotics in Healthy Host There are also reports that showed the potential of probiotics in healthy hosts, maintaining a balanced microbiota, which is an important key for health. The consumption of a probiotic product containing L. coryniformis CECT5711 and L. gasseri CECT5714 was analyzed in 30 children with no gastrointestinal pathology (Lara-Villoslada et al., 2007). An increase in fecal lactobacilli counts was shown at the end of the experimental protocol, and these findings were associated to enhancing the defense against gastrointestinal aggressions and infections and enhancing the immune function with increased IgA concentration in feces and saliva. A recent work reported a clinical trial that included 40 participants with no known digestive diseases. Laminaria japonica, a widely used ingredient in seaweed kimchi, and LAB of traditional fermented Korean food were given to volunteers and was related to increases in the number of some administered LAB species in their gastrointestinal microbiota (Ko et al., 2014).

7.6 CONCLUDING REMARKS Functional foods play an outstanding role in global markets, as demonstrated by their increasing demand derived from the increasing cost of health care, the steady increase of life expectancy, and the desire of older people for improved quality of their later years (Bigliardi and Galati, 2013). In this chapter, the use of LAB that can produce or enhance nutraceutical contents or possess innate beneficial (probiotic) effects has been discussed with the aims of explaining how these important microorganisms can be used as novel biotechnological tools to produce functional foods. The use of adequately selected LAB or genetically engineered strains shows great promise in the development of novel foods that

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not only provide health-promoting properties but also can greatly increase the economic value of different traditional foods.

Acknowledgments The authors would like to thank the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), and the Centro Argentino Brasilen˜o de Biotecnologı´a (CABBIO) for their financial support.

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C H A P T E R

8 Omics and Edible Vaccines Anjana Munshi1 and Vandana Sharma2 1

2

Central University of Punjab, Bathinda, India Indraprastha Apollo Hospital, New Delhi, India

8.1 INTRODUCTION: AN OVERVIEW OF EDIBLE VACCINES Vaccination is one of the standard and very important tools for administering medicine in clinical treatment. Experiments carried out by Edward Jerner and Louis Pasteur introduced this technique by merely exposing the individual to inactivated pathogen. The idea was to train the immune system to recognize a pathogen prior to the outbreak of infection and thus the disease is prevented from the attack of actual pathogen. The vaccine is a preparation of required medicinal compound unstable in another form or biological preparation to improve the immunity or to treat the disease. Conventional vaccines are synthesized from attenuated pathogens; the process may also involve mammalian cell culture and other techniques. Most of the vaccines contain a protein or a set of proteins derived from a pathogen of interest. After its administration into the body, a protective immune response is initiated. While in case of edible vaccines, these are taken as normal food items and the targeted protein for a specific purpose is delivered without any needle or injection. Potato was the first plant which was transformed to produce edible vaccine. The first edible vaccine against New Castle disease in chicken was approved by the USDA in 2006 (www.aphis.usda.gov). This plant vaccine was produced using tobacco plants. Multicomponent edible vaccines can also be prepared by crossing two plant lines of different antigens (Geetika and Sanjana, 2014). The field of omics strives to couple information from genomics, proteomics, metabolomics, and metagenomics, and facilitates its integration with biotechnology. Omics-based advanced technologies focus on desired key traits with precision. Additionally, it enables the expansion of agricultural research in multiple areas, such as food, health, energy, chemical feedstock, and especially chemicals, which help to improve and remediate the environment (Jeanette and Emon, 2016). These technologies can enhance the yield of crops

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and nutritional properties of food for the benefit of consumer, e.g., tomato with high content of lycopene and fruits with high quantity of vitamin and antioxidant properties (Ahmad et al., 2012). Omics-based technologies allow the visualization and monitoring of all the changes. Study of host genome using integrated omics-based technologies, including genomics, transcriptomics, proteomics, and metabolomics, not only helps in the identification of microbial antigens but also assists in the development of targeted vaccines.

8.1.1 Production of Edible Vaccines Using Genomics Genomics has helped in studying the host genome and in identification of microbial antigens. The completion of the sequencing of the first plant genome, reported to have 25,498 genes in Arabidopsis Thaliana. This research work has opened the horizons in the genomic era in plant research (Jeanette and Emon, 2016). By adding a specific gene to a plant, or knocking down a gene with RNAi, the desirable phenotype can be produced in a precise way as compared to traditional breeding techniques (Jeanette and Emon, 2016). Genomics provides controllable methods for molecular breeding and marker-assisted selection, and accelerates the development of new crop varieties (Jeanette and Emon, 2016). The science of genomics has assisted in the development of targeted vaccines of biopharmaceutical importance and industrial enzymes.

8.1.2 Production of Edible Vaccines Using Transcriptomics Transcriptomics is defined as the study of transcriptome—the complete set of RNA, also known as expression profiling, as it is a study of the expression levels of mRNAs in a given cell population. The genome is roughly fixed for a given cell line with the exception of mutations, whereas a transcriptome is dynamic as it is a reflection of the genes actively expressed at any given time under various conditions (Jeanette and Emon, 2016). It determines the pattern of gene expression changes due to internal and external factors such as biotic and abiotic stress (Jeanette and Emon, 2016). The high throughput techniques such as next-generation sequencing provide the capability for understanding the functional elements of the genome (Valdes et al., 2013).

8.1.3 Production of Edible Vaccines Using Proteomics Proteins in plants are responsible for many cellular functions. Proteomics can determine the expression of mRNA, resulting in protein synthesis to explain gene function. A variety of proteins in plants play key roles for the texture, yield, flavor, and nutritional value of virtually all food products (Roberts, 2002). Expression profiling helps in identifying proteins at a specific time and elucidates the function of particular proteins (Jeanette and Emon, 2016). Translational plant proteomics is further expansion of proteomics from expression to functional, structural, translation, and the manifestation of desired traits. By using translational proteomics, the outcomes of proteomics for food authenticity, food security and safety, energy sustainability, human health, increased economic values, and

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for maintaining ecosystem balance can be applied (Agrawal et al., 2012). Proteomics crucially helps in sensitively detecting and quantifying food allergens or multi allergens.

8.1.4 Production of Edible Vaccines Using Metabolomics Metabolomics explains the chemical processes, providing a linkage between genotypes and phenotypes (Aliferis and Chrysayi-Tokousbalides, 2011). It provides information about the expressed proteins that are metabolically active and identifies the biochemical processes and the active function of the various resulting metabolites. The dynamic nature of metabolome is subjected to environmental and other conditions such as biotic or abiotic stress. Metabolic profiling provides an immediate image of processes occurring within a cell, for example, during fruit ripening, key compounds responsible for imparting taste and aroma (Jeanette and Emon, 2016; Dixon et al., 2006). Metabolic profiling is done with the help of mass spectrometry and nuclear magnetic resonance analyses to ascertain metabolic responses to herbicides and to investigate the metabolic regulation and alterations due to environmental conditions of light, temperature, humidity, soil type, salinity, fertilizers, pests and pesticides, and genetic perturbations (Jeanette and Emon, 2016; Aliferis and Chrysayi-Tokousbalides, 2011; Dixon et al., 2006). Various advanced metabolomics profiling techniques have been used to analyze the safety and risk assessments of transgenic food (Jeanette and Emon, 2016; Ahmad et al., 2012; Valdes et al., 2013; Wang et al., 2013).

8.2 EDIBLE VACCINES These are genetically engineered into a consumable crop/plant to produce the protein of desired therapeutic value. After entering into the body, some of the protein enters into blood circulation after digestion. Once the enough amount of protein enters into circulation, immune response against disease-causing pathogens is initiated. These do not require large stainless steel tanks for cell culture and purification. They require only green houses. In the process of edible vaccine production, the desired antigen coding gene isolated from microbes and processed in two manners: (1) by genetic engineering, recombinant virus is introduced into plants. Chimeric virions are extracted and purified from these plants. The edible vaccines thus produced are used for immunological purpose, (2) another method is of transformation technique, vector is integrated with gene of interest (Geetika and Sanjana, 2014; Mishra et al., 2008; Kamenarova et al., 2005). A few examples of edible vaccines along with their application status have been listed in Table 8.1 (Daniell et al., 2009; https://www.crcpress.com). The transgene can be introduced into plant cell by many methods of biopharming. These have been described briefly in the following section.

8.2.1 Plasmid/Vector Mediated Agrobacterium tumefaciens is a soil bacterium, which is used to transfer a small segment of DNA into plant genome by the process known as transformation (Mishra et al., 2008).

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TABLE 8.1 Pharmaceutical Proteins Derived From Plants (PMP) Origin With Designated Medical Applications Host plant (expressing in plant)

PMP

Application (status)

Taliglucerase alfa; recombinant glucocerebrosidase (prGCD)

Carrot cell culture

Phase 3 completed in 2012; FDA approved 2012, used against Gaucher’s disease

Entrotoxigenic Escherichia coli heat labile toxin B (LT-B)

Potato, maize

Diarrhea (in Phase I clinical trial) immunogenic and protective in mice and in human

H5-VLP 1 GLA-AF Vaccine

Tobacco

Phase I clinical trial completed in 2014, against H5N1 strain and an H2N2 strain

Cholera toxin B subunit (CTB)

Potato, tomato, rice, fruits

Tested in mice, protective in cholera

ZMApp

Tobacco

Phase I and Phase II in 2012, against Fabry’s disease

Pfs25 VLP

Tobacco

Phase 1 (2015), against malaria

Accessory colonization factor subunit A (ACFA) or CTB-ACFA

Tomato leaves

Against cholera, current status not available

Diphtheria
tetanus
pertussis

Tobacco, carrot

Tested in mice, induced strong antibody response

Human insulin (CTB insulin)

Potato

Tested and found protective in mice, against insulin-dependent diabetes mellitus

Recombinant human insulin

Arabidopsis

Tested in vitro and in vivo on mice and mammalian cell culture and found active

SARS-CoV S protein (S1)

Tomato and tobacco leaf

Immunogenicity in mice, against SARS

HIV-1 subtype C p24 antigen

Arabidopsis

Immunogenicity in mice, when tested against HIV

Human glucocerobrocidase

Tobacco

Against Gaucher’s disease, has been progressed to Phase III clinical trial

IFN-α2b

Rice

Against hepatitis C assessed in a Phase IIa clinical trial

Gastric lipase

Maize

Phase II trial, pancreatic insufficiency

For more information, please refer to https://clinicaltrials.gov/.

The whole plant is regenerated from individual plant. In this method of gene transfer, the desired gene is inserted into T region of disarmed Ti plasmid of Agrobacterium. The recombinant DNA into Agrobacterium is cultured along with plant cells to be transformed (Streatfield, 2006). The testing of these antigens produced by transgenic plants in animal experiments showed that the genes successfully expressed in these plants (Mishra et al., 2008; Mariotti et al., 1989; Mercenier et al., 2001; Chikwamba et al., 2002; Yuki and Kiyono, 2003) Earlier this method was limited to tobacco and a few other species, but now it has been extended to vegetable species of agronomic interest like Graminae

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and Leguminosae (Chikwamba et al., 2002; Lee et al., 2001). This method has opened new prospects for the development of edible vaccines for humans as well as for veterinary use (Mishra et al., 2008).

8.2.2 Gene Gun or Biolistic Method This method is also known as microprojectile bombardment method, where selected DNA sequences are precipitated onto microparticals (coated with gold and tungsten) and particles are fired at plant cells with high velocity. The microparticals are released into the cell wall to release the foreign DNA into the cell where it gets integrated to nuclear genome of the plant. The molecular mechanism behind this integration is still not clear (Mishra et al., 2008). Transgenic plants thus produced are allowed to grow into new plants with the aim to produce the desired pharmaceutical or antigen protein.

8.2.3 Electroporation/Electrotransfection Electroporation is a technique in which pulse of high electric field/high voltage (0.5 mA or 25 mV for 15 minutes) is applied to increase the permeability of cell membrane. This method is used to cause some type of structural rearrangement of the cell membrane resulting in a temporary increase in porosity and providing a local driving force for ionic and molecular transport through the pores (Darbani et al., 2008). The most common application of electroporation is in vitro introduction of DNA into cells. Physical factors such as transmembrane potential generated by the imposing pulse of electric field, extent of membrane permeation, duration of the permeated state, mode and duration of molecular flow, global and local (surface) concentrations of DNA, form of DNA, tolerance of cells to membrane permeation, and the heterogeneity of the cell population may affect the electrotransfection efficiency of transgenic plant thus produced (Darbani et al., 2008; Hui, 1995; Weaver, 1995).

8.2.4 Lipofection This method involves a derivation of polyethylene glycol-mediated transformation known as liposome-mediated transformation technique. Liposomes are positively charged lipids and are used for DNA uptake due to their favorable interactions with negatively charged DNA and cell membranes (Darbani et al., 2008). In this approach external DNA is encapsulated in a spherical lipid bilayer (which is known as a liposome) to prepare lipoplexes (Gad et al., 1990). The endocytosis process makes the DNA free to recombine and integrate into the host genome Fukunaga et al., 1983). Viral vectors can also be used in this system. The successful transformation has been reported in various plants, e.g., in tobacco, wheat, and potato (Dekeyser et al., 1990; Zhu et al., 1993; Sawahel, 2002). Another example is transformation of intact yeast artificial chromosomes into plant cell, which was successfully achieved via lipofection-like particle bombardment. The lipofection
polyethylene glycol combination method was more efficient than other methods (Darbani et al., 2008; Wordragen et al., 1997).

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Other methods include polymer-based transfection or polyfection, microinjection-based method, wave- and beam-mediated transformation, and desiccation-based transformation. These methods have been reviewed in detail by Darbani et al. (2008).

8.3 MODE OF ACTION OF EDIBLE VACCINES Most of the time a disease-causing pathogen enters into body via mucosal surfaces lining the digestive, respiratory, or urinoreproductive systems (Mishra et al., 2008). The mucosal immune system is supposed to be the first line of defense and is the most effective target site for vaccination (Mor et al., 1998; Korban et al., 2002). The aim of edible or oral vaccine is to provide mucosal as well as humoral immunity against pathogens or infectious agents. When taken orally, edible vaccine undergoes mastication process and then enters into intestine for further degradation via enzymes and acids present in the gut. The antigens produced in transgenic plants are delivered through bioencapsulation in which the parts of plant are fed directly because the outer cell wall protects the antigens from gastrointestinal secretions. The antigen is released and taken up by microfold (known as M) cells in the intestinal wall present over Peyer’s patches (rich in lymphoid tissues) and gut-associated lymphoid tissues, passed onto the macrophages, antigen-presenting cells, local lymphocytes
producing serum immunoglobulins (IgG, local IgE), response and memory cells, and thereby neutralizing the attack of pathogen (Mishra et al., 2008; Ma et al., 1995).

8.4 CONVENTIONAL VACCINES VERSUS EDIBLE VACCINES Both conventional vaccines and edible vaccines are supposed to play the same role once they enter into body, i.e., to produce antibodies against disease-causing harmful pathogen. It has been observed that in case of vaccines produced from microbial system, there is a possibility of endotoxin contamination or problems with viruses or oncogenic DNA arises, e.g., Anthrax vaccine produced from fermenters could get contaminated by Bacillus anthracis toxin produced. However, if it is from transgenic plants, then it is free from toxin (Geetika and Sanjana, 2014). The edible vaccines provide more immunity when compared to conventional vaccines because in later case the sugar attached to animal vaccines was not reckoned to be beneficial. Conventional vaccines are expensive, need sterilization conditions, purification, refrigeration for proper storage, have poor mucosal response, and require cold chain and trained medical personnel when compared to edible vaccines. Children have to experience pain for immunization and vaccination; therefore, noncompliance is observed. The edible vaccines bypass all these conditions, need not be administered with a needle and syringe, can be eaten, and noncompliance is not observed. There is no need of trained medical personnel, have good stability, do not require refrigeration, shipping cost is eliminated, cold chained is not required, and contamination is also not found. Moreover, edible vaccines are economical as compared to conventional vaccines because of mass production, transportation, ease of separation, and purification from plant materials.

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8.5 DISADVANTAGES OF EDIBLE VACCINES There are chances of development of allergic reactions to plant protein glycans and other plant antigens, and contamination of plant and plant products by mycotoxins, pesticides, or endogenous metabolites (Doshi et al., 2013). Development of immunotolerance to vaccine peptide or protein, the consistency of dosage from plant to plant, is not similar, stability of vaccine inside the fruit is not known, dosage may be variable from plant to plant, and selection of the best plant is difficult, and certain foods, e.g., potato is not eaten raw and cooking may modify the properties of vaccine contained in it because the exact or required dose/amount of antigen cannot be measured in a plant as in case of syringe while using conventional vaccine. Moreover, this method is not convenient for infants (not able to eat). The uncertainty that tomato and banana do not have standard or perfect size and therefore people may consume too much vaccine that may be toxic or very less amount may lead to the outbreak of disease among population believed to be immune (www.pharmatutor.org).

8.6 APPLICATIONS OF EDIBLE VACCINES Successful edible vaccines prove to be a boon for medical sciences and can cure a number of diseases such as cancer, infectious diseases, and heart diseases with minimum effort. Many antigens of desired therapeutic value have been expressed successfully in transgenic plants and have been demonstrated to retain their native functional forms. A few of these have been discussed below.

8.6.1 Autoimmune Diseases Type I diabetes or insulin-dependent diabetes mellitus, affecting young adults and children, is a disease that results from autoimmune destruction of the insulin secreting beta cells in the pancreas. Injecting pancreatic glutamic acid decarboxylase (GAD67) prevented mice from diabetes. Transgenic plants, potato, and tobacco with the gene encoding for GAD67 have been developed (Geetika and Sanjana, 2014; Ma et al., 1995; Blanas et al., 1996). Studies have shown that feeding of diabetic mice with these vaccines helped in suppressing the autoimmune attack and delayed the rise in blood sugar levels (Ma et al., 1995; Travis, 1998).

8.6.2 Gastrointestinal Disorders According to WHO, cholera vaccine provides cross protection against enterotoxin Escherichia coli heat labile enterotoxin B (LT-B) and is quite effective in preventing cholera. Transgenic potatoes expressing LT-B gene fed to mice leads to the production of secretary as well as serum antibodies. In addition, cooking of raw potato did not inactivate the antigen leading to the conclusion of production of edible vaccine to be expanded from raw food plants to fruits (Mason et al., 1998; Lindblad and Holmgren, 1993; Richter et al., 1996).

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8.6.3 Malaria Malaria vaccine was developed by researchers using three antigens namely merozite surface protein (MSP) 4, MSP 5 from Plasmodium falciparum, and MSP4/5 from Plasmodium yoelli (Geetika and Sanjana, 2014; Wang et al., 2004). Mice were orally immunized with this recombinant antigen and co-administered with cholera toxin B subunit as mucosal adjuvant to induce immune response. However, oral delivery of plant-derived malaria vaccine inducing immune response is uncertain, because the expression level of antigens in plants is very low.

8.6.4 Measles Measles affected people usually become deaf or develop encephalitis or in adverse cases may lead to death as well. The available vaccine for measles is a live attenuated vaccine having certain disadvantages. For preparing edible vaccine, MV-H (measles virus hemagglutinin from Edmonston strain) antigen was introduced in tobacco plant using a plasmid vector. It was observed that serum antibodies induced immune response against the antigen. The fecal samples of immunized animal were also found to contain antigencontaining IgA antibodies. Therefore, transgenic rice, lettuce, and baby food have been developed against measles. If this was given along with CTB, 35
50 g of MV-H lettuce was enough, but a higher dose might show better results (Giddings et al., 2000).

8.6.5 Hepatitis B Hepatitis B is an infectious disease of liver caused by Hepatitis B virus. Initially the symptoms are yellowing of skin, vomiting, and abdominal pain, which eventually may develop into liver cancer and cirrhosis and death in 15%
25% cases. During this infection, the first detectable antigen is hepatitis B surface antigen (HBSAg). Vaccination is available against Hepatitis B infection. The transgenic plants were developed by CaMV (Cauliflower mosaic virus) plasmid cloned by HBSAg subtype and thus regenerated plants produced HBSAg from the transformed cells. Further experiments in potatoes showed higher concentration of expression antigen in roots than in leaf tissues and was not sufficient to be used for oral vaccine (Domansky, 1995). Further research was carried out to increase HBSAg levels using different promoters, e.g., patatin promoter and different transcriptional regulatory elements (Geetika and Sanjana, 2014). Artnzen and colleagues investigated a number of signaling peptides and 50 and 30 untranslated regions in constructs driven by normally constitutive CaMV 35s promoter to develop transgenic potato with higher levels of HBSAg antigen (Artnzen, 1997). When HBSAg expressed in tobacco plant was administered to animals as parental vaccine, it showed primary response equivalent to conventional vaccine. When animals were fed on tobacco plant with HBSAg expression, it showed better results as compared to conventional vaccine.

8.7 CLINICAL TRIALS AND RESEARCH STUDIES Many research studies and clinical trials are undergoing to establish the efficacy and usefulness of edible vaccines. For example, tomato plant expressing rabies could induce I. MICROBIAL AND PLANT TECHNOLOGIES

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antibodies in mice. The transgenic potato produced from transgenic plant with CTB gene of Vibrio cholera was found to be very effective in mice. Transgenic potato expressing Norwalk virus antigen has shown seroconversion. Human trials of potato-based hepatitis B virus have shown good results. A study carried out by Mason et al. (1996) investigated the antigenic or immunogenic effects of transgenic potato tubers and tobacco leaves, carrying a Norwalk virus capsid protein; in mice, this virus causes gastroenteritis in human. A successful expression of antigens in plants was achieved for rabies virus G-protein in tomato (McGarvey et al., 1995). Rotavirus VP7 has been expressed in transgenic plants, and oral immunization in mice was observed by inducing the production of mucosal IgA and serum IgG against VP7 (Wu et al., 2003). The development of systemic and mucosal antibody response was observed in response to LT-B in young and aged mice. The expression of a recombinant subunit antigen (ORF2), representing the carboxy-terminal 267 amino acids of the 660 amino acid hepatitis E virus (HEV) capsid protein of HEV was studied in tomatoes, and activity of protein or expressed antigen was tested using enzyme-linked immunosorbant assay method (Ma et al., 2003). In addition, efforts have also been made to develop edible vaccine against neurocysticercosis that occurs due to ingestion of contaminated food and water by Taenia solium (Lightwlers, 2003). Vaccine against severe acute respiratory syndrome coronavirus has also been developed by scientists and is under study. These examples show that plants are promising bioreactors in generation of therapeutically desired biopharmaceuticals.

8.8 SECOND-GENERATION EDIBLE VACCINES Second-generation edible vaccines are multicomponent vaccines which provide protection against several pathogens and have the ability to develop more than one antigenic protein. These are produced by crossing two cell lines containing different antigens. The adjuvant can be co-expressed with same antigen in the same plant. For example, a trivalent edible vaccine against cholera, ETEC or Enterotoxigenic E. coli, and rotavirus could initiate an immune response to these three successfully (Geetika and Sanjana, 2014).

8.9 CURRENT DEVELOPMENTS Edible vaccines or oral vaccines must be protected during their passage to gastrointestinal tract. To counter this problem, a large number of delivery systems have been developed and modified for presenting nonliving antigen to mucosal surfaces, which will allow these antigens to survive from acid and enzymatic attack from gastrointestinal tract. These include polylactide, polyglycolide, microsphere, liposomes, proteasomes, co-chelates, virus-like particles, and immune-stimulating complexes (Doshi et al., 2013). Even more palatable alternatives to potatoes have been developed, e.g., banana. Many solutions have been tried to overcome this limitation. These include techniques such as optimization of the coding sequence of bacterial or viral genes for expression as plant nuclear genes and defining the subcellular components to accumulate the product for optimal quantity and quality. Another method is to improve the immunogenicity of the orally delivered antigens by using mucosal adjuvants (Doshi et al., 2013; www.pharmatutor.org). I. MICROBIAL AND PLANT TECHNOLOGIES

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8.9.1 Banana, Tomato, and Potato The Boyce Thompson Institute for Plant Research at the United States is working on genetically engineered plants to produce vaccines in the fruits. Bananas have been used to develop vaccine against diarrhea. Bananas are perfect choice for this purpose because they grow widely in many parts of the developing countries and can be eaten raw especially by children. Other advantage is that bananas are sterile and genes do not pass from one banana to other banana plant, need no cooking, grow quickly, and are rich in vitamin to boost immune response. Tomatoes grow quickly, have high content of vitamin A, are heat stable, different batches can be blended to set the uniform dose of required antigen, and do not pass infection. Tomatoes have been used for developing edible vaccine against HIV, Alzheimer’s disease, SARS, anthrax, and respiratory syncytial virus. The other plants used for edible vaccine production are rice for cholera, flu, botulism, and hay fever; and tobacco for Crohn’s disease and against human papilloma virus causing cervical cancer. Soybean and Lettuce have also been used for producing edible vaccines, but readily spoilage is their main disadvantage.

8.10 PATENTS ON EDIBLE VACCINES The edible vaccine, reported to be efficacious in animal trials and patented first of all, was against transmissible gastroenteritis virus in pigs. Vaccine against porcine reproductive system, respiratory syndrome, and foot and mouth disease of animals has been investigated in clinical trials (Yang et al., 2007; Esmael and Hirpa, 2015). Several patents have been filed by biotechnology companies on edible vaccines, e.g., Prodigene, has claimed for vaccine produced in transgenic plants for the treatment of hepatitis and gastroenteritis. Found Advan Mil Med has patented antibacterial vaccine for the treatment of shigellosis (Khoudi et al., 1999). Ribozyme Pharm has developed nucleic acid vaccine used in the treatment of viral infection in plants, animals, or bacteria. Many institutes across the globe have developed potentially effective edible vaccines, e.g., vaccine against invertebrates (insects, arachnids, and helminths), and Hepatitis B virus core antigen recombinant vaccine have been developed by the University of Yale and University of Texas, respectively. Biosource (now large-scale biology) has developed and patented plant viral vector with the potential of anti-AIDS vaccine (www.unicef.org). The selected patents on edible vaccines have been summed up by Mishra et al. (2008).

8.11 FUTURE PROSPECTS Although edible vaccines are boon for medical sciences, yet researchers are grappling with many problems such as poor growth of transgenic plants when they initiate producing foreign protein. The studies carried out on animals and human so far have provided a proof of feasibility with consideration of certain issues.

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In many countries, plants producing edible vaccines fall under restrictive category set up to control genetically modified crop plants. This creates problems for the acceptance of edible vaccines especially in Europe. Another difficulty is the resistance towards genetically modified plants and crops. For example, Zambia had refused genetically modified maize in food aid from the United States despite a terrible draught (https://www.theguardian.com/science/2002/oct/17/gm.famine). Edible vaccines have many advantages over conventional vaccines, not only they cut the cost, shipping, refrigeration, and storage but also are safer and reduce adverse reactions, pain of injection, and improve patient compliance. Quality assurance, preclinical evaluation, efficacy, and environmental influence need to be taken into account before endorsing edible vaccines for human use. Random insertion of therapeutically effective desired genes into plants can destabilize the genome of plant species and may influence the balance of ecosystem. If the technology is enriched with invaluable scientific knowledge and with right regulatory framework, it may usher into a new era of eating vaccines instead of injecting them with a needle and syringe.

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Hui, S.W., 1995. Effects of pulse length and strength on electroporation efficiency. Methods in Molecular Biology. Plant Cell Electroporation and Electrofusion Protocols. Humana Press Inc., Totowa, NJ, pp. 29
30. Jeanette, M., Emon, V., 2016. The omics revolution in agricultural research. J. Agric. Food Chem. 64 (1), 36
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86. Khoudi, H., Laberge, S., Ferullo, J.M., Bazin, R., Darveau, A., Castonguay, Y., et al., 1999. Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnol. Bioeng. 64, 135
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2175. Lee, R.W.H., Strommer, J., Hodgins, D., Shewen, P.E., Niu, Y., et al., 2001. Towards development of an edible vaccine against pneumatic pasteurellosis using transgenic white clover expressing a Mannheimia fusion protein. Infect. Immun. 69, 5786
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515. Mishra, N., Gupta, P.N., Khatri, K., Goyal, A.K., Vyas, S.P., 2008. Edible vaccines: a new approach to oral immunization. Ind. J. Biotechnol. 7, 283
294. Mor, T.S., Gomez-Lim, M.A., Palmer, K.E., 1998. Edible vaccine: a concept comes of age. Trends Microbiol 6 (219), 226. Richter, L., Mason, H., Arntzen, C.J., 1996. Transgenic plants created for oral immunization against diarrheal diseases. Travel Med 3 (52), 56. Roberts, K.M., 2002. Proteomics and a future generation of plant molecular biologists. Plant Mol. Biol. 48, 143
154. Sawahel, W.A., 2002. The production of transgenic potato plants expressing human alpha-interferon using lipofection-mediated transformation. Cell Mol. Biol. Lett. 7 (1), 19
29. Streatfield, S.J., 2006. Mucosal immunization using recombinant plant based oral vaccines. Methods 38, 150
157. Travis, J., 1998. Scientists harvest antibodies from plants. Science News 5, 359. Valdes, A., Ibanez, C., Simo´, C., Garcia-Canas, V., 2013. Recent transcriptomics advances and emerging applications in food science. Trends Anal. Chem. 52, 142
154. Wang, L., Goschnick, M.W., Coppel, R.L., 2004. Oral immunization with a combination of Plasmodium yoelii merozoite surface protein 1 and 4/5 enhances protection against malaria challenge. Infect. Immunol. 72, 6172
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178. Wu, Y.Z., Li, J.T., Mou, Z.R., Fei, L., Ni, B., Geng, M., et al., 2003. Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA. Virology 313 (2), 337
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Yang, C.J., Liao, C., Lai, M., Jong, C., Liang, Y., Lin, N., et al., 2007. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes. BMC Biotechnol. 7, 62. Yuki, Y., Kiyono, H., 2003. New generation of mucosal adjuvants for the induction of protective immunity. Rev. Med. Virol. 13, 293
310. Zhu, Z., Sun, B., Liu, C., Xiao, G., Li, X., 1993. Transformation of wheat protoplasts mediated by cationic liposome and regeneration of transgenic plantlets. Chin. J. Biotechnol. 9 (4), 257
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Further Reading Langridge, W., 2000. Edible vaccines. Sci. Am. 283, 66
71. US National Institutes of Health Clinical Trial Home Page. https://clinicaltrials.gov.

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C H A P T E R

9 Plant Metabolic Engineering Neelam S. Sangwan, Jyoti S. Jadaun, Sandhya Tripathi, Bhawana Mishra, Lokesh K. Narnoliya and Rajender S. Sangwan CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, Uttar Pradesh, India

9.1 INTRODUCTION Metabolic engineering is a progressive technique by which the production of certain substances is increased within the cell. Metabolic engineering is mainly focused on metabolic intermediates or products, such as starch, vitamin E, amino acids, and enzymes. Metabolic engineering was differentiated from genetic engineering because it investigates the properties of integrated metabolic pathways and genetic regulatory networks, not with individual genes and enzymes (Muir et al., 2001; Woolston et al., 2013). Metabolic engineering can be inferred as the directed and useful manipulation of metabolic pathways in a living system by manipulating transporters, enzymatic, and regulatory functions of the cell using recombinant DNA technology (Bourgaud et al., 2001). Metabolic engineering may be approached through overexpression or upregulation of targeted gene such as, enhanced tocopherol production by overexpression of geranylgeranyl transferase gene in corn seeds (Cahoon et al., 2003). Similarly, down regulation of zeaxanthin epoxidase gene by antisense and cosuppression inhibition, resulted in an accumulation of zeaxanthin (Romer et al., 2002). Metabolic engineering also has distinct industrial dimensions because it aims on construction of microbes which can be further utilized as biocatalysts for the cost-effective production of pharmaceuticals, fuels and many other chemicals (Sangwan and Sangwan, 2007; Woolston et al., 2013).

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9.1.1 Metabolites Plants are the most predominant source of small phytomolecules, called as metabolites. Plant metabolites are organic compounds synthesized via enzyme-mediated processes called as metabolic pathways. These plant metabolites are important for both essential (proteins, lipids, and carbohydrates) as well as specific and specialized functions (anthocyanin, carotenoid, etc.). The primary metabolites are known to be the essential for the survival of the organisms and any alterations in their levels are likely to have severe major manifestations (Sangwan and Sangwan, 2014). However, secondary metabolites are considered to be the metabolites having specialized functions and playing a role in providing quality of life to the producer organism. Often, secondary metabolites are associated with defense related, antifeedent, insect attractant, and repellent functions. At present, in plant kingdom more than 100,000 natural products having low molecular mass have been identified so far (Trethewey, 2004). 9.1.1.1 Types of Metabolites On the basis of their importance, functions, and need in plants, these metabolites are classified into two types: primary and secondary metabolites. Primary metabolites like lipid, protein, nucleic acids, and carbohydrates are found universally in plant kingdom and directly involved in growth and fundamental metabolic pathways (Glycolysis, Krebs cycle, and Calvin cycle). The primary metabolites act as the precursors for secondary metabolites. Difference between primary and Secondary metabolites is that unlike primary metabolites secondary ones are generally not essential for growth and developmental process of plants but their absence results in long-term impairment of the organism’s survival ability and potential, not immediate death as in case of primary metabolite deficiency (Sangwan and Sangwan, 2014). On the basis of their biosynthetic pathways, plant secondary metabolites are usually classified into three main groups—terpenes (plant volatiles, sterols, carotenoids, and cardiac glycosides), phenolics (flavonoids, coumarin, tannins, lignin, stilbenes, and lignans), and nitrogen containing compounds (alkaloids and glucosinolates) (Bourgaud et al., 2001; Yadav et al., 2014: Jadaun et al., 2017a). 9.1.1.2 Importance of Secondary Metabolites Secondary metabolites are not essential for growth and development of plant but these are required for the survival of plants in unfavorable environment. Secondary metabolites are important for flower color or volatile, flavor of food, attraction of pollinator, interaction with symbiotic microorganisms, and tolerance against pest and diseases (isofalvonoids and phenylpropanoid derivatives). These are also take part in the mechanism of frost tolerance, nutrient storage, structural reinforcement, photo protective, UV
Vis absorption, signaling to mutualist, and in the adaptation of the plant under various environmental conditions and stress (Singer et al., 2003; Verpoorte et al., 2002). Presently, secondary metabolites (morphine, digitoxin, salicylic acid, etc.) are also important for their health improving effects and disease preventing activities such as antioxidant and cholesterol lowering (lovastatin) properties. Secondary metabolites such as terpene, used as antibacterial, insecticide (pyrethrin), antifungal, antitumoric (bleomycin), antimicrobial (pentacyclic terpene arjunolic acid), antibiotic (penicillin, vancomycin) and anticancer (taxol) compounds.

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Because of these properties, these molecules are used as fine chemicals such as drug, flavors, dye, fragrance, and insecticides (Table 9.1) (Verpoorte et al., 2002; Sangwan et al., 2001; Sangwan and Sangwan, 2014; Sangwan et al., 2007a,b). Some plants, such as Withania, Centella, Artemisia, Cymbopogon, etc. possess medicinally important phytoconstituents like withanolides, withaferin, withanone, asiaticoside, madecassoside, artemisinin, essential oil, etc. These compounds have tremendous significance as drugs (Chaurasiya et al., 2012; Sangwan et al., 1993, 2004, 2007a,b, 2008, 2010, 2013b; Sangwan and Sangwan, 2007, 2014). Moreover in the year 2015, in Physiology and Medicine Noble prize has been awarded to Youyou Tu for discovering the artemisinin, a potential antimalarial drug, isolated from Artemisia annua (Su and Miller, 2015).

9.2 METABOLIC ENGINEERING—A TOOL FOR CREATING DESIRED DIVERSITY Metabolic engineering is very important to improve or increase plant metabolites which are involved in their better survival and enhance their cellular and biological activities. Metabolic engineering can be utilized to enhance resistance against pests, insects, microorganism or various diseases. It is used to improve quality traits (color of fruits and flowers), improve taste or flavor of food, fragrance of flower, lowering of undesirable (toxic) compounds from food and fodder, increase the production of desired compounds (vitamin, antioxidant, etc.) in food (Verpoorte et al., 2002). Recently, attempts are in progress to produce desirable medicinal compounds such as artemisinin, taxol, etc. having immense medicinal value using metabolic engineering approaches. Biosynthesis of different types of secondary metabolites has been attempted for the metabolic engineering. Several reports in literature are available in this regards such as phenylylpropanoid biosynthesis (Guterman et al., 2002), terpenoid biosynthesis (Aharoni et al., 2004, 2006; Chaurasiya et al., 2012; Sharma et al., 2005, 2009), tropane biosynthetic (Zhang et al., 2004; Kushwaha et al., 2013a,b), carotenoid biosynthesis (Giuliano et al., 2008), xanthophyll biosynthesis (Dharmapuri et al., 2002), flavonoid biosynthesis (Muir et al., 2001), alkaloid biosynthesis (Glenn et al., 2013), sterol biosynthesis (Chappell et al., 1995), saponin biosynthesis (Lambert et al., 2011), terpenoid indole alkaloids biosynthesis (Liu et al., 2011), lignin biosynthesis (Satake et al., 2013), and benzenoid biosynthesis (Orlova et al., 2006) etc. (Fig. 9.1). Besides, to improve response such as the plant defense and pollinator interaction, volatile compounds are specifically targeted for metabolic engineering (Dudareva et al., 2013).

9.3 APPROACHES AND STRATEGIES 9.3.1 Systems for Metabolic Engineering Metabolic engineering in plants opens up new opportunities in the field of agriculture, environmental applications, chemicals production, and medicine. In metabolic engineering approaches, usually there are mainly three systems which are frequently used, such as whole plant, tissue cultured plant cell, and plant gene(s) in microorganism.

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TABLE 9.1 List and Structures of Some Important Plant Secondary Metabolites With Their Significance Plant Name

Secondary Metabolite

Vitis vinifera

Anthocyanin

Attracting pollinators, antioxidants, protecting cells from high-light damage, etc.

Allium cepa

Flavonols

Antioxidant, UV protection, flower color, etc.

Glycine max

Isoflavonols

Phytoalexin, stimulate soil-microbe rhizobium to form nitrogen-fixing root nodules, etc.

Petroselinum crispum

Flavones

Antioxidant, drug interactions properties, etc.

Mentha spp.

Linalool

Antimicrobial, antibacterial, antiviral effects, antiinflammatory, fragrance ingredient in cosmetics etc.

Rosmarinus officinalis

α-Pinene

Used in flavorings, fragrances, medicines, fine, chemicals and high-density renewable fuels, etc.

Red grapes

Stilbene

Phytoalexin, cancer chemoprotective agents, cardioprotective properties, etc.

Acacia spp.

Tryptamine

Serotonin releasing agent, serotonergic activity enhancer, neuromodulator or neurotransmitter, etc.

Withania somnifera

Withanolide A

Antiarthritic, antiaging, cognitive, antiinflammatory, anticancer, antitumor, etc.

Chemical Structure

Significance

(Continued)

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TABLE 9.1 (Continued) Plant Name

Secondary Metabolite

Chemical Structure

Significance

Withaferin A

Withanone

FIGURE 9.1 Elucidation of generalized biosynthetic pathways for production of plant secondary metabolites. BBE: Berberine bridge enzyme; PAL: L-phenylalanine ammonia-lyase; 4CL: 4-coumarate CoA ligase; C4H: cinnamate 4-hydroxylase; CHS: chalcone synthase; CHR: chalcone (polyketide) reductase; CHI: chalcone isomerase; STS: stilbene synthase; FOMT: flavanone 7-O-methyltransferase; 2-HIS: 2-hydroxyisoflavanone synthase (isoflavone synthase); IOMT: isoflavone 48-O-methyltransferase; IDMT: isoflavone/isoflavanone dimethylallyl transferase; IFR: isoflavone reductase, VR: vestitone reductase; BX1: indole-3-glycerol phosphate lyase; BX2-5: cytochrome P450 enzymes of DIMBOA biosynthesis; ACC: acetyl CoA carboxylase; DXPS: 1-deoxy-xylulose 5-phosphate synthase; HMGR: 3-hydroxy-3-methylglutaryl CoA reductase; SS: sesquiterpene synthase; SQE: squalene epoxidase; BAS: beta-amyrin synthase. I. MICROBIAL AND PLANT TECHNOLOGIES

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9.3.1.1 Plant Systems Plants can be used as chemical factories because they are the only organism which can capture solar energy and fix it with combing to the atmospheric CO2 as the feedstock (Ohlrogge, 1999). Recombinant DNA technology is the root of plant metabolic engineering through which produces transgenic plants. Classical agricultural techniques and equipments are well established for growing, maintaining, and harvesting of these transgenic plants. Specialized plant organs such as glands, trichomes, etc. are also tried for metabolite extraction (Sharma et al., 2003). Crop species could be used for the production of phytochemicals, especially in pharmaceuticals areas where it is grown for food consumption (Singh et al., 2014). Thus crops which do not hybridize with main crops are gaining important values for the purposes of genetic manipulation via metabolic engineering. More success can be envisaged while using whole plant system as it is complete network of pathways and only desirable key step is needed to manipulate (Fig. 9.2) (Sangwan et al., 2014). There are some drawbacks of this approach which need serious considerations such as requirement of large field space, trials and clearances, and long gestation time to grow. This is specifically pertinent in case of medicinal and aromatic plants where yield of valuable phytochemicals are extremely limited in many of the cases (Sangwan and Sangwan, 2014). Therefore alternate systems, requiring low space, resource, and with faster growth rate, are in demand for production of phytochemicals at industrial scale. 9.3.1.2 In vitro Cultures Nowadays, tissue culture is the widely used technique for obtaining the higher quantity of phytoconstituents in least time and space. Tissue culture offers controlled in vitro conditions for the production of phytochemicals (Mishra et al., 2013). Culture conditions can be manipulated in such a way (such as elicitor’s treatment and by using other stress inducers) that the appropriate gene arrays can be expressed and higher levels of metabolites are synthesized and accumulated (Radman et al., 2003; Mishra et al., 2014; Jadaun et al., 2017b). In tissue cultured cell lines, metabolic profiles can be altered by transforming genes which

Plant systems for metabolic engineering

Plant cell tissue culture

Whole plant

Callus culture

Cell suspension culture

Multiple shoot culture

Root culture

Plant genes in microorganism

Bacterial culture

FIGURE 9.2 Plant systems used for metabolic engineering.

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149

encode biosynthetic enzymes or regulatory factors of metabolic pathway of interest. Plant tissue culture also provides an open platform for screening of genes that can be further helpful for the production of transgenic plants with respect to particular trait. Tissue culture technique has been shown to be promising in expression of secondary metabolites in unconventional systems as well as in enhanced amounts in conventional systems (Sabir et al., 2013; Chaurasiya et al., 2012; Jadaun et al., 2017a). One such example is by using tissue cultured technique in Withania somnifera, the production of withanolides was increased (Chaurasiya et al., 2012; Sangwan et al., 2007a,b, 2008; Sabir et al., 2007, 2010, 2012, 2013) (Fig. 9.2). Thus for large-scale production of novel drugs, or unusual metabolites from rare plant species or transgenic plant, tissue culture is a very good alternative source for production of metabolites. If any gene, which alters the flux of pathway, transformed in plant via tissue culture leads to increased production of metabolites in whole plant or in cultures (Hallard et al., 1997; Guterman et al., 2002; Sabir et al., 2011). 9.3.1.3 Microbial Cells Microorganisms (e.g., Escherichia coli) contain biochemical profile similar to plant chloroplast and they possess short life cycle, so they grow very fast. Using this advantage, the results of genetic modification can be easily applied on these microbes which could be later tested in plants. In this technique, plant genes are cloned in a suitable vector and then transformed into the host microorganism (Fig. 9.3). Microorganisms have short life cycle so they increase their number rapidly. Production of the transgenic plant takes more time than culturing of microorganism. Carotenoid and isoprenoid biosynthetic pathways are genetically manipulated using this approach. This system can also be applied for identification of the function of a transgene encoding a putative enzyme

FIGURE 9.3 Diagrammatic representation of basic methodologies used for metabolic engineering in plants.

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(Matthews et al., 2003). This approach can be used to screen out genes which may influence metabolic pathway flux in either increasing or decreasing order (Gallagher et al., 2003). Pathway flux operated in plant plastid (like nonmevalonate pathway) can be easily manipulated via microbial strategies (Matthews and Wurtzel, 2000). Prominent and successful examples are available in literature exhibiting the tremendous potential of the microbial cell culture in the production of specialized metabolites.

9.3.2 Management and Modulation of Metabolic Flux All metabolites are synthesized via specific biosynthetic pathways. For enhancing the production of any preferred metabolite, first requirement is to have information about each and every step or factor which affects its biosynthetic pathway. Metabolic flux of desired metabolite could be increased by altering the steps or factors which influence the pathway. Recent thrust is that biologists are interested in discovering new genes and enzymes for better understanding of metabolites flux (Kolisnychenko et al., 2002; Sangwan et al., 2010). Some nontraditional modern strategies, by which metabolic flux could be increased, are gene identification through mining of transcriptome or genome, simultaneous expression profiling of multiple genes in plants, diverting flux by overexpression and silencing, diverting whole pathway flux by transcription factors (TFs) and diverting whole pathway flux by cis-regulatory elements (promoters). 9.3.2.1 Identification of Key Genes Change in the flux of plant secondary metabolites is possible through modulations in the participating genes of the related pathway. The first requirement is the identification of genes which are involved in the interested/desired pathway. There are several ways to identify genes but recent upsurge of transcriptomics and genomics approaches for gene identification is widely potential owing to ease for identification of many genes at a time and tools to analyze them. Many plant transcriptomes are sequenced like Arabidopsis rosescented, geranium, Centella asiatica, W. somnifera, Neem, etc. (Yamada et al., 2003; Narnoliya et al., 2017; Sangwan et al., 2013a; Gupta et al., 2013; Krishnan et al., 2011). Tissue specific expressed sequence tags (ESTs) information can also be generated by using techniques such as microarray, suppressive subtractive hybridization for identification of tissue specific genes and their metabolites (Narnoliya et al., 2014; Rajakani et al., 2014). After assembling the transcriptome or genome sequences data, a complete metabolic pathway can be annotated and identified via bioinformatics tools. Further, using recent biotechnological approaches identified gene or set of genes can be modulated and consequently pattern of metabolic flux will be altered (Sangwan et al., 2013a,b; Narnoliya et al., 2017). By using these approaches, cis-regulatory elements (promoters) and TFs could be identified, but there are some limitations, such as all identified genes, promoters, and TFs from transcriptome or genome have to be validated via wet lab for further functional characterization and their assignment in the pathway. Therefore some other approaches come out which overcome these limitations. Phytochemicals are biosynthesized via secondary metabolic pathways present in medicinal plants and all the pathways are regulated by some key enzymes of that particular pathway (Tiwari et al., 2014, 2016; Srivastava et al., 2015). Production of metabolites is at

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FIGURE 9.4 Some examples of metabolically engineered plants.

their basal level in plant but if key genes of that pathway are overexpressed in that plant then metabolites flux might increase, considerably. Genetic manipulation of plants by recombinant technologies has valuable impact on basic plant research and biotechnology (Yelin and Tzfira, 2007). For introducing genes in plant system, first of all, the genes of interest are cloned into suitable plant expression vector and transformed in Agrobacterium strain then further introduced into plant (Fig. 9.3). Transformed plants are cultured via tissue culture techniques and these cultures are maintained and evaluated for desired characteristics (Fig. 9.4). Heterologous genes could be overexpressed into plants and ensuring that transferred gene will be expressed at the maximum desired level to increase flux, as exemplified by increased levels of monoterpenoid alkaloid biosynthesis by coexpressing two genes—tryptophan decarboxylase and strictosidine synthase (Leech et al., 1998). Limitations could be minimized by sequence homology studies of transgenes and controlling copy number of transgene and therefore it prevents the accumulation of multigene in transgenic plants. By sequential crossing, accumulation of transgene was produces stable up to three genes but recently by cotransformation technique, simultaneously multiple genes can be inserted in a stable manner (Halpin and Boerjan, 2003). In tobacco plant, the transcription of three genes was regulated by single promoter, creating translational fusions of that multigene, separated by a spacer sequence of protease cleavage sites. This implies that the multigene approach results in a functional enzyme which constitutes a complete metabolic pathway (Bodman et al., 1995). Alternative ribosome entry sites can be used as spacer sequences between multigenes (Martinezsalas, 1999). 9.3.2.2 Redirecting Flux by Overexpression and Silencing of Genes Although multiple gene transfer technique is good but it is somehow complicated and it may take more time. To handle multiple gene at a time and their regulation is little bit

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difficult, so a new approach comes out which is easier than last one. This strategy involves either increase in the key gene’s expression or silencing of the expression of those genes which is responsible for decrease in the desired metabolite production. Flux of metabolic pathways can be diversified by these two approaches—overexpression and silencing. For increasing the level of phytoconstituents, first requirement is the complete understanding about the pathway which helps us in selection of targets for engineering and possible sites for modification. After selecting appropriate gene(s), it is overexpressed in suitable system and production of desired substance will be increased. It also has some limitations such as only few genes related to plant secondary metabolism are characterized so it becomes more difficult to reach up the satisfactory level of metabolites production through overexpression approaches. There is one way which can be considered, i.e., integration of microbial gene into plant when information about the gene from that plant is unknown. This approach can be exemplified by the production of salicylic acid by using microbial gene (Verberne et al., 2000). Decreasing metabolites flux is interesting in case of undesirable compounds like toxins, or in eliminating the whole pathway that is competing with the pathway of interest. Several strategies are currently being used for decreasing the regulation or to knockout a specific pathway step but nowadays RNA interference (RNAi) is used as a potential tool for this because it is giving more fruitful results than classic methods. There is one more different strategy that enzyme of the targeted pathway could be knocked out via overexpressing an antibody against it (Tang and Galili, 2004). The silencing and overexpression approaches can influence the secondary metabolites not in homologous but heterologous system as well. But when both gain and loss-of-function strategy is applied together then satisfactory level of metabolites flux can be reached. This approach has been very useful in modulation of traits related to lignin biosynthesis in aspen trees (Li et al., 2003). 9.3.2.3 Diverting Whole Pathway Flux by Regulation of Transcription Factors Sometimes, for controlling flux of metabolites by manipulating one or more genes of pathways, global approaches are required; herein more understanding about that particular pathway is necessitated. Generally, there are some regulatory proteins for controlling the pathway metabolism and these proteins are known as transcription factors. They regulate the transcription of particular gene by binding with the cis-regulatory element (promoters) of that gene, which is generally present in upstream region. Therefore TFs may be another target points for regulating the expression of single or multiple enzymes for controlling metabolite’s flux (Mitsuda et al., 2007; Borevitz et al., 2000). TFs are DNAbinding proteins, which are able to bind with specified DNA sequences (promoters or enhancers). All the TFs have DNA binding domain which help them to bind on DNA sequences. Transcription factor could bind to thousand bases upstream from transcription start site and can either stimulate or repress transcription of the related gene. One transcription factor can influence more than one gene. For resolving the problem of multiple gene transfer, a new strategy can be considered, the overexpression of transcription factor. Using this approach maize TFs, C1 and R (which controls level of anthocyanins in aleuronic layers), were expressed in Arabidopsis and successfully manipulated anthocyanin biochemistry and expression (Stracke et al., 2007; Skirycz et al., 2007). Transcription factors C1 and R together with a strong promoter were expressed which caused a massive

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TABLE 9.2 An Overview of Transcription Factors Implicated in Plant Metabolic Engineering Metabolic Pathways

Transcription Factors or Family

Plant Name

References

Flavonoids

MYB11/R2R3-MYB

Arabidopsis thaliana

Stracke et al. (2007)

Phenylpropanoids

DOF4; 2/C2C2-DOF

A. thaliana

Skirycz et al. (2007)

Benzenoids

ODO1/R2R3-MYB

Petunia hybrid

Verdonk et al. (2005)

Indole-derived glucosinolates

ATR1 (MYB34)/R2R3-MYB

A. thaliana

Celenza et al. (2005)

Lignin

NST3/NAC

A. thaliana

Mitsuda et al. (2007)

Anthocyanin biosynthesis

PAP1/PAP2 (R2R3-MYB)

A. thaliana

Borevitz et al. (2000)

Phlobaphene/flavonoids

P1 (R2R3-MYB)

Zea maize

Grotewold et al. (1994)

accumulation of anthocyanin pigment in Arabidopsis, probably by upregulating the whole flavonoid pathway (Lloyd et al., 1992). Such transcription factor expression experiments have great opportunity in the identification of transcriptional regulatory networking of the pathway (Table 9.2). Several TFs such as MYB11, MYB34, DOF4, NAC, and P1 are already tried for metabolic engineering and significant alterations in metabolites have been observed (Stracke et al., 2007; Skirycz et al., 2007; Verdonk et al., 2005; Celenza et al., 2005; Mitsuda et al., 2007; Borevitz et al., 2000). Recently, artificially synthesized TFs are also used along with natural TFs, like Zn-finger family with a DNA-binding specificities. Thus it can be used to either transcriptionally activate or repress motifs for altering the metabolites flux through whole metabolic pathways using a single transgene (Jantz et al., 2004). 9.3.2.4 Diverting Whole Pathway Flux by Using Cis-regulatory Elements Promoter or cis-regulatory element is a region of DNA at which transcription of particular gene initiates. Generally, promoter consists of three regions—core, proximal, and distal regions. Core region contains transcription start site and binding site for RNA polymerase and it is required for proper initiation of transcription. Proximal promoter part contains specific primary regulatory elements and distal part contains additional elements. Promoter is a key factor for proper functioning of any transgene. Different kind of promoters such as 35S, actin, ubiquitin, 2A11, Lhcb3, β-conglycinin, Opaque-2, and APase have been isolated from different sources for controlling the expression of genes depending on the situation whether it is constitutive or tissue specific (Lessard et al., 2002; Benfey and Chua, 1990; An et al., 1996; Plesse et al., 2001; Haaren and Houck, 1993; Ali and Taylor, 2001; Rossi et al., 1997) (Table 9.3). Polyunsaturated fatty acids like Omega-3 long-chain docosa-hexaenoic acid (DHA) play important role in proper maintenance of human health and development, deficiency of these causes problems such as cardiovascular and inflammatory diseases. Commonly, these fatty acids are mainly supplied from fish and algal oils, but due to its wide and urgent need, an alternative source is required. Sequences of fatty acid biosynthesis gene were selected from yeast/algae and then by using a seed specific promoter such as FAE1 (Arabidopsis origin) these genes were transformed in crops such as

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TABLE 9.3 List of Commonly Used Promoters in Metabolic Engineering of Plants Promoter name

Type of Promoter

Origin

Features

References

35S

Constitutive

Viral

High expression in vascular tissue

Benfey and Chua (1990)

Actin

Constitutive

Plant

High expression in almost all tissues

An et al. (1996)

Ubiquitin

Constitutive

Plant

High-level expression but vary during development

Plesse et al. (2001)

2A11

Fruit specific

Plant (Solanum lycopersicum)

High-level expression in fruit

Haaren and Houck (1993)

Lhcb3

Leaf specific

Plant (Arabidopsis thaliana)

High-level expression in leaf

Ali and Taylor (2001)

b-Conglycinin

Embryo specific Plant (Glycine max)

High-level expression in embryo

Chen et al. (1988)

Opaque-2

Endosperm specific

Plant (Zea maize)

It shows developmental regulation

Rossi et al. (1997)

APase promoter

Inducible

Plant (A. thaliana)

Phosphate-inducible expression in roots

Arabidopsis, Brassica, etc. These transgenic plants are good alternative source of polyunsaturated fatty acids. One hectare of a transgenic Brassica napus crop produces as much polyunsaturated fatty acids DHA as approximately equal to 10,000 fish. Its seed contain 12% DHA (Petrie et al., 2012). By using the above examples, expressing constitutive or tissue specific promoter in plants, the quantity of desired products could be altered.

9.3.3 Systems Biology in Plant Metabolic Engineering Development of lower-cost and higher-yield possessing process is the aim of metabolic engineering. Therefore the increased metabolic activities can be achieved more efficiently by utilizing the methods and techniques from recombinant and other molecular biology based methods. However, due to unenvisaged alterations the above conventional methods are not always successfully recommended. Thus to understand the global context of a metabolic system in a better way and to hypothesize the strategies of metabolic engineering more powerfully, the demand of systemic approaches has been tremendously increased. The basic objective of systems biology is to generate integrated multifaceted knowledge of operating biosynthetic routes in plant (Lee et al., 2005; Yoon et al., 2013). With recent advances in high-throughput technology, expeditious addition in information of biological data set at different levels, i.e., genomic, transcriptomic, proteomic, metabolomic, fluxomic, etc. had taken place (Ideker et al., 2001; Stephanopoulos et al., 2004). Consequently, for generating an extrapolative model, it is crucial to identify the enzymes of the related

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Some applications of transgenic crops

Increased disease resistance

Improved nutritional value

A

“Golden Rice”

D

Increase in shelf life

Bt cotton G

B

Flavonoid rich corn Bt Brinjal

C

Cold resistant grape fruit

F

Transformed N. benthiana

H

I

Flavor savor tomato

Transformed apples

Transgenic banana

Herbicide resistant plant

Enhanced secondary/ pharmaceutical products

J

Herbicide resistant coffee

Carnation M with color pattern

K

Herbicide resistant rice

N

L

Herbicide resistant soybean

Vaccinated bananas

Catharanthus culture for producing O anticancer drug

Stress tolerant plant

P

Heat stress tolerant Arabidopsis

Q

Freezing tolerant Arabidopsis

R

Salinity stress tolerant transgenic rice

FIGURE 9.5 Application of metabolic engineering in crop improvement. (A) Transgenic “Golden Rice” had more amount of β-carotene (www.goldenrice.org), (B) transgenic flavonoid rich corn (www.kidney-support.org), (C) cold resistant grape fruit (left) (ucanr.org), (D) Bt cotton (www.oeic.us), (E) Bt brinjal (www.ndtv.com), (F) transformed Nicotiana benthamiana, (G) transgenic flavor savor tomato showed more self life (plantsinaction.science.uq.edu.au), (H) apple transformed with nonbrowning gene showed delaying in browning (kfolta.blogspot.com), (I) transgenic banana (members.tripod.com), (J) transgenic herbicide resistant coffee (Ribas et al., 2006), (K) transgenic herbicide resistant rice (californiaagriculture), (L) herbicide resistant soybean (eoedu.belspo.be), (M) transgenic flower of carnation represents modified color pattern (plantsinaction. science.uq.edu.), (N) bananas having edible vaccine against hepatitis B (www.mnn.com), (O) Catharanthus used for production of vincristine and vinblastine (plantplaces.com), (P) heat stress tolerant Arabidopsis thaliana (greensonga.com), (Q) freezing tolerant Arabidopsis (Chen et al., 2008) and (R) salinity stress tolerant transgenic rice (lsuagcenter.com).

pathways where and at what time their accumulation take place in a cell (Sweetlove et al., 2003). Each of these levels of regulation requires an elaborated information about the signal transduction pathways in addition to the transcriptional regulation, protein trafficking, and much more. Metabolic engineering may be accomplished in a much better way through integration of wet lab experiments (high-throughput experiments) with the leads available from in silico approaches (Fig. 9.5). Successful reports have utilized the knowledge generated from all omic related approaches and shown the promise for future directions. 9.3.3.1 Strategies of Systems Biology The systems biology involves newer approaches of understanding high-throughput data retrieved from various omic approaches with computational simulation. (Lee et al., 2005). The process is iterated till the goal of developing improved organisms with desired traits is achieved (Grafahrend-Belau et al., 2009). This cyclic iteration involves the usage of transcriptomic data either from RNA-seq through next generation sequencing technology

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(NGS) or microarray, 2D or MS generated proteomic data, GC/MS, LC/MS, or NMR generated metabolic profiling data or metabolome and labeled isotopes substrates generated data from metabolic flux analysis (MFA) (Grafahrend-Belau et al., 2009; Fernie, 2003). The prediction of the next round of modification of single/multiple genes is then carried out by integrating the information from bioinformatics and modeling with the omics data (Grafahrend-Belau et al., 2009; Yoon et al., 2013). The introduction of the high-throughput NGS technologies along with the phenotyping tools for phenomics has enhanced the study of nonmodel plants via genome and transcriptome sequencing and computational biology (Grafahrend-Belau et al., 2009; Sangwan, 2012; Narnoliya et al., 2017). 9.3.3.2 Integration of High-Throughput Omics Experiments In the previous section we have discussed about the unprecedented high rate of generation of various biological data resulting in the rapid development in biotechnology and metabolic engineering. NGS platforms have tremendously decreased the analytical cost to reach the increased throughput. Further, microarrays have been replaced by the NGS driven transcriptomic sequencing (RNA-seq, for instance) as they do not require the reference gene sequences due to higher resolution (Metzker 2010). Although 25 plant species have their genome published, the only complete and finished genome sequences are from Arabidopsis and rice (Hamilton and Robin Buell, 2012). For other plant species the corrections for gaps and errors in the assembly are still ongoing and for some other plants sequencing is accelerating rapidly. Genetic information from the model plants could be used for understanding the physiological mechanisms in closely related plant species as in case of Medicago truncatula for nitrogen fixation of legumes, rice for the development of grains, and tomato for the study of development of fruits. For the studies of the pathways related with synthesis of the secondary metabolites which varies widely in different plant species and where no model plant exists, transcriptome sequencing has much value (Fernie 2003; Champagne and Boutry, 2013). Thus the recent techniques are revolutionizing the manner in which the high-throughput data is made available for the faster accomplishment of genome, transcriptome, proteome, metabolome providing valuable metabolic engineering tools and techniques (Zhang et al., 2004; Patil et al., 2004). The combination of these omic technologies presents holistic insights into cellular environment and various interactions thereon. These prospects will come to true realization by the correct integration of all these data by the use of truly-validated in silico modeling and simulation tools. 9.3.3.2.1 GENOME BASED ANALYSIS

For extracting the information important in understanding the diversity and uniqueness of metabolic pathways and identification of targets for plant improvement, analysis of genomes is a potential and effective method. The genes which are not important for the production of desired biochemical products could be identified in addition. These genes along with desired genes can be introduced for establishing the new pathways and enhancing the pathway fluxes (Kolisnychenko et al., 2002). Genomics knowledge of medicinal plants can speed up the drug development procedure based on natural products in addition to the collection of genotypes having desirable and productivity associated traits. Recently, many genome projects have been accomplished using NGS platform with Roche

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GS FLX, Illumina Hiseq, and Applied Biosystems (ABI) SOLiD are used (Metzker, 2010; Rastogi et al., 2014). 9.3.3.2.2 TRANSCRIPTOME BASED ANALYSIS

Broad and deeper sampling of transcriptomes is possible again using next-generation sequencing platforms, owing to the low cost together with the characteristic ability for de novo assembly and transcript abundance quantification. With this it has provided better understanding of diverse plant species, especially those having medicinal properties (Tang et al., 2011). The large-scale identification is possible by utilizing the information from deeply sequenced transcriptomes generated using next-generation sequencing platforms. There is a rapid improvement in the computational programs for transcriptome analyses (Sangwan et al., 2013a,b; Gupta et al., 2013; Krishnan et al., 2011). For analysis, various programs are available which utilize reference genomic information. Examples of such packages include well-known and utilized software like Bowtie (Langmead et al., 2009), TopHat (Trapnell et al., 2009), and Cufflinks (Trapnell et al., 2010). These programs help in the alignment of the sequence reads to the genome and help in the statistical assessment of transcript abundances. Then the assembly programs and quality assessment programs (PHRED, CONSED, and PHRAP) are highly useful for RNA-seq based analysis. One more way of transcriptome assembly is the application of multiple k-mer approach and assemblers utilizing this approach are Trans-ABySS and Oases (Robertson et al., 2010; http:// www.ebi.ac.uk/Bzerbino/oases/). 9.3.3.2.3 PROTEOME BASED ANALYSIS

To attain a closer understanding of cellular metabolic status proteome profiling is the best option which can be performed by gaining the information from the metabolic reactions catalyzed by different enzymes. The analysis starts with the separation of the proteins by 2D electrophoresis and then separated protein spots is characterized by mass spectrometry (Han et al., 2001). Moreover, under two or more variable conditions or genotypic backgrounds, comparative analysis of protein spots with altered intensities can be examined easily by this technique. There are many examples showing successfully demonstrated improvement in different traits by this approach. Traditionally, proteins were analyzed by two-dimensional protein gel electrophoresis associated with matrix-assisted laser desorption ionization mass spectrometry (Han et al., 2001; Nordhoff et al., 2001). Now an untargeted and high-throughput method, shotgun proteomics, has thus been established to overcome the limitations of two-dimensional gel protein analysis which is time and labor consuming with low sensitivity. Proteins are digested without gel separation and LC
MS could be employed for identifying the fragmented peptides. The generated peptide sequences are subjected to database search. This method is advantageous and fast (200
800 protein analysis per days) over the two-dimensional gel analysis (Weckwerth, 2008). Thus these approaches may strengthen proteomics and its role in systems biology and engineering design. 9.3.3.2.4 METABOLOME BASED ANALYSIS

In spite of the enormous list of plant metabolites (5000
25,000) chemical structures of only small portions are identified and known. Near about 3300 compounds are known in

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case of Arabidopsis (Trethewey, 2004). The main focus of metabolomics is the identification of metabolites and quantitative determination of their concentrations. A number of highthroughput and high-resolution tools have been developed for identification and analysis of cellular metabolites revealing metabolic status of cells (Sauer, 2004; Yadav et al., 2013; Yoon et al., 2013). Combinations of techniques are usually preferred owing to its better resolution and applicability. GC
MS is appropriate for the primary metabolites analysis in central metabolic pathways, like amino acids, sugars, organic acids in tricarboxylic acid cycles, and free fatty acids, whereas LC
MS is suitable for secondary metabolites, phosphate sugars, and cofactors. Similarly, mass imaging technique also provides a promising approach by using NMR in which mass signals produced by tissues scanned with different ionization methods are collected, and the localized metabolites are visualized (Yoon et al., 2013). These technologies permit the detection and quantitative estimations of a myriad of classes of metabolites with structural complexities and close similarities (Fernie 2003, Chaurasiya et al., 2009). 9.3.3.3 In silico Modeling and Simulation of Plant Metabolism Representation of large metabolomics data sets is possible by the construction of models which can be further analyzed (Lee et al., 2005). Many approaches have been tried for in silico modeling and simulation studies for exploring the targeted metabolic systems. 9.3.3.3.1 KINETIC MODEL-BASED ANALYSIS

The kinetic model is considered as underdynamic approach and is capable of providing a complete description of the dynamic behavior along with the structure of the metabolic network. More commonly the kinetic information for the reactions of interest is collected either from the works published in the literature or from the publically available databases (Sivakumaran et al., 2003). However, it may lead to the often unreliable solutions because of the inconsistency of the system caused by the parameters collected from different sources. Thus software packages for kinetic modeling and estimation of various kinetic parameters are available for such dynamic simulations such as GEPAS, GENESIS, DBsolve, STOCKS, Dynetica, etc. (You et al., 2003; Kierzek 2002; Mendes 1993; Sivakumaran et al., 2003). Previously, metabolic models are focused on pathway flux due to the abundance of theoretical approaches which can be used to obtain flux control structures. Nowadays, a large number of models related to plant metabolism follow the theorems of metabolic control analysis (Thomas et al., 1997). Basics of such models have been applied for understanding the regulation of plant metabolic pathways; however, limited information is available as concerns the studies related with metabolism. 9.3.3.3.2 FLUX MODEL-BASED ANALYSIS

MFA can be used for the manifestation of complex metabolic regulation by gene expression and enzyme activity (Schuster et al., 2000). MFA could be described as the quantification of metabolite flow (flux) and enzymatic rate in the constructed metabolic pathways. Traditional MFA derives information from mass balances with reaction stoichiometry is unable to distinguish between cyclic and parallel reactions whereas, the 13C-based MFA, another alternative to this overcomes this limitation and has been used in various plant systems to inspect physiological changes subjected to different growth temperatures as in

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case of soybean embryos, in lipid synthesis of Arabidopsis and also mutations in sucrose synthase (SuSy) of maize. The next computational approach for MFA is flux balance analysis (FBA) with genome-scale metabolic models. Given an objective function, FBA is constrained by mass balance and reaction stoichiometries. Thus stoichiometric analysis is an interesting approach that has become more popular recently. This approach basically defines an elementary flux models- nondecomposable subnetwork that is responsible for every flux feasible within the network (Schuster et al., 2000). 9.3.3.3.3 GENOME-SCALE MODEL-BASED ANALYSIS

These are the models based on genome sequences with stoichiometric reactions. These global metabolic pathway models are used for understanding metabolism and predicting phenotypes, identifying essential genes, determination of targets for metabolic engineering. Though many genome-scale models of microorganisms are available, these models are still limited in case of plant owing to compartmentation within the cell with distinct tissues and organs (Poolman et al., 2009; Grafahrend-Belau et al., 2009; Hay and Schwender, 2011; Saha et al., 2011). Nevertheless, it is very important to test the model experimentally and validate it for its promising application in plant metabolic engineering. Some examples may be studied regarding the application of microbial genome-scale models in metabolic design and it was achieved by the application of OptForce a computational, multilevel optimization procedure which predicted the complete set of metabolic modifications (knockout, upregulate, downregulate) in E. coli leading to the overproduction of the target chemicals (acetyl CoA and malonyl CoA) approximately four times more than wild type. An integrated flux technology is thus capable of providing more specific targets quantitatively (Xu et al., 2011). Experimentally, assignment of only 13% of plant genes is achieved with computational assignment of a few genes and rest being still unknown (Collakova et al., 2012). 9.3.3.4 Tools and Databases for In silico Modeling and Simulation To accomplish various objectives of systems biology using modeling and simulation, several tools and program packages have been developed (Table 9.4). These may broadly study as follows. 9.3.3.4.1 INTEGRATED METABOLIC DATABASE SYSTEM

These metabolic databases contain all information relevant to the metabolic pathways, reactions, and enzymes required for the explanation of the metabolic and physiological features of an organism. Presently, available popular metabolic databases are listed in Table 9.4. Biolsilico, an integrated metabolic database system was developed to ensure proper handling of the redundant and heterogeneous data sources scattered over different sites (Hou et al., 2004). 9.3.3.4.2 INTEGRATED METABOLIC NETWORKS

In the view of importance of analysis of metabolic flux in understanding metabolic engineering, it becomes important to have a user-friendly computer program which can

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TABLE 9.4 Various Databases and Packages for Systems Biology Study With Their Corresponding URL Links GENOMIC DATABASES DDBJ

http://www.ddbj.nig.ac.jp

EMBL

http://www.ebi.ac.uk/embl.html

Entrez

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db Z Genome

GOLD

http://www.genomesonline.org/

MICROARRAY DATABASES GEO

http://www.ncbi.nlm.nih.gov/geo/

ArrayExpress

http://www.ebi.ac.uk/arrayexpress

PROTEOMIC DATABASES SWISS-PROT

http://www.expasy.org/sprot

SWISS-2DPAGE

http://www.expasy.org/ch2d/

MIPS

http://mips.gsf.de/

STRING

http://www.bork.embl-heidelberg.de/STRING

METABOLIC PATHWAY DATABASES ENZYME

http://www.expasy.org/enzyme/

BRENDA

http://www.brenda.uni-koeln.de

BioSilico

http://biosilico.kaist.ac.kr

BioCyc

http://biocyc.org

Biocarta

http://www.biocarta.com/genes/allPathways.asp

ERGO-LIGHT

http://www.ergo-light.com/

KEGG

http://www.genome.ad.jp/kegg

MetaCyc

http://metacyc.org/

PathDB

http://www.ncgr.org/pathdb

PROGRAM PACKAGES MetaFluxNet

http://mbel.kaist.ac.kr/

INSILICO Discovery

http://www.insilico-biotechnology.com/

FluxAnalyzer

http://www.mpi-magdeburg.mpg.de/proejcts/fluxanalyzer

FBA

http://systemsbiology.ucsd.edu/downloads/fba.html

analyze metabolic fluxes quantitatively. This will be of great help to the researchers less familiar with the detailed computational methods. Therefore a package MetafluxNet was developed which presented an important systems biology platform for metabolic characterization and engineering (Lee et al., 2003).

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9.4 APPLICATIONS OF METABOLIC ENGINEERING The eventual aim of metabolic engineering is to understand the regulation of cell typespecific biochemical pathways and usage of this information for generating the technologies which can be able to synthesize products valuable for industry, medical, and also for food application (Fig. 9.5). In this section we will discuss the major applications of plant metabolic engineering in different sectors.

9.4.1 In Industry Metabolic engineering of industrially important oils, waxes, and other plant lipids is showing an increased rate of advancement due to significant improvement in the underlying technologies. This can be exemplified by production of safflower (Carthamus tinctorius L.) with super high-oleic acid in which the seed oil has been metabolically engineered. In this case, RNAi-mediated silencing techniques were used to target fatty acid thioesterase and FAD2 genes, expressed in seed and this experiment was resulted in the development of safflower oil with approximately 95% oleic acid with only 2% linoleic acid as a remnant with no harmful effects on agronomic performance (Vanhercke et al., 2013). This has paved the way for greater industrial application of oleic acid as a chemical feedstock as well as lubricants and in transformer fluids with direct industrial implication presenting a landmark application of metabolic engineering of highly pure oleochemical in plant oils to be used as industrial feedstock (Skoric et al., 2008). Lavender (Lavandula latifolia red.) is an important component of oil industry, cultivated all over the world with limonene as a minor constituent. A significant increase of limonene content in developing leaves of spike lavender has been observed when the limonene synthase genes isolated from Mentha spicata were overexpressed under control of CaMV 35S constitutive promoter as compared to the control leaves (Mun˜oz-Bertomeu et al., 2008). Like limonene, the level of other terpenoids (linalool, menthol, geraniol phytosterol, and sesquiterpene) is also increased successfully (Schnee et al., 2006; Diemer et al., 2001; Lucker et al., 2004; Davidovich-Rikanati et al., 2007; Lewinsohn et al., 2001; Lucker et al., 2001; Lavy et al., 2002; Aharoni et al., 2006; Mahmoud et al., 2004; Mahmoud and Croteau, 2001; Kim et al., 2010; Bhauso et al., 2014; Schaller et al., 1995; Dharmapuri et al., 2002). Another example of directed alteration in plant metabolism with industrial utilization is the modification of fatty acid composition and triglycerides (Voelker et al., 1992).

9.4.2 In Food and Neutraceuticals Carotenoid, another group of secondary metabolites in plants apart from their importance in color carotenoid compounds in flowers, fruits, and food, have also antioxidant property and are the main precursors of vitamin A as β-carotene. A successful chapter in metabolic engineered manipulation comes from genetic alteration of carotenoid biosynthetic pathway by the introduction of lycopene β-cyclase, (LYCB) and Erwinia phytoene desaturase gene to produce Golden Rice accumulating β-carotene (Ye et al., 2000).

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Afterwards Golden Rice 2 was also developed which contains maize phytoene synthase (psy) gene instead from daffodils. It increased β-carotene content by 23 times. This transgenic line is being launched in Philippines and India in rice breeding program to create rice cultivars having higher provitamin A content (Paine et al., 2005). Such crops could help combating vitamin A deficiency, a common problem claiming 0.6 million children lives with age less than 5 years in developing countries. In another case, increased production of monoterpenoid flavor compound S-linalool was obtained by overexpression of S-linalool synthase transgene in tomatoes (Steele et al., 1998). Isoflavonoids have a great significance as pharmaceutical and nutraceutical (Liu et al., 2002). Isoflavonoid level in plants could be increased by enhancing the expression of genes related to flavonoid biosynthesis and this approach is successfully applied to various plants (Deavours and Dixon, 2005; Li et al., 2006; Muir et al., 2001; Yu et al., 2003; Bavage et al., 1997). Further, vanilin is an important aromatic flavor component of foods, perfumes, beverages, and pharmaceuticals. The term white biotechnology has been given to the production of vanillin (Priefert et al., 2001). More emphasis has been given to its production from natural source rather than organic synthesis. One more case has shown the genetic modification of vitamin E levels in Arabidopsis seed oil and level was increased 10-fold by overexpression of gamma-tocopherol methyl transferase (Shimoda et al., 2008).

9.4.3 In Pharmacy and Medicine Artemisia annua has been an attention of researchers since 1980s being an important source of antimalarial drug artemisinin and continuous efforts are underway to increase the production of the metabolite within the plant and in heterologous and homologous systems (Sangwan et al., 1993; Jadaun et al., 2017b). Higher production of alkaloids has also been achieved up to significant level by transferring the targeted gene from one source to another source (Goddijn et al., 1995; Berlin et al., 1993; Chavadej et al., 1994; Geerlings et al., 1999; Facchini et al., 1999; Hallard et al., 1997; Burtin and Michael, 1997; Alia et al., 1998; Dechaux and Boitel-Conti, 2005; Yang et al., 2011; Moyano et al., 2002; Frick et al., 2007) Resveratrol, a representative of polyketide group of stilbenes, is a constituent of red wine which have biological activities as inhibitor of inflammation, angiogenesis and metastatis, tumor promotion, anticancer agent, and regulation of cell cycle promotion. Characterization of the biosynthetic pathway and the enzymes involved in this plant has been performed with the application of metabolic engineering strategies in plants, microbes, etc. E. coli cells have shown significant production of resveratrol from its precursor tyrosine and pathway genes, such as tyrosine, by utilizing enzymes phenylalanine ammonia lyase (PAL), stilbene synthase, 4-Coumarate CoA ligase, and acetyl CoA carboxylase enzymes. PAL was derived from yeast Rhodotorula rubia, 4-Coumarate CoA ligase from actinomycete, S. coelicolor acetyl CoA carboxylase, and stilbene synthase from Arachis hypogea (Oliver and Joseph, 2008). Further, enhanced production of pharmaceutically important polyunsaturated archidonic acid and eicosapentaenoic acid was observed by utilizing nine genes combined together to analyze a large number of condensation and denaturation reactions (Staunton and Weissman, 2001).

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9.4.4 In Agriculture A study showing the transfer of entire biosynthetic pathway to heterologous plant species for increasing resistance against pests was attained by using plant metabolic engineering approaches for the biosynthesis of cyanogenic glucoside from Sorghum bicolor (Katsuragi et al., 2010). Additionally terpenoids engineering has shown the possibility to alter the insect behavior as shown by transgenic tobacco with higher levels of cembratrienol (a diterpenoid). In transgenic Arabidopsis plants, producing linalool, also aphid behavior was found to be altered. More convincing results were found in case of transgenic Chrysanthemum which repelled western flower thrips (Frankidiniella occidentalis) by producing linalool (Aharoni et al., 2006). Enhanced resistance to cutworm Spodoptera litera herbivory was found to be observed in transgenic tobacco overexpressed with three Coffea methyltransferase genes namely—CaMXMT, CaXMT, and CaDXMT—for the biosynthesis of caffeine playing an important role as potential defense compound. The property of the plant was expected to be due to the caffeines having putative role as a defense compound (Ogita et al., 2004). Pasture bloat, a potentially lethal and serious problem associated with excessive methane production in grazing cattle could be controlled by increased proanthocyanidin accumulation. An attempt for increased production of proanthocyanidin is made by overexpression of anthocyanidin reductase gene tissues of Nicotiana and Medicago (Xie et al., 2006).

9.5 CURRENT STATUS AND LIMITATIONS Present scenario of metabolic engineering depends on the rapid growth in molecular biology techniques and development of new methods for metabolic profiling. There are several distinct examples of transgenic lines of plants produced by altering the primary and secondary metabolic pathways. Metabolic engineering approaches and experimentations are on the steady rise since the last decade. Recent upsurge in the quantum of data arriving due to tremendous information about the transcriptome, genome, and proteome of various crops including various medicinal and aromatic plants is sharpening the efforts towards metabolic engineering. Consequently, there are several examples of transgenic plants having altered metabolic profile such as alkaloid, terpenoids, and flavonoids (Tables 9.5
9.7). However, main limitation in plant transformation is the subcellular localization of different enzyme although we can use this point in our favor by using specific promoters targeted to particular organelles. We can control the flux of metabolites to a targeted subcellular compartments and this will help in isolation and extraction of desired compounds. For example, berberin produced in culture is toxic but when it is produced in Coptis this remains in controlled manner and does not seem to be toxic (Sirikantaramas et al., 2008). Another reason for reliability of tissue specific promoters is that in several cases use of constitutive promoter resulted in alteration of phenotype due to low availability of precursors to the branching pathways, for example increased expression of FaNES protein in plastids exhibited the retardation in nerolidol production while increased expression in mitochondria leads to enhanced level of nerolidol (Aharoni et al., 2004). Several reports are available on application of different tissue specific, developmental, or

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TABLE 9.5 List of Some Transgenic Plants With Targeted Enzyme and Alteration in Metabolite Related to Alkaloid Biosynthesis. TDC: Tryptophan Decarboxylase; STR: Strictosidine Synthase; CO: Choline Oxidase; TYDC: Tyrosine Decarboxylase; H6H: Hyoscyamine 6 β-Hydroxylase; CYP80B3: Cytochrome P450 Dependent Monooxygenase (S)-N Methylcoclaurine 30 -Hydroxylase; ADC: Arginine Decarboxylase Genetically Engineered Enzyme

Transformed Plant Species

Source of Gene(s)

Targeted Alkaloids

Alteration in Metabolites

References

TDC

Catharanthus roseus

C. roseus

Tryptamine

Upregulated

Goddijn et al. (1995)

TDC

Peganum harmala

C. roseus

Serotonin

Upregulated

Berlin et al. (1993)

TDC

Brassica napus

C. roseus

Indole glucosinolates

Downregulated Chavadej et al. (1994)

TDC and STR

Cinchona officinalis

C. roseus

Quinoline alkaloid

Upregulated

Geerlings et al. (1999)

TYDC

B. napus

Papaver somniferum

Tyramine

Upregulated

Facchani et al. (1999)

ADC

Nicotiana tabacum

Avena sativa

Agmatine

Upregulated

Burtin and Michael (1997)

CO

Arabidopsis thaliana

Arthrobacter globiformis

Glycine betaine

Upregulated

Alia et al. (1998)

H6H

Datura innoxia Hyoscyamus niger

Scopolamine

Upregulated

Dechaux and Boitel-Conti (2005)

H6H

Atropa belladonna

H. niger

Hyoscyamine and scopolamine

Upregulated

Yang et al. (2011)

H6H

Duboisia

N. tabacum

Tropane or pyridine-type alkaloids

No alteration

Moyano et al. (2002)

CYP80B3

P. somniferum

P. somniferum

Morphine

Upregulated

Frick et al. (2007)

inducible promoters affording better flexibility to the accumulator system. Another level for engineering in metabolite is the regulation of regulatory elements mainly TFs. TFs are the most prominent candidate for globally switching on the metabolic turnover in crops. TFs regulates the basic machinery of cell which is involved in developmental process, tolerance against the drought, cold, disease, and in nutrient efficiency so these are the key points to modify the expression of target genes. A brief description is given about the prominent TFs that have been tried in metabolic engineering (Table 9.2). Recent and current approach of plant metabolic engineering is “Inverse Metabolic Engineering.” In this approach a mutagenic treatment is applied to a target set of population and this population is further screened for desired trait. Clones exhibiting the desired phenotypes were isolated and evaluated genetically.

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TABLE 9.6 List of Transgenic Plants With Targeted Enzymes and Alteration in Metabolites Related to Terpenoids Pathway. TPS: Terpene Synthase; LS: Limonene Synthase; MS: Methofuran Synthase; GS: Geraniol Synthase; ND: Nerolidol Synthase; SLS: S-Linalool Synthase; L3H: Limonene-3Hydroxylase; DXPR- 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase; FPS: Farnesyl Diphosphate Synthase; MPDH: Mannitol 1-Phosphate Dehydrogenase; HMGR: 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases; b-Lcy: Lycopene β-Cyclase; b-Chy: β-Carotene Hydroxylase; MS: Monoterpene Synthases; CYPH: Cytochrome P450 Hydroxylase Genetically Engineered Enzyme

Transformed Plant Species

Source of Gene(s)

Targeted Terpenoids

Alteration in Metabolites

References

TPS

Zea mays

Arabidopsis cytosol

Bergamotene, sesquiterpene, β-farnescene

Upregulated

Schnee et al. (2006)

LS

Mentha arvensis and Mentha piperita

Mentha spicata

(1)-limonene

Monoterpene level altered

Diemer et al. (2001)

MS

Mentha piperetta

Mentha piperetta

Pulegone, menthol, and methofuran

Upregulated, upregulated, downregulated

Lucker et al. (2004)

GS

Ocimum basilicum

Lycopersicon esculentum plastid

Geraniol and its derivative

Upregulated

Davidovich-Rikanati et al. (2007)

SLS

Clarkia breweri

Lycopersicon esculentum plastid

Linalool, 8-hydorxy linalool

Upregulated

Lewinsohn et al. (2001)

SLS

C. breweri

Petunia plastid

Linalool glycosides

Upregulated

Lucker et al. (2001)

SLS

C. breweri

Carnation plastid

Linalool, linalool oxide

Upregulated

Lavy et al. (2002)

SLS/ND

Fragaria x ananassa

Potato plastid

Hydroxylated and glycosylated linalool, linalool

Upregulated

Aharoni et al. (2006)

SLS/ND

L. esculentum

C. breweri

Linalool

Upregulated

Lewinsohn et al. (2001)

L3H

Mentha piperita

Mentha piperita

Limonene

Upregulated

Mahmoud et al. (2004)

DXPR

Mentha piperita

Mentha piperita

Menthol

Upregulated

Mahmoud and Croteau (2001)

FPS

Centella asiatica

Panax ginseng

Phytosterol and triterpene

Upregulated

Kim et al. (2010)

MPDH

Peanut

Bacterial gene mtlD

Mannitol

Upregulated

Bhauso et al. (2014)

HMGR

Nicotiana tabacum L.

Hevea brasiliensis

Sterol

Upregulated

Schaller et al. (1995)

b-Lcy, b-Cyc

Tomato

Capsicum annuum

β-Carotene, β-cryptoxanthin and zeaxanthin

Upregulated

Dharmapuri et al. (2002)

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TABLE 9.7 List of Transgenic Plants With Targeted Enzymes and Alteration in Metabolites Related to Flavonoid and Other Metabolic Pathway. IS: Isoflavone Synthase; CSL: Chalcone Isomerase; F3H: Flavanone 3-Hydroxylase; DFR: Dihydroflavonol Reductase Genetically Engineered Enzyme

Transformed Plant Species

Source of Gene(s) Medicago sativa

Targeted Metabolites

Alteration in Metabolites

Isoflavonoids, genistein glucosides

Upregulated

Deavours and Dixon (2005)

References

IS

Medicago truncatula

CS

Salvia involucrata S. involucrata

Apigenin

Upregulated

Li et al. (2006)

CS

Solanum lycopersicon

Petunia

Flavonols

Upregulated

Muir et al. (2001)

F3H

Glycine max

G. max

Isoflavone

Upregulated

Yu et al. (2003)

DFR

Lotus corniculatus

Antirrhinum majus

Tannins

Upregulated

Bavage et al. (1997)

Scope of plant metabolic engineering is tremendous and new dimension of agriculture and industry can be achieved such as better varieties in horticulture, improved nutritional quality (Golden Rice), postharvest quality (flavor savor tomato), improved resistant varieties (BT brinjal), and high-value pharmaceutical compound (plant vaccines) (Figs. 9.4 and 9.5). But along with easy and cheaper product, this technology creates a lot of ethical, socioeconomical and environmental issues. As in case of soybean transformed with Brazil nut gene shows allergic reactions after feeding by cattle and such observations require a stricter scrutiny.

9.6 FUTURE ASPECTS OF METABOLIC ENGINEERING Metabolic engineering is an effective technology and has a great potential. It is beneficial in production of desired chemicals or products in desirable amount at any time without the constraints of time and season. By using metabolic engineering strategies and approaches, native compounds can be formed in higher quantities and also newer forms can be envisaged. Instead of targeting a single enzymatic step in the metabolic pathways, targeting the multiple genes simultaneously may be more fruitful because effect of overexpression or suppression of one gene can be nullified by other limiting steps. Emerging knowledge of the TFs impart the new insights. TFs represent an impressive control on the metabolic flux whether it is in homologous or in heterologous system. Use of TFs along with alteration in gene expression may be a more promising strategy in metabolic engineering. Recent advances in transcriptome and genomes will probably add a milestone for the future of metabolic engineering. Transgenic crops should be evaluated under different environmental conditions. Transgenic plants should be screened against different combination of biotic and abiotic stress. These approaches can contribute in food and nutritional quality over worldwide areas (Sabir et al., 2012, 2013). Actually metabolic engineering is a

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process of permutation and combination of diversified techniques. It can be performed at numerous transcriptional and posttranscriptional levels to enhance the productivity and agronomic traits as well as medicinal properties of plants. It is a challenge to choreograph of all variables at a time after considering their limitations. Transgenic plants are the model systems to study the myriad aspects of molecular biology such as loss of function or gain of function. Now research is focussed on the production of edible vaccine in plants. Banana and tomato are successfully tried for this purpose. There is a need to invent new technologies for rapid isolation and identification of metabolites to boost up the growth of genetically modified plants.

9.7 CONCLUSIONS With the growing knowledge about the genes related to metabolic pathways and with refined technology in plant molecular biology, plant metabolic engineering can change the dynamics of whole world. New emerging computational biology facilitates the genetic engineering projects by reducing the time consumed in manual analysis. Alteration in gene expression, it may be at transcriptional or posttranscriptional level, affect the entire metabolic profiling of plants. For the production of pharmaceutically and industrially valuable compounds, genetic engineering is a magical tool. Plant metabolic engineering is a bona fide tool to fulfill the requirement of food and nutrition of increasing populations. There is a requirement of more work on the safety issues of genetically modified crops in concern to health and environment.

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3131. Aharoni, A., Jongsma, M.A., Kim, T.Y., Ri, M.B., Giri, A.P., Verstappen, F.W.A., et al., 2006. Metabolic engineering of terpenoid biosynthesis in plants. Phytochem. Rev. 5, 49
58. Ali, S., Taylor, W.C., 2001. The 30 non-coding region of a C4 photosynthesis gene increases transgene expression when combined with heterologous promoters. Plant Mol. Biol. 46 (3), 325
334. Alia, Hayashi, H., Sakamoto, A., Murata, N., 1998. Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine. Plant J. 16, 155
161. An, Y.Q., Huang, S., McDowell, J.M., McKinney, E.C., Meagher, R.B., 1996. Conserved expression of the Arabidopsis ACT1 and ACT 3 actin subclass in organ primordia and mature pollen. Plant Cell 8, 15
30. Bavage, A.D., Davies, I.G., Robbins, M.P., Morris, P., 1997. Expression of an antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird’s foot trefoil). Plant Mol. Biol. 35 (4), 443
458. Benfey, P.N., Chua, N.H., 1990. The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250 (4983), 959. Berlin, J., Rugenhagen, C., Dietze, P., Frecker, L.F., Goddijn, O.J.M., Hoge, J.H.C., 1993. Increased production of serotonin by suspension and root cultures of Peganum harmala transformed with a tryptophan decarboxylase cDNA clone from Catharanthus roseus. Transgenic Res. 2, 336
344. Bhauso, T.D., Radhakrishnan, T., Kumar, A., Mishra, G.P., Dobaria, J.R., Patel, K., et al., 2014. Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol. Sci. World J. 2014.

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Bodman, S.B., Domier, L.L., Farrand, S.K., 1995. Expression of multiple eukaryotic genes from a single promoter in Nicotiana. Biotechnology 13, 587
591. Borevitz, J.O., Xia, Y., Blount, J., Dixon, R.A., Lamb, C., 2000. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12, 2383
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6791.

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C H A P T E R

10 Biocontrol Technology: Eco-Friendly Approaches for Sustainable Agriculture Ratul M. Ram1, Chetan Keswani2, Kartikay Bisen1, Ruchi Tripathi1, Surya P. Singh2 and Harikesh B. Singh1 1

Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India 2 Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India

10.1 BIOPESTICIDES VERSUS CHEMICAL PESTICIDE: FACE TO FACE Plant pests and diseases have detrimental effects on crop production causing as much as 31% 42% annual crop loss globally (Agrios, 2005). Historically, various plant diseases caused by a broad range of phytopathogens have created catastrophic losses such as Irish famine (1845), Great Bengal famine (1943), Southern corn leaf blight epidemic (1970), and Coffee rust (1970). Therefore, effective management of phytopathogens has been a great challenge for agriculturists, and till recently only hazardous and toxic chemical pesticides have been widely employed for plant disease management. However, the indiscriminate use of chemical pesticides over the past few decades has seriously affected the environmental and human health by shifting the pest management practices towards eco-friendly and sustainable approaches (Table 10.1). Currently, due to rising awareness of the health benefits of organically grown produce, biological control is a preferred option for plant disease control by employing biopesticides that offer a long-term control of pests once established in the field conditions. Also, biopesticides are host-specific in nature thereby acts against the targeted pathogen only. Therefore, this sustainable and an eco-friendly approach is ideally suited for integration with most other plant protection measures used in integrated disease management program.

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TABLE 10.1 Developmental Parameters and Risk Assessment of Chemical Pesticides and Biocontrol Agents Chemical Control

Biological Control

Developmental costs

US$ 150 million

US$ 2 million

Success ratio

1:200,000

1:10

Developmental time

10 years

,10 years

Benefit/cost ratio

2:1

20:1

Risks of resistance

Large

Very small

Specificity

Less

High

Harmful side-effects

Many

Nil/few

Modified from http://www.iobc-global.org/download/IOBC_InternetBookBiCoVersion6Spring2012.pdf.

10.2 BIOCONTROL: THERAPY IN ORGANIC FARMING Organic farming is the production of food and fiber without the use of any synthetic chemical fertilizers, pesticides, herbicides, or genetically modified organisms. The emphasis is mainly on the application of naturally occurring antimicrobial organic substances as tools for sustainable productivity and management of pests, diseases, and weeds. Although, organic farming practices are highly prone to various disease outbreaks, hence, apart from employing early preventive measures, there are soaring demands for nonchemical pesticides, i.e., either the application of antagonistic microorganism (i.e., biocontrol agents) or various antimicrobial botanicals such as neem (Azadirachta indica), garlic (Allium sativum), eucalyptus (Eucalyptus globules), turmeric (Curcuma longa), tobacco (Nicotiana tabacum), and ginger (Zingiber officinale). According to Garret (1965), “Biological control of plant disease may be precisely defined as any condition or practice whereby survival or activity of a pathogen is reduced through the agency of any living organism with the result that there is reduction in incidence of the disease caused by the pathogen.” The microbes present in the plant rhizosphere and pathogens undergo interaction that may be either synergistic or antagonistic (Mishra et al., 2015). Various free living soil and rhizospheric microbes have been identified and commercialized as potential antagonists for controlling seed and soil-borne phytopathogens. Biocontrol of plant pathogens relies on two approaches, viz., management of resident population of organisms (the black box approach) and the introduction of specific organism to reduce disease (the silver bullet approach). Later on, it has been observed that application of single antagonistic strain often results in inconsistent control. The best way to overcome such obstruction is to combine the application of different microbes in a single unit which have synergistic association with each other. Such combinations may result in more extensive colonization of the rhizosphere along with the expression of defense responses in varying ecosystems (Duffy and Weller, 1995; Bashan, 1998). The successful outcome of such combinations has resulted in the development of formulations involving microbial consortium (Pierson and Weller, 1994; Budge et al., 1995;

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TABLE 10.2 List of Microorganisms Permitted for Commercialization for Agricultural Application Included in the Gazette of India on January 26, 1999 Bacteria

Fungi

Virus

Pseudomonas fluorescens

Trichoderma sp.

Beauveria bassiana

Nuclear polyhedrosis viruses

Agrobacterium tumefaciens

Gliocladium sp.

Metarhizium anisopliae

Granulosis viruses

Agrobacterium radiobacter Fusarium oxysporum strain 84 (nonpathogenic)

Verticillium lecanii

Bacillus subtilis

Penicillium islanidicum (for groundnut)

Verticillium chlamydosporium

Streptomyces lydicus

Aspergillus niger—strain AN27

Paecilomyces lilacinus

Burkholderia cepacia

VAM (fungus)

Nomuraea rileyi

Erwinia amylovora

Candida oleophila

Hirsutella species

Alcaligenes sp.

Pythium oligandrum

Photorhabdus luminescences akhurustii strain K-1

Serratia marcescens GPS 5 Chaetomium globosum

Myrothecium verrucaria

Streptomyces griseoviridis

Ampelomyces quisqualis

Piriformospora indica

Coniothyrium minitans

Phlebia gigantea

Duffy et al., 1996). Multiple organisms boost the level and constancy of control through multiple mechanisms of action and finally provide a better stability over a wide range of environmental conditions (Pandey and Maheshwari, 2007, Jain et al., 2013a,b; Bisen et al., 2015). In addition to plant pathogens, biological control is also successfully exhibited in controlling insect pests and nematodes. Various fungi viz., Beauveria bassiana, Metarhizium anisopliae, and Verticillium lecanii are used extensively for controlling important insect pests of several crops (Keswani et al., 2014). Fungi such as Arthrobotrys oligospora, Dactylaria spp., Dactylella spp., and Paecilomyces lilacinus act as important natural enemies of plant parasitic nematodes. Realizing the role of such microbes as potential biopesticides, the Government of India has permitted the mass production and commercialization of 34 such microbes (Table 10.2).

10.3 MECHANISMS EMPLOYED BY BIOCONTROL AGENTS FOR PLANT DISEASE MANAGEMENT The biocontrol activity is exerted either directly through antagonism of soil-borne pathogens or indirectly by the elicitation of induced systemic resistance response in plants

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FIGURE 10.1 Schematic representation of (A) mycoparasitism of various fungal pathogens (P) by BCA, (B) antibiosis of pathogens by antimicrobial secondary metabolites secreted by BCA, and (C) elicitation of induced systemic response in host plant by BCA.

(Fig. 10.1). The various mechanisms employed by biological control agents have been listed below.

10.3.1 Antibiosis The process is defined as “the interactions involving a low-molecular weight compound or an antibiotic produced by a biocontrol agent (BCA) having a direct effect on the growth of plant pathogen” (Weller, 1988; Keswani et al., 2017). The mode of action of antibiotics varies depending on their biochemical nature which primarily acts as metabolic inhibitors or block protein synthesis (translational) pathways (Keswani et al., 2014). Examples of some potent antibiotics are Bacillomycin D produced by Bacillus subtilis AU195, Gliotoxin by Trichoderma virens, Iturin A by B. subtilis QST713, 2,4-diactylphloroglucinol (DAPG) by Pseudomonas fluorescence F133, and so on (Table 10.3). Several biocontrol strains are known to produce multiple antibiotics that can repress one or more pathogens thus enhancing the biocontrol efficacy of the BCA. Bacillus cereus strain UW85 produces both Zwittermycin and Kanosamine (Pal and Gardener, 2006). Genetically engineered Pseudomonas putida WCS358r strain produces phenazine and DAPG displaying improved capacities to suppress plant diseases in wheat-cultivated fields (Glandorf et al., 2001).

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TABLE 10.3

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List of Antibiotics Produced by Various Biocontrol Agents Disease Controlled

Reference

Agrobacterium tumefaciens

Crown gall

Kerr (1980)

Bacillus amyloliquefaciens strain FZB42

Fusarium oxysporum

Wilt

Koumoutsi et al. (2004)

Xanthobacin A

Lycobacter sp. Strain K88

Aphanomyces cochlioides

Damping off

Islam et al. (2005)

2,4-Diacetylpholoroglucinol

Pseudomonas fluorescence F113

Pythium sp.

Damping off

Shanahan et al. (1992)

Gliotoxin

Trichoderma virens

Rhizoctonia solani

Root rot

Wilhite et al. (2001)

Bacillomycin D

Bacillus subtilis AU195

Aspergillus flavus

Aflatoxin contamination

Moyne et al. (2001)

Herbicolin

Pantoea agglomerans C91

Erwinia amylovora

Fire blight

Sandra et al. (2001)

Mycosubtilin

Bacillus BBG100

Pythium aphanidermatum

Damping off

Leclere et al. (2005)

Iturin A

Bacillus subtilis QST713

Botrytis sp., Rhizoctonia solani

Damping off

Moyne et al. (2001)

Zwittermycin A

Bacillus cereus UW85

Pythium aphanidermatum

Damping off

Smith et al. (1993)

Antibiotic

BCA

Target Pathogen

Agrocin 84

Agrobacterium radiobacter

Bacillomycin D

10.3.2 Mycoparasitism Mycoparasitism is the most important form of antagonism involving direct physical contact with the host mycelium (Pal and Gardener, 2006). It involves tropical growth of biocontrol fungus mycelium towards the target pathogen followed by extensive coiling and secretion of various hydrolytic enzymes leading to dissolution of pathogen’s cell wall or membrane (Tiwari, 1996) (Table 10.4). Mycoparasitism can be classified as a four-step process. The first step includes chemotropic growth of antagonistic fungal mycelium toward the phytopathogenic fungi followed by recognition. The third and fourth steps involve direct attachment and cell wall degradation of phytopathogenic fungus followed by penetration of host fungal cell. It is one of the main mechanisms employed by Trichoderma sp. to kill phytopathogenic fungi (Sharma, 1996). Trichoderma harzianum exhibits tremendous mycoparasitic activity against Rhizoctonia solani (Altomare et al., 1999). Several mycoparasites can attack a single fungal pathogen, e.g., Acremonium alternatum, Acrodontium crateriforme, Ampelomyces quisqualis, and Gliocladium virens are a few fungi having capacity to parasitize powdery mildew pathogen (Kiss, 2003).

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TABLE 10.4 List of Biocontrol Agents Displaying Mycoparasitism Biocontrol Agent

Target Pathogen

Coniothyrium minitans

Sclerotinia sp.

Sporidesmium sclerotivorum

Sclerotinia minor

Trichoderma sp.

R. solani, Fusarium sp., Sclerotinia sclerotiorum, Sclerotium rolfsii, Uncinula necator

Pythium nunn

Pythium sp.

Aspergillus niger

Macrophomina phaseolina

Ampelomyces quisqualis, Acrodontium crateriforme

Powdery mildew fungi (Erysiphe sp., Uncinula sp.)

TABLE 10.5 Major Competitive Mechanisms Employed by BCAs Against Various Phytopathogens Biocontrol Agent

Metabolite

Target Pathogen

Crop

Pseudomonas fluorescens 3551

Siderophores

Pythium ultimum

Potato

Pseudomonas putida WCS 358

Siderophores

Fusarium oxysporum f. sp. raphani

Radish

Pseudomonas putida N1R

Volatile substances

P. ultimum

Pea, soybean

Enterobacter cloacae Inactivation of stimulants of pathogen germination

P. ultimum

Cotton, cucumber

Trichoderma harzianum

Rhizoctonia solani, Fusarium sp., Sclerotinia sclerotiorum, Sclerotium rolfsii

Grapevine

Nutrient and space

10.3.3 Competition Soil and living plant surfaces provide a nutrient limited environment to soil microbes leading to severe competition among the resident populations for essential nutrients and space. Competition is considered to be an indirect interaction between the pathogen and the biocontrol agent, whereby the pathogens are eliminated by the depletion of food source and niche exclusion (Lorito et al., 1994) (Table 10.5). Due to limited availability of micronutrients such as iron and manganese, biocontrol agents have developed unique transport system for solubilization and chelation of these micronutrients mainly iron and referred to as siderophores (Kloepper et al., 1980).

10.3.4 Induced Resistance in Host Plants Induction of local and systemic resistance in host plants is an indirect mechanism of biocontrol agents to protect plants from pathogen invasion. Salicylic acid and I. MICROBIAL AND PLANT TECHNOLOGIES

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pathogenesis-related gene (NPR1) are key factors in systemic acquired resistance. Certain strains of Trichoderma penetrate root tissues and induce a chain of biochemical and morphological changes to induce defense responses in the host (Bailey and Lumsden 1998; Singh et al., 2011; Bisen et al., 2016; Keswani et al., 2016a). Inoculation of T. harzianum in rhizospheric region of grapes offers control against Botrytis cinerea in leaves (Desmukh et al., 2006). Trichoderma spp. secretes various signaling molecules in interaction zone resulting in the induction of resistance in plants (Keswani et al., 2013a,b, 2016b), among which proteins with enzymatic or other activity such as xylanase, cellulases, and swollenins play a significant role (Martine et al., 2001).

10.4 STRAIN IMPROVEMENT OF BIOCONTROL AGENTS Lower competence and consistency of biocontrol agents against phytopathogens in fields is one of the major limiting factors in their commercial application in comparison to chemical pesticides. However, these limitations can be overcome by using genetically improved strains of biocontrol agents which display greater antagonistic potential against targeted phytopathogens. Desired performance of a biocontrol agent may be achieved either by providing conducive environment for biocontrol agent (which is rarely the case in field conditions) or through genetic advancement of the BCA. The genetically improved strains of biocontrol agents offer not only an increased biocontrol activity against phytopathogens but also an enhanced plant growth promoting activities. Genetic manipulations of biocontrol agents are needed primarily to improve the antagonistic potential against broad spectrum of phytopathogens, to increase the production and secretion of antimicrobial secondary metabolites, and to enhance tolerance against various abiotic stresses. Improved strains of different biocontrol agent can be attained through various approaches viz. mutation through chemical and physical agents, sexual hybrids, and genetic manipulation, e.g., directed mutagenesis, protoplast fusion, transformation, and recombination.

10.4.1 Mutagenesis It is a process of inducing mutation in an organism for the generation of new biotypes with enhanced biocontrol potential. It is either achieved by physical (X rays, γ-rays, UVrays, and radioisotopes) or by chemical mutagens (ethyl methyl sulfonate, sodium nitrate, 5-bromouracil, etc.). UV mutagenesis is being widely used for developing Trichoderma mutants having enhanced chitinolytic activity displaying enhanced biocontrol of Aspergillus flavus and A. parasiticus (Patil, 2012). The F113 mutant of P. fluorescens was found to have improved competitive colonization ability and better biocontrol activity against various fungal root pathogens (Barahona et al., 2011). Similarly Hrp2 mutants of Pseudomonas solanacearum are active against tomato bacterial wilt pathogens. Mutation through UV light is employed to improve the production of various antibiotics such as phenazine, phloroglucinol, and pyrrolnitrin, and also to improve siderophore production in P. fluorescens against damping-off pathogens (Fusarium solani, F. oxysporum f. sp. lycopersici, and R. solani) in tomato. These mutants also demonstrate increased antibiosis potential in comparison to the wild-type strains (Haggag, 1999). I. MICROBIAL AND PLANT TECHNOLOGIES

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10.4.2 Protoplast Fusion In this approach, the protoplasts from two or more genetically different somatic cells are fused to obtain a hybrid protoplast either by the action of fusion-inducing agents or spontaneously. Through this technique, possibilities of transferring various genes governing beneficial characters such as disease resistance, nitrogen fixation, rapid growth rate, enhanced protein quality, frost hardiness, enhanced secondary metabolite secretion, drought resistance, herbicide resistance, and heat and cold resistance are enhanced. Consequently, this approach for the production of recombinants and hybrid strains has gained popularity in various avenues of microbial biotechnology owing to its narrow gap of genetic exchange. This approach may be used to produce interspecific or even intergeneric parasexual hybrids. Numerous examples of better strains of diverse microorganisms generated through protoplast fusion are commercially available. The intergeneric fusants between Trichoderma reesei and Saccharomyces cerevisiae help in the bioconversion of cellulosic materials to ethanol (termed as bioethanol). Protoplast fusion of T. reesei and Aspergillus niger has a high yield of cellulase enzyme (Ahmed and Barkly 2006). Rygielska (2004) reported interspecific triple fusants of starch-fermenting yeasts Schwanniomyces occidentalis ATCC 48086, Saccharomyces diastaticus ATCC 13007, and S. cerevisiae which displayed high tolerance to cycloheximide (0.001%), enhanced ability to grow at 40 C, and improved ability to synthesize and secrete amylolytic enzymes. Although, fusants of Penicillium chrysogenum and Cephalosporium acremonium are reported to produce a novel beta-lactam antibiotic.

10.4.3 Transformation It is the process of developing genetic variation in a cell through direct uptake and incorporation of exogenous genetic material (foreign DNA) from surrounding environment. This phenomenon occurs naturally in few bacterial genera but can also be induced in desired strains by various artificial means. This phenomenon was first demonstrated in 1928 by British bacteriologist Frederick Griffith in Streptococcus pneumoniae. It is being used extensively to improve the biocontrol and biofertilizer activity of Trichoderma atroviride.

10.5 OMICS IN BIOCONTROL TECHNOLOGY 10.5.1 Genomics The term “genomics” was coined by Dr. Tom Roderick, who defined it as the systematic study of the sum total of all the genes in an organism. The presence of avirulence genes (Avr) in pathogen and the resistance genes (R) in the host could be determined through their genomic study. It renders identification of the key genes involved in resistance and pathogenicity in an organism. Identification of the R-genes of a host would not only help us in developing transgenic crops carrying the desired resistance gene against a pathogen but it would also facilitate to knockdown those genes responsible for susceptibility towards pathogen. Besides, the study would also help in understanding several

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intragenomic phenomena’s such as heterosis, epistasis, and pleiotropy. The complete genome sequencing of several biocontrol agents such as T. atroviride, T. virens, T. reesei, Bacillus brevis, Pantoea vagans, Microbacterium barkeri, and P. fluorescence F113 are currently available. The most studied species is T. reesei, as it secretes cellulolytic enzymes having wide industrial application.

10.5.2 Proteomics Proteins are considered as the work horses of the cell catalyzing function. Unlike the genome that contains the fixed number of genes, the quantification of all proteins of a particular cell is a mammoth task as proteins are continuously produced within the cell in response to external stimuli. Proteome is the complete set of proteins of a particular tissue produced by the genome at any point of time. Phenotype of an organism is completely dependent on the proteome rather than the genome. The first stage of gene expression involves transcription of genes closely followed by the translation of messenger RNA to produce linear proteins which are then folded to produce fully functional proteins. The study of the proteome is essential because proteins represent the actual functional molecules in the cell and are ultimately affected with real-time changes in host biochemistry. Study of proteomic has become a significant tool to monitor the precise picture of metabolic/physiological scenario within the cell/tissue. The changes during the growth and development of an organism or in response to various biotic and abiotic stresses cannot be figured out through proteomic approaches. In this regard the role of proteomics in biocontrol agent and pathogen interaction has been critically reviewed (Chinnasamy, 2005). However, the induction of changes in gene expression and protein is less studied in plant pathogen interactions. Recognition of various differentially expressed proteins provides a clear picture of the plant pathogen cross talk. The proteomic study of agriculturally important microbes has become popular since last decade. In order to develop efficient and eco-friendly plant disease management strategies, the molecular study of fungal biology and their interaction with host is absolutely vital. Combination of proteomics with other molecular techniques offers a tool to study the pathogenicity and virulence of phytopathogens. Mass spectrometry and liquid chromatography tandem mass spectrometry has been conducted to improve protein identification in T. harzianum. In many biocontrol strains of Trichoderma, such as T. harzianum and T. atroviride, proteomic analysis has offered the identification of various key protein factors involved in the interaction between Trichoderma and pathogen and host plant. Proteomics of T. harzianum has revealed surprising facts about their responses during symbiosis, antagonism, saprophytism, etc. (Grinyer et al., 2004). Proteomics also plays an important role in characterizing pathogens and development of biomarkers in host pathogen interaction.

10.5.3 Metabolomics Metabolomics is the study and analysis of all metabolites of a cell in a given set of conditions. Various substances such as peptide, nucleotide, oligonucleotide, sugar, and

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organic acids can act as metabolites. Because gene expression data and proteomic analyses do not expose the complete molecular image of the cell, metabolomics approach offers a clear picture of the physiology of cell in real time. According to their major mode of action, efficacy of biocontrol agents can be enhanced for better agricultural applications. Production of antagonistic secondary metabolites, antibiotics, toxins and lytic enzymes, etc., is one of the principle mechanisms. Trichoderma strains produce various types of compounds such as lytic enzymes, metabolic intermediates, and hormone and other signaling molecules. Various secondary metabolites having different biological functions have also been reported. Direct interaction between biocontrol agent derived secondary metabolites and phytopathogens changes the proteome and transcriptome profile of plants. Hence, profiling of metabolites is a precise and well-established approach to study the plant responses to various stresses (Sanchez et al., 2008). The well-studied plant pathogen A. tumefaciens induces the synthesis of opines that cannot be utilized by plants. Likewise, Sclerotinia sclerotiorum induces the conversion of plant carbohydrates into fungal polyols (Jobic et al., 2007). Similarly, biocontrol agent mediated defense response against phytopathogens in the host results in the induction of different metabolic pathways that might play an important role in defense against pathogen attack (Jain et al., 2012; Singh et al., 2012). In many cases inoculation of biocontrol agent results in alteration in plant metabolic profile including significant changes in sugar, polyamines, amino acids, and citric acid, which ultimately leads to the activation of plant defense response (Brotman et al., 2012). Such defense mechanism involves physiological and biochemical responses such as cell wall modifications and formation of reactive oxygen species. To unravel the strategies involved in the infection process and pathogen biocontrol agent interaction, the metabolomic study is essentially required.

10.5.4 Secretomics The wide adaptability of fungi permits them to associate with their host plants in various ways. While interacting with a living host, the fungal pathogen invades its host by secretion of various digestive enzymes to derive nutrition from it. The term secretome was coined by Tjalsma et al. (2000) and defined as the “global study of proteins that are secreted by a cell, a tissue or an organism” (Chenau et al., 2008). However, Agrawal et al. (2010) redefined secretome as “the global group of secreted proteins into the extracellular space by a cell, tissue, cell, organ or organism at any given time and conditions through known and unknown secretory mechanisms involving constitutive and regulated secretory organelles.” The study of secretome became crucial when recent researches brought into focus the importance of secreted proteins that act as key factors in initializing the interactions between pathogenic or symbiotic fungi and their plant hosts. Secretomics may help to contribute to the food security by identifying the principle toxins secreted by pathogens and exploring the ways to interrupt its interactions with host leading to crop yield enhancement. Secreted proteins play a key role in various physiological processes, such as cell signaling and matrix remodeling, and are also associated with invasion and metastasis of pathogenic cells. Secretomic analysis of many biocontrol agents such as Trichoderma sp.

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and Pseudomonas sp. has been completed so far. In Fusarium graminearum, secretomic studies revealed the proteins involved in its interaction with barley and wheat.

10.6 CONCLUSION AND FUTURE PROSPECTS Soaring crop losses confronted in last century have had their heavy toll on human health. As a result, scientist toiled to find all possible alternatives to control plant diseases. The last century witnessed the rise of chemical pesticides and fertilizers, but only recently the hazardous effects of the injudicious use of these toxic pesticides and fertilizers have been observed. As an eco-friendly alternative, biopesticides and biofertilizers were brought into the picture. But only limited success was observed as their performance was severely compromised in adverse field conditions. Thus it almost became a necessity to explore either the stress-tolerant stains of BCAs or to produce them through biotechnological interventions. Structural and functional genomics analyses are unraveling the molecular intricacies of BCAs for plant disease control. Currently BCAs are subjected to various investigations aiming at translational research utilizing omics’ generated understanding for improved plant disease management. Future prospects of BCAs include the following: • Sequencing of both agriculturally important and harmful genes of BCAs is advocated for strain improvement. • Assessment of ecological impact of mass application of BCAs for safe use. • The BCA plant molecular interaction should be instigated, which will lead to functional understanding of underlying molecular mechanisms of biocontrol. • Knowledge generated through “omic research” would lead to production of novel transgenic strains of BCAs for effective management of various phytopathogens. • Currently, biggest hindrance in commercialization of BCAs is their poor shelf life. Thus, it would be of utmost importance to develop formulations with improved shelf life.

10.7 SUMMARY Organic farming emphasizes integration of natural pest management strategies, rather than simply relying on toxic chemical pesticides. Among these natural pest management strategies, biocontrol technique is an important approach in sustainable agriculture. As a potential alternative of hazardous chemical pesticides, agriculturally important microorganisms have gained popularity throughout the globe. Recent advances in “omics”-based researches have laid down a significant impact on application of biocontrol agents. Application of various strain improvement strategies leads to the production of many BCAs with increased antagonistic activity against a wide range of phytopathogens.

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Acknowledgments CK is grateful to Banaras Hindu University for providing financial assistance through DST-PURSE (5050) program. RMR is thankful to UGC for providing financial support through Rajiv Gandhi National Fellowship.

References Agrawal, G.K., Jwa, N.S., Lebrun, M.H., Job, D., Rakwal, R., 2010. Plant secretome: unlocking secrets of the secreted proteins. Proteomics 10, 799 827. Agrios, G.N., 2005. Plant Pathology, Fifth ed. Academic press, New York. Ahmed, M., Barkly, E.L., 2006. Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulase. Appl. Biochem. Biotechnol. 135, 117 132. Altomare, C., Norvell, W.A., Bjorkman, T., Harman, G.E., 1999. Solubilization of phosphate and micro nutrients by the plant growth promoting fungus Trichoderma harzianum Riafi. Appl. Environ. Microbiol. 65, 2926 2933. Bailey, B.A., Lumsden, R.D., 1998. Direct effects of Trichoderma and Gliocladium on plant growth and resistance to pathogens. In: Kubicek, C.P., Harman, G.E., Ondik, K.L. (Eds.), Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Taylor and Francis, London, pp. 185 204. Barahona, E., Navazo, A., Martı´nez-Granero, F., Zea-Bonilla, T., Pe´rez-Jime´nez, R.M., Martı´n, M., et al., 2011. Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens. Appl. Environ. Microbiol. 77 (15), 5412 5419. Bashan, Y., 1998. Inoculants of plant growth promoting bacteria for use in agriculture. Biotechnol. Adv. 16, 729 770. Bisen, K., Keswani, C., Mishra, S., Saxena, A., Rakshit, A., Singh, H.B., 2015. Unrealized potential of seed biopriming for versatile agriculture. In: Rakshit, A., Singh, H.B., Sen, A. (Eds.), Nutrient Use Efficiency: From Basics to Advances. Springer, India, pp. 193 206. Bisen, K., Keswani, C., Patel, J.S., Sarma, B.K., Singh, H.B., 2016. Trichoderma spp.: efficient inducers of systemic resistance in plants. In: Chaudhary, D.K., Verma, A. (Eds.), Microbial-Mediated Induced Systemic Resistance in Plants. Springer, Singapore, pp. 185 195. Brotman, Y., Lisec, J., Merit, M., Chet, I., Willmitzer, L., Viterbo, A., 2012. Transcript and metabolite analysis of the Trichoderma induced systemic resistance response to Pseudomonas syringae in Arabidiopsis thaliana. Microbiology 158, 139 146. Budge, S.P., McQuilken, M.P., Fenlon, J.S., Whipps, J.M., 1995. Use of Coniothyrium minitans and Gliocladium virens for biological control of Sclerotinia sclerotiorum in glasshouse lettuce. Biol. Control 5, 513 522. Chenau, J., Michelland, S., Seve, M., 2008. Secretome: definitions and biomedical interest. La Revue de Medecine Interne 29, 606 608. Chinnasamy, G., 2005. A proteomics perspective of biocontrol and plant defense mechanism. In: Siddique, Z.A. (Ed.), PGPR: Biocontrol and Biofertilization. Springer, Netherlands, pp. 233 255. Deshmukh, S., Hueckelhoven, R., Schaefer, P., Imani, J., Sharma, M., 2006. The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley. Proc. Natl. Acad. Sci. USA 103, 18450 18457. Duffy, B.K., Weller, D.M., 1995. Use of Gaeumannomyces graminis var. graminis alone and in combination with fluorescent Pseudomonas spp. to suppress take all disease of wheat. Plant Dis. 79, 907 911. Duffy, B.K., Simon, A., Weller, D.M., 1996. Combination of Trichoderma koningii with fluorescent Pseudomonas for control of take all disease of wheat. Phytopathology 86, 188 194. Glandorf, D.C., Verheggen, P., Jansen, T., Jorritsma, J.W., Smit, E., Leefang, P., et al., 2001. Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat. Appl. Environ. Microbiol. 67, 3371 3378. Grinyer, J., McKay, M., Nevalainen, H., Herbert, B.R., 2004. Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum. Curr. Genetics 45, 163 169. Haggag, W.M., 1999. Enhancement of suppressive metabolites from Pseudomonas flourescence against tomato damping-off pathogens. Arab. J. Biotechnol. 2, 1 14.

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Islam, T.M., Hashidoko, Y., Deora, A., Ito, T., Tahara, S., 2005. Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soil borne peronosporomycetes. Appl. Environ. Microbiol. 71, 3786 3796. Jain, A., Singh, S., Sarma, B.K., Singh, H.B., 2012. Microbial consortium mediated reprogramming of defence network in pea to enhance tolerance against Sclerotinia sclerotiorum. J. Appl. Microbiol. 112, 537 550. Jain, A., Singh, S., Singh, B.N., Singh, S., Upadhyay, R.S., Sarma, B.K., et al., 2013a. Biotic stress management in agricultural crops using microbial consortium. Bacteria in Agrobiology: Disease Management. Springer, Berlin, Heidelberg, pp. 427 448. Jain, A., Singh, S., Singh, S., Singh, H.B., 2013b. Microbial consortium induced changes in oxidation stress markers in pea plants challenged with Sclerotinia sclerotiorum. J. Plant Growth Regul. 32 (2), 388 398. Jobic, C., Boisson, A.M., Gout, E., Rascle, C., Fevre, M., Cotton, P., et al., 2007. Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by Sclerotinia sclerotiorum. Planta 226, 251 265. Kerr, A., 1980. Biological control of crown gall through production of Agrocin 84. Plant Dis. 64, 25 30. Keswani, C., Singh, S.P., Singh, H.B., 2013a. Beauveria bassiana: status, mode of action, applications and safety issues. Biotech. Today 3, 16 20. Keswani, C., Singh, S.P., Singh, H.B., 2013b. A superstar in biocontrol enterprise: Trichoderma spp. Biotech. Today 3 (2), 27 30. Keswani, C., Mishra, S., Sarma, B.K., Singh, S.P., Singh, H.B., 2014. Unraveling the efficient applications of secondary metabolites of various Trichoderma spp. Appl. Microbiol. Biotechnol. 98 (2), 533 544. Keswani, C., Bisen, K., Singh, V., Sarma, B.K., Singh, H.B., 2016a. Formulation technology of biocontrol agents: present status and future prospects. In: Arora, N.K. (Ed.), Bioformulations for Sustainable Agriculture. Springer, India, pp. 35 52. Keswani, C., Bisen, K., Singh, S.P., Sarma, B.K., Singh, H.B., 2016b. A proteomic approach to understand the tripartite interactions between plant-Trichoderma-pathogen: investigating the potential for efficient biological control. In: Hakeem, K.R., Akhtar, MohdSayeed (Eds.), Plant, Soil and Microbes Vol. 2. Mechanisms and Molecular Interactions. Springer International Publishing, Cham, Switzerland, pp. 79 93. Keswani, C., Bisen, K., Chitara, M.K., Sarma, B.K., Singh, H.B., 2017. Exploring the role of secondary metabolites of Trichoderma in tripartite interaction with plant and pathogens. In: Singh, J.S., Seneviratne, G. (Eds.), AgroEnvironmental Sustainability. Springer International Publishing, Cham, Switzerland, pp. 63 79. Kiss, L., 2003. A review of fungal antagonists of powdery mildews and their potential as bio agents. Pest Manag. Sci. 59, 475 483. Kloepper, J.W., Leong, J., Teintze, M., Schroth, M.N., 1980. Pseudomonas siderophores: a mechanism explaining disease suppression in soils. Curr. Microbiol. 4, 317 320. Koumoutsi, A., Chen, X.H., Henne, A., Liesegang, H., Gabriele, H., Franke, P., et al., 2004. Structural and functional characterization of gene clusters directing non ribosomal synthesis of bioactive lipopeptides in Bacillus amyloliquefaciens strain FZB42. J. Bacteriol. 86, 1084 1096. Leclere, V., Bechet, M., Adam, A., Guez, J.S., Wathelet, B., Ongena, M., et al., 2005. Mycosubtilin over-production by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Appl. Environ. Microbiol. 71, 4577 4584. Lorito, M., Hayes, C.K., Zonia, A., Scala, F., Del, S.G., Woo, S.L., et al., 1994. Potential of genes and gene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides. Mol. Biotechnol. 2, 209 217. Martine, C., Blanc, F., Le, C.E., Besnard, O., Nicole, M., Baccou, J.C., 2001. Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum. Plant Physiol. 127, 334 344. Mishra, S., Singh, A., Keswani, C., Saxena, A., Sarma, B.K., Singh, H.B., 2015. Harnessing plant-microbe interactions for enhanced protection against phytopathogens. Plant Microbes Symbiosis: Applied Facets. Springer, India, pp. 111 125. Moyne, A.L., Shelby, R., Cleveland, T.E., Tuzun, S., 2001. Bacillomycin D: an Iturin with antifungal activity against Aspergillus flavus. J. Appl. Microbiol. 90, 622 629. Pal, K.K., Gardener, B.M., 2006. Biological control of plant pathogens. Plant Health Instruct. 2, 1117 1142. Pandey, P., Maheshwari, D.K., 2007. Two-species microbial consortium for growth promotion of Cajanus cajan. Curr. Sci. 92 (8), 1137 1142.

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Patil, A.S., 2012. Strain improvement of Trichoderma harzianum by UV mutagenesis for enhancing its biocontrol potential against aflatoxigenic Aspergillus species. Experiment 4 (2), 228 242. Pierson, E.A., Weller, D.M., 1994. Use of mixtures of florescent Pseudomonas to suppress take all and improve the growth of wheat. Phytopathology 84, 940 947. Rygielska, J.K., 2004. Obtaining hybrids of distillery yeasts characterized by the ability of fermenting starch. Electronic Journal of Polish Agricultural Universities: Series Biotechnology 7 (2). Sanchez, D.H., Siahpoosh, M.R., Roessner, U., Udvardi, M., Kopka, J., 2008. Plant metabolomics reveal conserved and divergent metabolic processes to salinity. Plant Physiol. 132, 209 219. Sandra, A.I., Wright, C.H., Zumoff, L.S., Steven, V.B., 2001. Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro. Appl. Environ. Microbiol. 67, 282 292. Shanahan, P., O’Sullivan, D.J., Simpson, P., Glennon, J.D., O’Gara, F., 1992. Isolation of 2, 4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl. Environ. Microbiol. 58, 353 358. Sharma, G. (1996) Studies on the integrated management of banded leaf and sheath blight of maize caused by Rhizoctonia solani, MSc (Ag.), Thesis submitted to G.B. Pant University of Agriculture and Technology, Pantnagar, India, pp. 65. Singh, A., Sarma, B.K., Upadhyay, R.S., Singh, H.B., 2012. Compatible rhizosphere microbes mediated alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid activities. Microbiol. Res. 168, 33 40. Singh, B.N., Singh, A., Singh, S.P., Singh, H.B., 2011. Trichoderma harzianum mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defense against Rhizoctonia solani. Eur. J. Plant Pathol. 131, 121 134. Smith, K.P., Havey, M.J., Handelsman, J., 1993. Suppression of cottony leak of cucumber with Bacillus cereus strain UW85. Plant Dis. 77, 139 142. Tiwari, A.K. (1996) Biological controls of chickpea wilt complex using different formulations of Gliocladium virens through seed treatment. Ph.D. Thesis submitted to G. B. Pant University of Agriculture and Technology, Pantnagar India, pp. 167. Tjalsma, H., Bolhuis, A., Jongbloed, J.D., Bron, S., Van Dijl, J.M., 2000. Signal peptide dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol. Mol. Biol. Rev. 64, 515 547. Weller, D.M., 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Ann. Rev. Phytopathol. 26, 379 407. Wilhite, S.E., Lunsden, R.D., Strancy, D.C., 2001. Peptide synthetase gene in Trichoderma virens. Appl. Environ. Microbiol. 67, 5055 5062.

Further Reading Elad, Y., David, D.R., Levi, T., Kapat, A. and Kirshner, B. (1999) Trichoderma harzianum T-39-mechanisms of biocontrol of foliar pathogens. In: Modern fungicides and antifungal compounds II. 12th International Reinhardsbrunn Symposium, Friedrichroda, Thuringia, Germany, 24 29 May, 1998, pp. 459 467, Intercept Limited. Howell, C.R., Beier, R.C., Stipanovi, R.D., 1980. Production of ammonia by Enterobacter cloacae and its possible role in the biological control of Pythium pre-emergence damping off by the bacterium. Phytopathology 78, 105 1078. Ordentlich, A., Elad, Y., Chet, I., 1988. The role of chitinase of Serratia marcescens in the biocontrol of Sclerotium rolfsii. Phytopathology 78, 84 92. Roco, A., Perez, L.M., 2001. In vitro biocontrol activity of Trichoderma harzianum on Alternaria alternata in the presence of growth regulators. Electron. J. Biotechnol. 4 (2), 1 6. Sharma, R., Joshi, A., Dhaker, R.C., 2012. A brief review on mechanism of Trichoderma fungus: use as biological control agents. Int. J. Innov. Biosci. 2 (4), 200 210. Singh, B.N., Singh, A., Singh, B.R., Singh, H.B., 2014. Trichoderma harzianum elicits induced resistance in sunflower challenged by Rhizoctonia solani. J. Appl. Microbiol. 116 (3), 654 666. Singh, H.B., Keswani, C., Ray, S., Yadav, S.K., Singh, S.P., Singh, S., et al., 2015. Beauveria bassiana: Biocontrol beyond Lepidopteran pests. Biocontrol of Lepidopteran Pests. Springer International Publishing, Cham, Switzerland, pp. 219 235.

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C H A P T E R

11 Bioengineering Towards Fighting Against Superbugs Faria Khan1,2, Amjad Ali1,2 and Alvina Gul1,2 1

National University of Science and Technology (NUST), Islamabad, Pakistan 2 Polish Academy of Sciences, Warsaw, Poland

11.1 INTRODUCTION Antimicrobial resistance (AMR) is the phenomena whereby microbes incur resistance towards the preexisting antimicrobial agents. These antimicrobial drugs include antibiotics, antifungals, antivirals, and antimalarials. When resistant organisms emerge, the treatment via these drugs becomes ineffective. As a result, the infection persists in the patients and also spread to others (WHO, 2013). Emergence of multidrug resistant (MDR) pathogens is becoming a huge concern for healthcare industry as these “superbugs” are major contributors towards morbidity. At the same time, the cost of healthcare industry is increasing due to the extra efforts required to control such pathogens at global level (BioMerieux, 2013; Morris et al., 1998). AMR emerges via natural selection to increase microbe survival chances. The resistant traits can be exchanged between pathogens which are accelerated due to two primary reasons: misuse of antimicrobial drugs and poor infection control practices (WHO, 2013). It was also noted that the extensive use of antibiotics for viral infections and self-medication practices has increased the development of antibiotic resistance (CDC, 2009). There is a dire need to control the prevalence and spread of these MDR microbes by providing new therapeutic options through biotechnological interventions (Laxminarayan et al., 2013). MDR occurs when pathogens gain resistance to the multiple classes of drugs to which it was originally sensitive, while multiple drug-resistant organisms (MDRO) are the pathogens that have gained resistance over time towards varying classes of drugs (BioMerieux, 2013). The causes of antimicrobial drug resistance are complex and

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associated with the human activities at social level. Another factor that contributes towards emergence of these resistant strains is microbial mutations followed by natural selection pressure; this gives a competitive advantage to resistant pathogens (Laxminarayan et al., 2013). Both of these factors are discussed in detail in the following section of this chapter.

11.1.1 Global Perspective of Microbial Drug Resistance The consequences of superbug emergence are significant, which requires an immediate attention as the infectious diseases they caused hold a large burden of morbidity and mortality worldwide (Bisht et al., 2009). Examples include vancomycin-resistant Staphylococcus aureus and fluoroquinolone (FQ)-resistant Escherichia coli and Neisseria gonorrhea (Levy, 2005). Due to the emergence of more threatening diseases such as AIDS, Ebola, and cancer, modest policies have been adapted by different countries to regulate the use of antibiotics. Such uncontrolled use without surveillance hampers the control of infectious diseases (Ash, 1996). The word “apocalypse” has been used to describe the AMR particularly while referring to the emergence of MDR in the case of bacteria causing tuberculosis. The emergence of such resistant strains threatens the return to pre-antibiotic era and thus further increases the chances of apocalyptic outbreak of uncontrollable disease, such is the threat posed by extensively drug-resistant tuberculosis (Upshur et al., 2009). The emergence of resistant microbes is also contributing towards the increase in healthcare expenditure (Tomasz, 1994; Sipahi, 2008). The emergence of MDRO is in fact jeopardizing the healthcare gain of the society. The “miracle drugs” to treat various classes of infectious diseases is becoming exhausted; hence there is a dire need to halt the resistant process or to provide alternate therapy (Brown and Layton, 1996). Another major issue foresighted as a result of accelerated spread of resistant pathogens is the threat posed to health security, trade, and economics; Salmonella can be taken as an example, where ampicillin, chloramphenicol, and trimethoprimsulfamethoxazole resistant strains has emerged (D’Aoust, 1994). Occurrence of such incidences can severely hamper the trade and economics for the fear of transfer of resistant strains to other countries where the resistant strain has not yet arrived (Rudholm, 2002). Fig. 11.1 summarizes all the major problems associated because of the global spread of AMR organisms.

11.1.2 Human Actions Contributing Towards MDR Development In the worldwide emergence of MDRO, human behavioral and social issues are contributor in the development of “superbugs.” This situation is varying in high- and lowincome countries (WHO, 2001). The challenge faced by developing and low socioeconomic societies in response to MDR development is greater primarily due to the high prevalence of infectious diseases (Okeke et al., 2005). The evolution of more resistant pathogens is further increased in these regions due to poor hygiene conditions, unavailability of safe

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Ant imicrobial resistance: a global issue Increased cost of healthcare

Hampered control of infect ious diseases

Jeopardized healthcare gain of the society Threatened health security, economics & trade

Increased morbidity & mortality Threat of return to pre-ant ibiot ic era

FIGURE 11.1 Highlights of six predominant factors that increases a global concern over the AMR emergence.

FIGURE 11.2 Summary of the human factors contributing towards the emergence of microbial resistance, which vary in high-income countries (HICs) and low, middle-income countries (LMICs).

drinking water, social issues such as lack of availability of medical facilities, malnutrition, and poverty (Ramanan and David, 2012). In hospital settings, misdiagnosed and empiric therapy (the continual use of medicine without any effective response) is key factor contributing towards MDRO originations. Similarly, overprescription of antibiotics and self-medication practices further increase the emergence of these superbugs as explained in Fig. 11.2 (Sosa and Byarugaba et al., 2010). Social issues are also contributing towards the emergence of resistant microbes, which includes assembly and overcrowding of sick individuals at hospitals, nursing homes, schools, and community settings (Mitscher et al., 1999; WHO, 2013).

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11.2 MOLECULAR BASIS OF RESISTANCE Molecular mechanism of resistance involves acquisition of resistance followed by biochemical inactivation of drug by bacterial factors. The molecular resistance mechanism developed by superbugs can be categorized into several groups.

11.2.1 Acquired Resistance Acquired resistance refers to a mechanism whereby the intrinsic or innate bacterial factors that are involved in generation of drug-resistant microbes via acquisition of resistant genes. Generally acquired resistance arises from the following: 1. Chromosomal Mutations: Gene mutations in cells, which then lead to cross-resistance. 2. Plasmid Transfer: Gene transfer from one microorganism to other of same species (conjugation or transformation, horizontal gene transfer). 3. Transfer via jumping genes: transposons (conjugation). 4. Gene transfer via nonbacterial hosts: Integrons and Bacteriophages (transduction) (Giedraitiene et al., 2011). Fig. 11.3 gives a brief description about the mechanisms adopted by bacteria to acquire resistance and the processes involved at each stage. The mutations on the chromosome of bacteria can arise as a part of natural process owing primarily to gene amplifications, inversions, spontaneous mutations, duplication, insertions, deletions, conjugation, or other types of mutations. These mutated parts can contain resistant genes that can then be exchanged between same species of bacteria. 11.2.1.1 Biochemical Inactivation of Drugs Biochemical inactivation of drugs involves the use of defense mechanisms by bacteria via release of biochemical factors to overcome the drug response against it. These strategies can be categorized into three main groups. FIGURE 11.3 The molecular mechanisms involved in the acquiring of resistance in MDR bacteria. The processes that are involved at each level of mutation are also highlighted. Four main processes Transduction, Transformation, Conjugation, and Chromosomal Mutations are involved to pass on the resistant genes via gene transfer agent, i.e., chromosomal mutations, transposons, plasmid transfer, and integrons and bacteriophages.

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FIGURE 11.4 Biochemical mechanisms that are adapted to counter the effect of antimicrobial drugs by resistant pathogens in (A) tetracyclines (B) aminoglycosides and B-lactams, and (C) FQ and RMP. (A) Failure of penetration into the target cells. (B) Inactivation of the drug by enzymatic attack. (C) Alteration of the drug target within the cell.

1. Failure of drug penetration into target cells due to altered permeability via changes in outer membrane permeability (aminoglycosides) and new membrane transporters synthesis (e.g., chloramphenicol). 2. Inactivation of drug by enzymatic attack which results in antibiotic inactivation due to interference with cell wall synthesis (β-lactams and glycopeptide) or by “bypassing” metabolic pathway primarily due to inhibition of metabolic pathway (e.g., trimethoprim-sulfamethoxazole). 3. Alteration of drug target within the cell by inhibition of synthesis of protein (e.g., macrolides and tetracyclines) or by interference with nucleic acid synthesis (e.g., FQ and rifampin (RMP)) (Giedraitiene et al., 2011; Mitscher et al., 1999). These mechanisms are explained in detail in Fig. 11.4. At molecular level, this resistance emerges because of selection pressure caused by microenvironmental settings, the drug in question, and the pathogenic microorganism.

11.3 INDUSTRIALLY IMPORTANT DRUG-RESISTANT PATHOGENS 11.3.1 MDR in Tuberculosis Tuberculosis an infectious disease often associated with Mycobacterium tuberculosis affects lungs; however, it has the tendency to target other body parts as well (Dalton et al., 2012). The emergence of Multiple drug resistance tuberculosis (TB) is primarily because of resistance to treatment drugs such as isoniazid (INH) and rifampicin (RMP). This resistance has been observed in the MDR strains as a result of either inappropriate treatment or incomplete course, which allows the strains to cope with the stress and emerging fast through chromosomal alterations, highlighted in Fig. 11.5 (Yew, 2011).

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FIGURE 11.5 The indicators that lead to emergence of drug-resistant strains of mycobacterium tuberculosis (TB) (Marahatta, 2010).

FIGURE 11.6 Reaction in the absence and presence of mutations leading to development of drug-resistant strains and, where PZA is not activated, the killing is prevented.

11.3.1.1 Group 1 They are conventionally called the first line of drugs against tuberculosis. Few of them are described here by discussing the mechanism of action of these drugs and how resistance get develop against such drugs. INH: A prodrug, activated by katG gene, which kills the cell after activated using the enzyme coded by this gene to produce reactive oxygen species (ROS). The strains resistant to INH loses the catalase activity of KatG gene, which renders the drug inactivated (Zhang and Yew, 2009). RMP: By adhering to B subunit of RNA polymerase, it stops the mRNA synthesis thereby blocking the protein expression at transcription level (Zhang and Yew, 2009). Pyrazinamide (PZA): This drug is used in combination with other drugs and serves mainly in the reduction of treatment time from 12 to 6 months by killing persistent strains under acidic conditions but has nearly no activity at neutral pH. Like INH, PZA is also a prodrug activated into pyrazinoic acid. It is shown by the reaction in Fig. 11.6.

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11.3.1.2 Group 2 (Streptomycin/Capreomycin) Functions by jamming the 30S subunit of ribosome result in the abnormal pathogenic protein folding. Mutations in rpsL and rrs gene and 16S RNA, respectively, makes the cell Streptomycin-resistant (SMR). Modification in 16S and 23S rRNA by methyltransferase causes the strains Capreomycin-resistant (CPMR). This occurs in the drug-related stress conditions, which causes the upregulation of tlyA gene, resulting in expression of methyltransferase (Zhang and Yew, 2009). 11.3.1.3 Group 3 (FQ) They inhibit the function of DNA topoisomerases that play an important function of supercoiling of DNA strands particularly preventing the activity of topoisomerase II (DNA gyrase) and topoisomerase IV. DNA gyrase consists of four units 2A and 2B subunits. gryA gene codes for A subunit involves in breaking and uniting DNA and gryB codes for subunit B with ATPase activity. FQ-resistant strains have QRDR (quinolone resistance determining region) in gryA and gryB region. 11.3.1.4 Group 4 (Ethionamide/Prothionamide, and Thiomides) Like INH and PZA, ethionamide (ETH) is also a prodrug activated by monoxygenase coded by EtaA/EthA blocking the same target as that of INHs. EtaA is flavin adenosine dinucleotide oxidizing both ETH and prothionamide (PTH), thereby activating the prodrug into toxic form hence killing the cells. This is prevented by the mutations in the etaA/ethA causing thiomides-resistant strains (Zhang and Yew, 2009) (Table 11.1). TABLE 11.1 Drug class

Mechanism of Resistance by Mycobacterium tuberculosis Against Various Drugs

Examples

Mechanism of action

Resistance against drugs

Group 1 Conventional first-line drugs INH and rifampin (RMP), oral drugs, e.g., PZA

INH: Prodrug: activated by katG gene. RMP: Blocks transcription by blocking RNA polymerase PZA: Prodrug activated by pncA gene

Resistance by mutations in katG gene and mabA and inhA and in PZA/nicotinamidase pncA gene

Group 2 Injectable agents includes aminoglycosides, e.g., SM and CPM

SM: Blocks translation CPM: Blocks translation

Mutations in rpsL gene and rss gene activation and upregulation of tlyA gene

Group 3 FQ, moxifloxacin, and levofloxacin

Inhibit functions of DNA gyrase and topoisomerase IV

Quinolone resistance determining region in gryA and gryB gene

Group 4 Conventional second-line drugs: ETH, PTH, and thiomides

Prodrug activated by etaA/ethA

Mutations in etaA/ethA gene (point mutations and substitution)

Group 5 Linezolid and clofazimine

Blocking guanine basis Increases phospholipase A activity

—

The modern classification of drugs for the treatment of MDR-TB (in ascending order) and the mechanism of action of these drugs (Shim and Jo, 2013).

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11.3.1.5 Group 5 (Linezolid and Clofazimine) Linezolid incurs anti-TB responses by inhibiting bacterial protein synthesis. It binds to 23S ribosomal RNA (rRNA) thereby incurring loss in bacterial protein synthesis. Bacterial gene mutations affecting the 23s rRNA confer high-level resistance to linezolid. The mechanisms for low-level drug resistance are still unknown. At present, clinical usage of linezolid has been restricted due to potential toxicity (Dey et al., 2013). Clofazimine is a fat-soluble riminophenazine dye that is conventionally used in the treatment of leprosy. Although clofazimine is listed as a group 5 drug for MDR-TB, its potential side effects, contribution in MDR-TB development and efficacy against TB disease, are still uncertain (Hillemann et al., 2008).

11.4 MDR IN PSEUDOMONAS AERUGINOSA Pseudomonas aeruginosa poses a great challenge towards the development of drugs, despite its simplicity with respect to Klebsiella pneumonia and other Enterobacter species owing to few multiresistant plasmids; it poses a bigger challenge due to its inherent nature of resistance which is contributed by acquiring mutations quickly (Livermore, 2002). Drug uptake restriction or its efflux, and drug modification and target alteration are the mechanisms which an organism exploits to elicit resistance for survival as summarized in Fig. 11.7 (Lambert, 2002). Drugs applied against Pseudomonas infection requires to cross the cell membrane barriers which in this case resist the entry of drugs into cell. This Alginate (polysaccharide) restricts the diffusion of aminoglycosides e.g., amikacin, tobramycin, and gentamicin (Ciofu et al., 2001). Aqueous channels also play an important role, e.g., oprD (a drug channel protein on membrane) absence increases the required drug concentrations during treatment (Staczek et al., 1998). Four efflux systems are currently reported to exhibit drug resistance including mexAB-oprM, which expel the quinolones, mexXY-oprM extruding aminoglycosides, and mexEF-opN, which has developed the ability to expel carbapenems. Increased expression of these gene systems have direct effect on AMR development in Pseudomonas (Lambert, 2002)

FIGURE 11.7 Strategies adopted by Pseudomonas aeruginosa towards development of AMR against clinically used antibiotics.

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Inactivation of drugs is another strategy adopted by specific strains. Upregulation of serene-based metalloenzymes (e.g., carbapanemases) has been reported to cause modification of antibiotics thereby causing inactivation through transfer of different groups (acetyl, phosphate) to the amino hydroxyl groups (MacLeod et al., 2000).

11.5 DRUG RESISTANCE IN CANDIDA ALBICANS Candida Albicans is an opportunistic microorganism, which is responsible for causing skin/mucous membrane (superficial) and invasive (life-threatening) infections (Spampinato and Leonardi, 2013). It can be spread through various mechanisms, summary of which is provided in Fig. 11.8. Azole class of drug is a commonly used antifungal agent against Candida, which includes the miconazole and fluconazole. They function by inhibiting the ergosterol, an important component of cell membrane, by blocking the enzyme in the endoplasmic reticulum involved in its synthesis. Echinocandins make the cell wall porous making its way to cause osmotic lysis by inhibiting glucan synthase. Polyenes, on the other hand, disrupt the ergosterol, and flucytosine inhibits the thymidine kinase (TK) enzyme. Ally amines are responsible for cell membrane disruption, and griseofulvin prevents the microtubule formation thereby preventing apoptosis. It can be further explained in Fig. 11.9. Table 11.2 provides the mechanism of resistance adapted by fungus to overcome antifungal responses.

FIGURE 11.8 The possible risk factors that can lead towards life-threatening conditions during Candidiasis infection (Pfaller and Diekema, 2004).

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FIGURE 11.9

Highlights of five mechanisms of action of antifungal drugs that help fight against fungal diseases at molecular level.

TABLE 11.2 Mechanism of Drug Resistance Adapted by Candida Species Group of drugs

Examples

Mechanism of action

Resistance developed

Azoles

Miconazole/ clotrimazole/ fluconazole

Inhibits ergosterol synthesis by inhibiting lanosterol 14 alpha demethylase (Hof, 2006)

Point mutations in MRR1 and TAC1, ERG1, ERG3. Inactivation of C5 Sterol desaturase (Ribeiro et al., 2005)

Echinocandins

Caspofungin/ micafungin, anidulafungin

Inhibits 1-3-beta-D-glucan synthase and makes cell wall vulnerable causing osmotic lysis (Grover, 2010)

Point mutations in FKS1 and FKS2 (Balashov et al., 2006)

Polyenes

Amphotericin B/ Disrupts ergosterol resulting in nystatin formation of aqueous pores (Sanglard and Odds, 2002)

Point mutations in ERG3 and ERG6 (Kontoyiannis and Lewis, 2002)

Nucleoside analogs

Flucytosine

Inhibits thymidylate synthase, blocks synthesis of DNA (Vermes et al., 2000)

Point mutations in FCY1, FCY2, and FUR1 (Vandeputte et al., 2011)

Allylamines

Amorolfine/ naftifine/ terbinafine

Disrupts cell membrane by inhibition of squalene epoxidase (SEPO), which is responsible for ergosterol synthesis (Sanglard et al., 2009)

Overexpression of CDR1 (Candida drug resistant) gene (Loeffler and Stevens, 2003)

Thiocarbamates Tolciclate/ tolnaftate

Inhibits SEPO

CDR1,2,3 overexpression

Antibiotics

Disrupts spindle formation and microtubule synthesis, mitosis is prevented (Francois et al., 2005)

Prolonged transport systems that are energy dependent and are absent

Griseofulvin

This table summarizes the classes of drugs used against the fungal infections, their respective mechanism through which they inhibit/kill fungus, and the strategies adapted by Candida sp., to counter these drugs by the modification of genes either by upregulation or by mutations in the genes.

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11.7 MDR IN HERPES SIMPLEX VIRUS (HSV)

TABLE 11.3

Drug Resistance Mechanism Adapted by Plasmodium falciparum

Examples of compounds

Mechanism of action

Aryl amino alcohol

Chloroquine, primaquine, quinine, halofantrine

Antifolates

Artemisinin derivatives

Class

Resistance against drugs

Consequence

It inhibits the heme products to form dimers

Point mutation in pfCRT, pfmDR1, and pfcg2 gene

The drug is excreted out of the parasite immediately

Pyrimethamine, proguanil, trimethoprim sulfadoxine

Pyrimethamine inhibits DHFR, sulfadoxine inhibits DHPS

Point mutations in DHFR gene. Drug affinity decreased

Drug loses the target due to low affinity and expelled out

Arteether, dihydroartemisinin, artemisinin artesunate

Inhibition of SERCA and generation of ROS

Drug efficacy reduces owing Interference with heme to low affinity, ACT is given detoxification pathway (Mita and Tanabe, 2012), mutations in to prevent the progress of SERCA gene infection

This table highlights the mechanism of antimalarial drugs and the resistance strategies adapted by the P. falciparum. DHFR, dihydrofolate reductase; pfCRT, transporter protein in parasitic vacuole; DHPS, dihydropteroate synthase; SERCA, Sarco-endoplasmic reticulum Ca21 ATPase; ACT, artemisinin-based combination therapy.

11.6 MDR IN MALARIA Malarial drugs are unable to treat effectively owing to the resistance developed by Plasmodium species. Three drug groups have been classified, which are commonly used in combination with each other (Parija and Praharaj, 2011). The combination is used to depend on the clinical condition of a patient. In the last two decades MDR species of Plasmodium has been increased. Table 11.3 discusses the classes of drugs and the developing AMR (Le Bras and Durand, 2003). Immediate attention towards AMR is needed to check the parasites developing resistance. Most successful treatment to date is by artemisinin-based combination therapy (ACT); however, ACT-resistant strains are emerging (Mita and Tanabe, 2012) (Fig. 11.10).

11.7 MDR IN HERPES SIMPLEX VIRUS (HSV) Herpes Simplex Viruses (HSV) 1 and 2 cause oral and genital infections associated with mucous membranes (Piret and Boivin, 2011). Currently acyclovir and penciclovir drugs are being given for the management of infection, which are activated by TK that activates these prodrugs and thus killing the virus by producing cytotoxic compounds (Strasfeld and Chou, 2010). Different classes of antiviral drugs currently available in the market against HSV are highlighted in Fig. 11.11. With the emergence of mutations in TK genome due to stress generated by antiviral drugs, we are left only with the options for combination therapies. This problem can be tackle down with effective management to develop the strategies intelligently to counter the emergence of MDR HSV serotypes as summarized in Table 11.4. Furthermore, there

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FIGURE 11.10 The in vitro studies done to check the resistance by detecting the molecular markers which are present in the strains resistant to a class of drug. Finally, by checking how much effective our therapeutic drug would be, we can predict about the evolution of AMR species.

FIGURE 11.11

Different classes of antiviral agents used as a treatment against HSV.

TABLE 11.4 Drug Resistance Mechanism by HSV Drug

Mechanism of action

Resistance developed

Acyclovir/ penciclovir

Phosphorylated and activated by TK to triphosphate form, causes inhibition of DNA polymerase (Gilbert et al., 2002)

Mutations in TK (frame shift mutations) or in DNA polymerase

Ganciclovir

UL7 kinase of CMV and TK of VZV and HSV causes its monophosphorylation and resulting in same mode of action exhibited by acyclovir

Absence of TK, either the gene is absent or it is not expressing

Foscarnet

Analog of pyrophosphate, no need of activation by viral enzyme. Blocking the action of DNA polymerases. By binding the specific site preventing DNA elongation in replication phase

Point mutations in the binding sequence of DNA polymerase

Cidofovir

Activated by TK, inhibits action of DNA polymerase (Strasfeld and Chou, 2010)

TK mutation, defective activation of prodrug

This table highlights strategies adapted by HSV to develop antiviral drug resistance and the mechanism of action of these drugs. VZV, Varicella Zoster Virus; CMV, Cytomegalovirus.

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is a need for finding the novel compounds against which the resistance has not been developed thus for the progress of therapies against HSV infections.

11.8 STRATEGIES TO CONTROL AMR Strategies to prevent the development and spread of AMR factors depend primarily on the understanding of pathogens type and associated diseases with them (Schwartz et al., 1997). To develop pathways through which AMR can be countered, knowledge of the ways through which resistance has occurred has to be clearly understood. As mentioned, these pathways differ from pathogens and the settings in which these resistant variables of microbes have emerged (Tenover and McGowan, 1996; World Health Organization, 2012; Burke, 1998). Several domains for containment of resistant pathogens have been identified, some of which have been summarized in the flow diagram in Fig. 11.12.

11.8.1 Infection Prevention and Control at Personal and Community Level From social perspectives, changes in lifestyle and attitude are required at individual and community level to prevent the spread of AMR. These may include the optimized use of existing antimicrobial drugs and, wherever possible, resort to alternative therapeutic interventions (Sipahi, 2008). It is important that both patients and doctors must reduce their expectations from antibiotics. Instead of prescribing unwanted medicines, it is the duty of healthcare practitioners and doctors to educate the patients about the prominent threat of AMR and the spread of pathogens to the community (Wise et al., 1998).

FIGURE 11.12

Main domains that must be targeted and to develop strategies for control of AMR.

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11.8.2 Policy, Cost, and Surveillance of MDR Pathogens: Political Commitment To raise awareness and to halt the AMR development, there is a need to organize Multidisciplinary Antimicrobial Management Teams at government and political levels. Furthermore, control over trade and economics to monitor the spread of resistant pathogens has to be considered, and all of these require an extensive political involvement and devotion (Struelens, 2003).

11.8.3 Fostering Innovations There is a need to look for more therapeutic interventions using biotechnological approaches to counter the immediate threat posed by AMR to avoid serious public health problems; research and innovations regarding new strategies to counter AMR has to be developed at academia, biotechnology, and biopharmaceutical industries. New drug targets should be discovered and innovation and research to counter infectious diseases have to be promoted (Norrby et al., 2005). New strategies that are currently under research to counter the AMR are also identified (Mesaros et al., 2007) with the new drugs in pipeline such Doripenem, a derivative of meropenem, an antibacterial agent which has the potential to counter the resistance associated with beta-lactam drugs (Dalhoff et al., 2006).

11.9 BIOTECHNOLOGICAL INTERVENTIONS TO COUNTER MDR 11.9.1 Nano-Silver: Antimicrobial Agents Medical nanomaterials can function as therapeutic drugs, drug carriers, vaccines, biomolecular recognition devices, biosensors and diagnostic instruments, and biomedical implants with the control and release of these nanomaterials at nanolevels (Power, 2001; Wang et al., 2009). Silver nanoparticles are the most extensively engineered nanomaterials with established antimicrobial properties and plays an evident role in biomedical applications (Behra et al., 2013). It is observed that antibacterial activity is perhaps due to the partial release of Ag 1 ions from AgNps, hence induction of strong antibacterial effect in the absence of capping agent (Xiu et al., 2011). The extent of potent bactericidal effect of silver nanoparticles could be observed through immobilized silver nanoparticles in the form of nanostructures where predominant antibacterial activity was observed through controlled release of Ag1 from fine Ag-nanoparticle composite of an average 10 nm size (Sotiriou and Pratsinis, 2010). The exact mechanism of Ag nanoparticle as an antimicrobial is not known; however, it is suggested that Ag nanoparticles mediate size, mobility, and composition-dependent bactericidal effect (Quang Huy et al., 2013). It is also suggested that nanoparticles in 1 10 nm size range present direct interaction with bacteria to produce bactericidal effect (Morones et al., 2005). Furthermore, silver nanoparticles also exhibit a potent anti-biofilm potential by hydrated polymeric matrix destruction; the anti-biofilm potential is also noted to be speciesindependent, hence quiet beneficial for futuristic application of these silver nanoparticles to control the spread of resistant microbes (Kalishwaralal et al., 2010). Exact mechanism through

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FIGURE 11.13

205

Proposed mechanisms through which antibacterial effects are induced by silver and zinc oxide

nanoparticles.

which Ag nanoparticles induce antibacterial effect is not yet known; however, many methods have been proposed as summarized in Fig. 11.13 (Prabhu and Poulose, 2012; Rai et al., 2012).

11.9.2 Zinc Oxide Nanoparticles as Synergic Antimicrobials Zinc oxide nanoparticles exhibit many potential biomedical benefits and have established bacteriostatic and bactericidal effects. Hence it is another potential antimicrobial agent along with silver nanoparticles for future biotechnological applications (Narayanan et al., 2012). The US Food and Drug Administration (FDA) has listed ZnO among five zinc compounds that are safe to use as drugs (Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation). Many modes of action of zinc oxide nanoparticles to induce bactericidal effect have been proposed. It is now known from several studies that Zinc oxide induces production of ROS inside bacterial cell (Raghupathi et al., 2011). Others further suggest that the interaction of ROS such as H2O2 in the presence with ZnO with cell surface membrane induces some changes in cell membrane structure and phospholipid bindings. Hence ZnO accumulation around cell membrane induces ROS production and exhibits both bactericidal and bacteriostatic effects on targeted bacteria (Nagarajan and Kuppusamy, 2013). These mechanisms of ZnO-mediated bacterial cell destruction are summarized in Fig. 11.13.

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11.10 THE ANTIBACTERIAL MECHANISM OF NANOPARTICLES Fig. 11.13 shows 10 mechanisms through which silver nanoparticles and three mechanisms through which zinc oxide nanoparticles may induce bactericidal effects. All the suggested mechanisms are discussed below: 1. Ag nanoparticle may adhere to bacterial cell wall to induce conformational changes in its structure and result in subsequent damage to bacterial cell (Sondi and Salopek-Sondi, 2004). 2. Cell membrane permeability may be altered by silver and zinc oxide nanoparticles both through direct interaction with phospholipid bilayer or through release of ROS, which then alters membrane permeability and results in bactericidal and bacteriostatic effects (Morones et al., 2005; Sondi and Salopek-Sondi, 2004). 3. Silver ions released in the presence of silver nanoparticles may interact with some thiol (sulfur containing) groups in enzymes to form Ag-S bonds, which then alters the function of the bacterial enzymes (Feng et al., 2000; Matsumura et al., 2003). 4. Both zinc oxide and silver nanoparticles internalize accumulate within bacterial cell to induce the formation of “pits” on bacterial membranes with subsequent bacterial cell lysis (Morones et al., 2005; Nair et al., 2009). 5. Ag 1 acting like a weak acid intercalates with DNA to form a weak acid base interaction which then results in bacterial cell damage. Similarly, ZnO nanoparticles interfere with bacterial DNA to switch on genes that can subsequently increase ROS formation inside bacteria cell (Klueh et al., 2000). 6. Some peptides present inside bacterial proteins and enzymes are rendered ineffective when Ag binds with them to cause a subsequent change in downstream cellular signaling pathways inside bacteria. Self-apoptosis can then be induced by bacterial cell (Yamanaka et al., 2005). 7. Enzyme inhibition induced by Ag-S bonds also interferes with transmembrane energy generation inside bacterial cells which in turn induces the formation of ROS (Yamanaka et al., 2005). 8. ROS production is induced by both Ag and ZnO nanoparticles through oxidative stress which in turn alters cell membrane permeability (Hwang et al., 2008; Xie et al., 2011). 9. Ag ions may also induce bacterial apoptosis by binding with 30S ribosomal subunit, which would then deactivate the complex and halt protein translation (Yamanaka et al., 2005). 10. With altered cellular signaling and induction of stress response such as “DNA conglomeration defense mechanism” by bacterial cell following Ag-nanoparticle treatment, quorum sensing between biofilm-forming bacterial cells might be inhibited.

11.11 CONCLUSION AND FUTURE PERSPECTIVES AMR is posing a great challenge in disease management as resistant strains are emerging at an alarming pace. Poor disease management along with the increased use of antibiotics is increasing drug resistance over the years. This is making disease diagnosis

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difficult and clinical conditions complicated by rendering existing range of drugs inefficient. Finally, there is an immediate need at one end for developing efficient management strategies to combat this global concern and on the other end there is a need of finding novel compounds using modern biotechnology tools such as through nanomaterials. These compounds may be deemed effective against these infectious agents and target eradication of drug-resistant pathogens.

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Kalishwaralal, K., BarathManiKanth, S., Pandian, S.R.K., Deepak, V., Gurunathan, S., 2010. Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis. Colloids Surf. B Biointerfaces 79 (2), 340 344. Klueh, U., Wagner, V., Kelly, S., Johnson, A., Bryers, J.D., 2000. Efficacy of silver-coated fabric to prevent bacterial colonization and subsequent device-based biofilm formation. J. Biomed. Mater. Res. 53 (6), 621 631. Kontoyiannis, D.P., Lewis, R.E., 2002. Antifungal drug resistance of pathogenic fungi. Lancet 359 (9312), 1135 1144. Lambert, P.A., 2002. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J. R. Soc. Med. 95 (Suppl. 41), 22 26. Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A.K.M., Wertheim, H.F.L., Sumpradit, N., et al., 2013. Antibiotic resistance? The need for global solutions. Lancet Infect. Dis. 13 (12), 1057 1098. Le Bras, J., Durand, R., 2003. The mechanisms of resistance to antimalarial drugs in Plasmodium falciparum. Fundam. Clin. Pharmacol. 17 (2), 147 153. Levy, S.B., 2005. Antibiotic resistance—the problem intensifies. Adv. Drug Deliv. Rev. 57 (10), 1446 1450. Livermore, D.M., 2002. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clin. Infect. Dis. 34 (5), 634 640. Loeffler, J., Stevens, D.A., 2003. Antifungal drug resistance. Clin. Infect. Dis. 36 (Suppl. 1), S31 41. MacLeod, D.L., Nelson, L.E., Shawar, R.M., Lin, B.B., Lockwood, L.G., Dirk, J.E., et al., 2000. Aminoglycosideresistance mechanisms for cystic fibrosis Pseudomonas aeruginosa isolates are unchanged by long-term, intermittent, inhaled tobramycin treatment. J. Infect. Dis. 181 (3), 1180 1184. Marahatta, S.B., 2010. Multi-drug resistant tuberculosis burden and risk factors: an update. Kathmandu Univ. Med. J. 8 (29), 116 125. Matsumura, Y., Yoshikata, K., Kunisaki, S.-I., Tsuchido, T., 2003. Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate. Appl. Environ. Microbiol. 69 (7), 4278 4281. Mesaros, N., Nordmann, P., Ple´siat, P., Roussel-Delvallez, M., Van Eldere, J., Glupczynski, Y., et al., 2007. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin. Microbiol. Infect. 13 (6), 560 578. Mita, T., Tanabe, K., 2012. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance. Jpn. J. Infect. Dis. 65 (6), 465 475. Mitscher, L.A., Pillai, S.P., Gentry, E.J., Shankel, D.M., 1999. Multiple drug resistance. Med. Res. Rev. 19 (6), 477 496. Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramı´rez, J.T., et al., 2005. The bactericidal effect of silver nanoparticles. Nanotechnology 16 (10), 2346. Morris, A., Kellner, J.D., Low, D.E., 1998. The superbugs: evolution, dissemination and fitness. Curr. Opin. Microbiol. 1 (5), 524 529. Nagarajan, S., Kuppusamy, K.A., 2013. Extracellular synthesis of zinc oxide nanoparticle using seaweeds of Gulf of Mannar, India. J. Nanobiotechnol. 11 (1), 39. Nair, S., Sasidharan, A., Divya Rani, V.V., Menon, D., Nair, S., Manzoor, K., et al., 2009. Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells. J. Mater. Sci. Mater. Med. 20 (1), 235 241. Available from: https://doi.org/10.1007/s10856-008-3548-5. Narayanan, P., Wilson, W.S., Abraham, A.T., Sevanan, M., 2012. Synthesis, characterization, and antimicrobial activity of zinc oxide nanoparticles against human pathogens. BioNanoScience 2 (4), 329 335. Norrby, S.R., Nord, C.E., Finch, R., 2005. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect. Dis. 5 (2), 115 119. Okeke, I.N., Laxminarayan, R., Bhutta, Z.A., Duse, A.G., Jenkins, P., O’Brien, T.F., et al., 2005. Antimicrobial resistance in developing countries. Part I: recent trends and current status. Lancet Infect. Dis. 5 (8), 481 493. Prabhu, S., Poulose, E.K., 2012. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int. Nano Lett. 2 (1), 1 10. Parija, S.C., Praharaj, I., 2011. Drug resistance in malaria. Indian J. Med. Microbiol. 29 (3), 243 248. Pfaller, M.A., Diekema, D.J., 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42 (10), 4419 4431. Piret, J., Boivin, G., 2011. Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management. Antimicrob. Agents Chemother. 55 (2), 459 472.

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12 Nanotechnology in Bioengineering: Transmogrifying Plant Biotechnology Anu Kalia Punjab Agricultural University, Ludhiana, India

12.1 INTRODUCTION The present-day plant biotechnology (PBT) stipulates alteration of the genetic pattern of plants to increase their value. This value addition to a crop/plant depends on a variety of factors. The modern PBT can be fathomed down to experimentation regarding plant callus cultures followed by genetic manipulation of these tumorous undifferentiated cell masses (Sussex, 2008). It could be defined as application of a collection of scientific tools and techniques to screen beneficial traits in plants so as to develop useful and beneficial plants thereby helping in delivering tangible benefits to the growers and consumers without significantly affecting the environment, a highly anticipated goal particularly for the developing countries. PBT is thereby considered to shorten the valuable time required to identify and introgress desirable traits in plants as performed by plant breeders, and it helps in transgressing the “kingdom” constraints to introduce genes from entirely different genera or species.

12.1.1 What is Plant/Crop Bioengineering? Plant or crop bioengineering (PB) involves techniques for identification and isolation of desirable gene(s) from varied hosts (a virus, bacteria, animal, or even different plant) followed by its introduction in the genome of host plant to develop genetically edited or modified plants possessing novel characteristics. However, it goes better and bigger beyond the genetically engineered or modified (GE or GM) crops as it may even involve engineering the biosynthetic pathways for several single or multitude of traits, metabolites like secondary compounds, enzymes, or other proteins. With the alarming concerns rising

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for environmental pollution due to escape or bioaccumulation of agri-inputs, climate extremes expected to be faced by the crop plants, need for higher productivity per unit arable land to feed growing human population, and requirement of multinutrient balanced diet for all, crop bioengineering seems to be an appropriate refuge. Unlike the timeconsuming traditional plant breeding approaches of trait selection, cross hybridization, and incorporation in desired plant, it is rather a rapid and more precise technique for raising transgenics in a short time span with possibly expressing all desirable traits in one plant.

12.2 WHERE NANOTECHNOLOGY CAN HELP IN PB? Nanoscience and technology (NS and NT) is an innovative cutting-edge discipline encompassing the tools, phenomena, and processes of physical, chemical, material sciences, and engineering to fabricate novel nanomaterials (NMs) or integration of myriad of nanocomponent(s) to create platforms/devices with unique physical, chemical, and biological interaction properties. NMs, one of the major products of nanotechnology, are the matter that exists in a transitional/interstitial stage between atom/molecules and bulk having all (“zero dimensional or 0-D”), either (1-D) or any one (2-D/3-D) among the three (xyz) dimensions constrained between nanoscale, i.e., 1 100 nm. As per the elemental composition, these could be composed of any material, i.e., metal/metal oxide, nonmetal/ nonmetal oxide, pure carbon, such as C60/C70 fullerenes, fullerols, and carbon nanotube (CNT) (single, double, and multiwall CNTs (MWCNTs)), radionuclides, organic polymers (natural/ synthetic) and composites exhibiting varied morphologies (particles, rods, wires, tubes, lattices, and films), and size dimensions. These could be synthesized by several topdown and bottom-up approaches that may involve the use of physical methods, wet chemistry techniques, and biological reduction methods. Nevertheless, the synthesis of NM starts as the individual atoms and molecules assemble together by many forces playing a pivotal role followed by nucleation and then coalescence of the smaller particles to form larger aggregates (Fig. 12.1). There are plethora of techniques involved in PBT and crop bioengineering including tissue culture, recombinant DNA technology including PCR and gene cloning, genetic transformation, and transgenics (Fig. 12.2). There is a scope of enhancing the efficacy and potential of these different techniques by the intervention of a new science and technology called “Nanotechnology” that was conceptualized by Dr Richard Feynman in late 1950s and emerged in mid 1980s by the elaborate work of Eric C. Drexler, Richard Smalley, and Gerd Bining.

12.2.1 Nanocides: NMs as Explant Sterilants in Plant Tissue Culture The most efficient approach of micropropagation technology, “plant tissue culture,” is a unique crop improvement biotechnological intervention that involves culturing explants/ plant cells under aseptic controlled nutritional and environmental conditions to develop clones having similar characteristics to original explants or may even exhibit somaclonal

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FIGURE 12.1 Mechanism of formation of higher dimensional NM (0-D to 1-D or 2-D) by bottom-up molecular assembly approach.

FIGURE 12.2 Possible applications of engineered NMs in PBT and crop bioengineering.

or gametoclonal variations for regeneration into complete plants. The explants used for culturing may exhibit the presence of microflora due to internal or external pathogenesis, i.e., contamination, which may hamper the growth of the cells or even lead to tissue death. To get rid of these contaminations, the explant is treated with sterilants like 0.01% 0.1% mercuric chloride, 5% 10% bleach, bactericidal (hydrogen peroxide, silver nitrate) or

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fungicidal compounds (bavistin, benomyl), antibiotics (tetracyclin, rifampcin, gentamicin, carbenicillin, 1% cetrimide, and chlorhexidine) (Eziashi et al., 2014), surface tension decreasing compounds (70% alcohol, detergents like sodium dodecyl sulfate, tween 20/80), and broad spectrum biocide like plant preservative mixture (contains methylchloroisothiazolinone, methylisothiazolinone, MgCl2, Mg(NO3)2, potassium sorbate, and sodium benzoate) (Colgecen et al., 2011). Silver ions released from its bulk form are known to possess antimicrobial (antiviral, antibacterial, and antifungal) activity (Spacciapoli et al., 2001). However, nanoparticulate silver formulations are more effective at lower concentrations as these ensure deeper penetration in the explants surface tissues due to their nanosize and greater dissolution and release of silver (Ag1) ions over a longer period of time due to larger surface area to volume ratios (Rai et al., 2012a,b). Hence, the released Ag1 ions may interact with the proteins (via the sulfydryl (-SH) group containing cysteine amino acid) particularly those involved in respiration process (Abdi et al., 2008), exclusive binding with the DNA bases (bridges noncanonical sites in guanine and replaces at N3 proton site in cytosine) and not to the sugar phosphate backbone (Swasey et al., 2015; Ihara et al., 2009), which may lead to DNA unwinding at the molecular level. These molecular level interactions may culminate to curbing of an array of cellular processes, for instance, interruption of cell wall synthesis and damage to cell envelopes, which may cause inhibition of cell division (Abdi et al., 2008). The extent or rate of antimicrobial activity though depends on many factors but primarily the shape (anisotropic particles are more effective than spherical particles of same size), size (smaller nanoparticles (NPs) act better than larger counterparts), concentrations of released Ag1 ions, contact time, temperature, and the developmental/growth stage of microbial cell (vegetative cells most vulnerable followed by spores or cysts) play a vital role (Abdi et al., 2008). But interestingly, as the NP biomacromolecule interaction studies are being more elaborately explored, the actual hydrodynamic size of the NPs was observed to predominately govern their antimicrobial efficacy than the physical or crystallite sizes (Khurana et al., 2014). Therefore, the smaller the hydrodynamic size of the NP, the more would be its antimicrobial activity. There are several reports stating the use of silver NPs as a plant tissue sterilant; however, the time of incubation of the test tissue and the concentration used varies among these reports (Table 12.1).

12.2.2 Nanovehicles: NMs as Gene/Protein Delivery Vehicles 12.2.2.1 Plant Gene Transformation: What Are the Techniques? The gene transformation could be defined as stable incorporation, integration, and expression of introduced genes in plant nuclear genome without involving fusion of gametes or other cells. However, the genetic transformation of plants and organisms bearing an outer microfibrillar amorphous matrix or cell wall is little tricky than the animal cell counterparts. As the cell wall acts as an extra barrier for the delivery vehicle to dislodge its payload at the target site, nucleus of the cell, it become essential to bypass this barrier. There are two major categories of gene transformation in plants on the basis of the type of involvement of a vector, i.e., the Direct (or vector less/vector independent and includes both physical and chemical methods like biolistics, microinjection,

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TABLE 12.1

Application of Silver Nanoparticles (Ag NPs) as a Novel Tissue Sterilant in Different Plants

Plant Species

Type of Tissue Disinfected

Concentration Used (in mg L21)

Gerbera jamesonii

Excised immature capitulum

120 mg L21

Valeriana officinalis L.

B0.5 1.0 cm stem cuttings

100 mg L21





150 mg
L

21

Melissa officinalis L.




Olea europaea L. (Olive mission)

Single node explants for surface sterilization

100 400 mg L21

Olea europaea L. (Olive mission)

Incorporation in MS media to curb internal contamination

4 mg L21

Solanum tuberosum

Leaf explants

100 mg L21

Lycopersicon esculentum cv. Micro-Tom

Cotyledon explants

50 mg L21

Nicotinia tabacum cv. Xanthi

Incorporated in MS media

5 100 mg L21













Incubation Time (in minutes) Reference 15

Fakhrfeshani et al. (2012)

180

Abdi et al. (2008)

15

Mohebalipour et al. (2012)

60

Rostami and Shahsavar (2009)

For many days

Rostami and Shahsavar (2009)

5 20

Mahna et al. (2013)

1 5

Mahna et al. (2013)

For many days

Safavi (2012)

MS, Murshigae and Skoog.

electroporation, temperature-mediated, electrophoresis, silicon carbide mediated, and pegylation/protoplast fusion for the two, respectively) and Indirect (Agrobacterium and virus-mediated) methods (Husaini et al., 2010). 12.2.2.2 Nano-enabled Plant Gene Transformation The plant genetic transformation by NMs started with the report of mesoporous silica nanoparticles (MSN) (Torney et al., 2007). However, the last half decade has witnessed impetus among researchers to use these novel vehicles for accomplishing genetic/metabolic engineering in plants (Rafsanjani et al., 2012; Sokolova and Epple, 2008). The nanovehicles are quite versatile for delivery of a variety of payloads including nucleic acids, proteins, and other compounds in a plant cell. The efficacy of a NM to function as a nanovehicle to carry a particular payload is determined by size, topography (corrugated, layered, sutured, and porous), surface physical properties, and the surface chemistry of the NM. Interestingly, these NM surface properties, both physical and chemical, can be tempered by deposition of new ligands/functional groups, i.e., “functionalization” on the surface (Sokolova and Epple, 2008). This provides an efficient mean to get attach molecules/compounds/ligands of different types on a single NM (Fig. 12.3). If the NM is porous (MSN) (Torney et al., 2007), has laminated structure (graphene sheets), array of tubes or pores (nanocrystalline aluminosilicates zeolites), or has the ability to enclose and package payload (liposomes, lipofectin, dendrimers or other polymeric capsules) (Rafsanjani et al., 2012), then the payload could be sequestered I. MICROBIAL AND PLANT TECHNOLOGIES

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FIGURE 12.3 Coating/adsorption/functionalization of biological macromolecules on NMs for fabricating NM-based plant gene transformation vehicles.

or encapsulated inside the NM. Such sequestered or encapsulated payloads could be delivered to the specific sites in the cell with higher safety as it will help in bypassing the exonuclease enzymatic attack in cytoplasm and greater accuracy as there will be enhanced endocytotic vesicle formation that will fuse with the nuclear membrane or release payload near the nuclear pore. 12.2.2.3 Nano-enabled Vectorless or Direct Physical Methods 12.2.2.3.1 NM-ENABLED TRANSFORMATION

As discussed in the above sections, ultra small size of the NPs equips them with the ability to trespass the cell wall and membrane barriers without any forcible entry techniques. The internalization of the payload functionalized NMs in a plant cell may occur either by passive diffusion through the cell wall in the apoplast region provided the NM size is smaller than 5 nm followed by traversing through the transmembrane channels or through the phospholipid bilayer or by active process of endocytotic vesicle formation (Serag et al., 2013). Due to the surface active functional groups or the surface chemistry, these NMs may be involved in a cascade of reactions even after the delivery of the payload. They may be internalized in the vacuole and later reach the plasmalemma by exocytotic vesicular system and become embedded in the cell membrane or wall as a component (Giraldo et al., 2014; Serag et al., 2013). Mere incubation of the intact plant cells in buffer solution containing the NM vehicle carrying payload can result in uptake and translocation of the NM to the cell cytoplasm and then to nucleus. The major NMs reported to deliver genes include the silica NPs (Chang et al., 2013; Xia et al., 2013), gold NPs, single-walled carbon nanotubes (SWCNTs) (Liu et al., 2009), and MWCNTs (Serag et al., 2013), hydroxyapatite or calcium phosphate NPs (Naqvi et al., 2012), magnetic NPs (Wang et al., 2010), iron oxide NPs (Jiang et al., 2013), zinc oxide and sulfide NPs (Fu et al., 2012), Cadmium sulfide or Quantum dots or Qdots (Wang et al., 2011), functionalized magnesium phyllosilicate or aminoclay NPs I. MICROBIAL AND PLANT TECHNOLOGIES

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FIGURE 12.4 Type of NMs applied for accomplishing genetic transformation in plants.

(Kim et al., 2014), polymeric NPs (liposomes, poly-L-lysine, poly-ethyleneimine, dendrimers, cellulose NPs) (Sokolova and Epple, 2008), and many more (Fig. 12.4). 12.2.2.3.2 NANOBIOLISTICS OR NANOPROJECTILE-BASED GENE GUN TECHNIQUE

Biolistics technique involves delivery of nucleic acid ballistically using gun powder discharge or regulated gas pressure including the use of nitrogen, compressed air, or inert gas helium for accelerating the gold or tungsten microprojectile particles (size range 0.7 1.0 µm) in plant cell (Ganeshan and Chibbar, 2010). It is being transformed to Diolistics (O’Brien and Lummis, 2007) and recently to nanobiolistics (O’Brien and Lummis, 2011) involving introduction of specific dyes and payload carrying NPs ballistically in a cell. Usually, metal or metal oxides are utilized for biolistics as nonmetal oxides are much lighter or less dense at nanoscale than the formers. However, Kim et al. (2011) have reported the development of nanobiolistic gun usable MSN Type II particles made heavier by capping with gold NPs to deliver DNA/protein in plant cell. Similarly, MartinOrtigosa et al. (2012) have tried to optimize the experimental parameters by using gold nanorods and gold-plated MSN, co-bombardment with microparticles and enhanced DNA-NP attachment protocol to deliver DNA into onion epidermis tissue and maize and tobacco leaf explants.

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12.2.2.4 Other Nano-enabled Direct Techniques The other direct techniques involve electroporation-mediated plant gene transformation (PGT) the efficiency of which could also be enhanced by using NMs (Silva et al., 2010; Wang et al., 2010). Similarly, magnetoporation involves the use of magnetic NPs and employment of static and oscillating magnetic field to enhance genetic transformation in cells (Jenkins et al., 2011). Another way of PGT is sonoporation involving application of shear forces of the sound energy for rendering passive diffusion of exogenous particles (DNA/RNA/proteins) into cell cytoplasm due to formation of transient sporadic pores in cell membrane as an impact of acoustic cavitation microbubbles and microstreaming (Tomizawa et al., 2013). The extent of gene delivery and transformation is expected to enhance if target plant cells are treated with NP/NM-loaded DNA or plasmid payload in the presence of sonication (Tomizawa et al., 2013). However, now optoporation or micropuncture is getting popular. It involves in situ or in vitro laser (ultraviolet laser diode, picosecond laser, femtosecond near infrared (NIR) pulsed laser) irradiation of either single plant cell(s) or callus cultures (Schinkel et al., 2008) for noninvasive permeabilization to create a fine and precise hole in the cell wall (size may vary from several nanometers to 5 µm) so as to develop a concentration gradient to facilitate movement of biologically relevant molecules from exterior to inside of cell (LeBlanc et al., 2013; Mitchell et al., 2013). 12.2.2.5 Nano-enabled Chemical Techniques The chemical techniques include delivery of genes in presence of chemicals like polyethylene glycol, dextran sulfate, artificial lipids, or polymers as polycationic ones or proteins with a possibility of extension to virtually all plant species. A direct Green Fluorescent Protein (GFP)-encoding plasmid delivery was successfully attempted by using poly(amidoamine) dendrimers into turfgrass cells (Pasupathy et al., 2008), which showed transformation observed as green fluorescence signal through confocal laser scanning microscopy. 12.2.2.6 Nano-enabled Vector-mediated or Indirect Gene Transformation Techniques Agrobacterium tumefaciens is a gram-negative soil rhizospheric bacteria known to cause “crown gall” disease in dicot plants and has been scientifically applauded for its unique natural genetic engineering potentials (McCullen and Binns, 2006). Though this bacterium has been harnessed to genetically modify many crops, still there are few limitations of low gene size, degradation of the introduced DNA after transformation, problem regarding regeneration of the infected tissue, and incompatibility for gene transfer in monocot plants, i.e., Agrobacterium recalcitrance (Husaini et al., 2010) gives the scope for application of fine tuning pre- as well as postinfection protocols to maximize transformation efficiency. NMs can enhance the gene transformation efficacy of plant cell vector-mediated techniques primarily involving A. tumefaciens. Accomplishment of a transgene delivery has been reported in microspores of monocot triticale plants by preparing a DNA protein nanocomplex in the presence of a cell-penetrating peptide using Agrobacterium (Ziemienowicz et al., 2012).

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12.2.3 Nanosequencing: Nanopore-based Gene or Protein Sequencing Tools/Techniques The nanotechnological products and processes have revolutionized the way rapid, high throughput, fully automated, and low cost sequencing of the nucleic acids (DNA/RNA), and even proteins can be performed within less than a day (Branton et al., 2008) on a miniaturized portable sequencing device system available even as a single-use handheld system MinION (Feng et al., 2015). The sensitivity of this fourth generation sequencing/ analytical technique is to the level of one nucleoside to as large as kilobase length ssDNA or RNA that can be sequenced without the need for PCR amplification or labeling with radioactive, organic, or fluorescent labels (Feng et al., 2015; Srinivasan and Batra, 2014). This concept later developed to a practical technique for measurement of modulation in electric current in response to nucleotide/nucleoside bases in sequential order on electrophoretically mobilizing ssDNA or RNA across a biological (mostly protein) or solid-state nanopore of suitable diameter (Wang et al., 2015a,b; Srinivasan and Batra, 2014). The next generation sequencing techniques having proficiency of long read lengths (Steinbock and Radenovic, 2015) are now being aptly utilized for the rapid sequencing of plant genomes spanning over grain and legume crops, model plant Arabidopsis, vegetables to tree species (Shangguan et al., 2013), and making genetic characterization of the plant genomes affordable such that a larger repository of these sequences help in working on several plants as “model” plants (Egan et al., 2012).

12.2.4 Nanobioimaging: NMs for High-Resolution Real-Time Imaging The structural and functional unit of life, “the Cell,” has always flared curiosity to discern its ultrastructural details including the subcellular components, multiple functions that it can perform, and myriads of inter/intracellular communication or interactions it undergoes for maintaining its existence since times immemorial. The major tools to study these aspects of a cell, the microscopes, have also evolved over decades. There are variants for optical, electron, x-rays, laser-based, probe-based, vibrational, and other microscopies equipped with better digitized image capturing (charge coupled devices (CCD), electron multiplying CCD (EMCCD), intensified CCD/IEMCCD), image analysis softwares (Image J; Fiji, Cell Profiler, MatLab) (Domozych, 2012), and even image analysis databases to chalk out probable software(s) required to obtain the best possible solutions (Lobet et al., 2013). The nanotechnological interventions are endowing microscopes with super high resolutions as there are far improved nano-enabled silicon-based solid-state fabrication techniques available now to manufacture compact yet robust portable microscopes. Partly, development and application of improved nanotags (NPs of Au, Ag, silica, CdS quantum dots) (Wolfbeis, 2015; Ho¨tzer et al., 2012) vis-a`-vis real-time imaging techniques have modified the data collection, analysis, and interpretation of the microscopic images (Li et al., 2014). The NP plant soil continuum interaction studies could involve landmark techniques to discern and segregate the molecular mechanistic events for specific physiological/genetic alterations leading to requirement of quantitative information on development of a peculiar

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phenotypic trait in a test plant as a response to NP amendment. Such studies can be made feasible by use of the latest automated, “Plant phenomics” approaches (Li et al., 2014). The plant scientists are also eager to elucidate real-time nondestructive imaging information for the phenotypic, biochemical, physiological, and metabolic changes happening in plant at different growth and developmental stages as well as in response to different stress (abiotic or biotic) conditions. Such data could be generated by transcending the use of high-resolution imaging techniques quite routinely used in biomedical and other disciplines. A new protocol has been reported involving immersion of plant tissue in phosphotungstate (for deeper penetration in thick tissue) or osmium tetraoxide (for thin or open tissue) to enhance tissue contrast in high-resolution low X-ray CT imaging for a 3-D image reconstruction later using appropriate software (Staedler et al., 2013). Similarly, another common biomedical noninvasive and nondestructive imaging technique, magnetic resonance imaging (MRI), has now being proposed to be utilized to study plant organ development as well as to detect in vivo metabolic and physiological changes in response to abiotic stress. A rice root biology study involving assessment of effect of different concentrations of gadopentaacetic acid (Gd-DTPA) was performed on rice plant. The results showed Gd-DTPA at 5 mmol to be a safe dose to work as an MRI contrast agent in rice root in vivo studies (Liu et al., 2014). The application of designer conjugate NMs opens new vistas for in vivo plant cell biochemical imaging studies. The designer carbon shell encapsulating 4-mercaptobenzoic (4-MBA) acid functionalized composite Au-Ag NPs were syringe infiltrated on tobacco abaxial surface, and surface-enhanced Raman spectroscopy (SERS) studies were performed to trace the SERS signal for 4-MBA in real-time nondestructive mode even after 10 days of growth of the tobacco plant (Shen et al., 2011). The conjugate spectroscopy imaging techniques are also versatile to discern the plant nutrient status. Hyper Spectral Imaging makes nondestructive real-time spatial localization of a particular macro or micronutrient in plant organs feasible (Yu et al., 2014). This will revolutionize our understanding on nutrient partitioning, translocation, and redistribution among different plant organs at various developmental stages of the plant. Evidently, such tools will be beneficial in developing the nutrient application schedules for the genetically edited crops allowing the use of precision agriculture techniques. Moreover, the high-speed integrated cytometry in vivo studies regarding photosynthate assimilation, attack and spread of infectious agents, and even movement of applied NPs in plant vasculature could be helpful in discerning the ultimate fate of the applied nutrients and NPs to the plant system (Nedosekin et al., 2011).

12.2.5 Nanotheranostics: Nano-based Therapy and Diagnostic Products for Plant Pests and Pathogens Theranostics, a biomedical terminology, will be entrenching the plant biological sciences in the coming era. Plant theranostics may be defined as a combinatorial technique of precise and sensitive pathogen identification (virus, fungus, and bacteria), i.e., diagnostics, which may involve spectroscopy or imaging tools followed by its tailor-made remediation, i.e., therapeutics using a single or augmented agent(s). The up-coming field of “Plant

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Nanotheranostics” will deal with developing nanotechnology derived low-cost products/ platforms for highly sensitive, rapid, and reliable plant pathogen detection to ensure early disease diagnostics (Rai et al., 2012a,b) for optimal treatment or therapy simultaneously or in a stepwise manner. The nano-enabled diagnostics is a need-of-hour technology as the unique properties of NMs are expected to be helpful in developing low cost, robust, user-friendly, energy-efficient, miniaturized, or may be handheld portable modules that could perform an on-site ex-planta or in-planta rapid and ultra-sensitive detection of single or multiple known or unknown plant pathogens with laboratory scale accuracy without requirement of special pretreatment protocols and scientific prowess (Nezhad, 2014). Moreover, these may be employed for deciphering the intricate plant pathogen and plant other microbe interactions apart from detecting pathogen population genetics for better monitoring and control. Such quick point-of-care on-site portable diagnostic systems may vary in one or several aspects such as sample loading to capture and detection mechanisms and could fall in categories as nano-enabled kits, sensors, biosensors, barcodes, microarrays, and many others (Khiyami et al., 2014). There are plenty of reports documenting the antibacterial, antifungal, and antiviral activities of diverse types of NMs (Yah and Simate, 2015; Botequim et al., 2012), but development of novel theranostic agents will be more desirable (Lim et al., 2015). These NMs will not only possess the surface ligands targeting them to reach a particular site using the plant apoplast and symplastic pathways but could also be induced by certain stimuli like optical (infrared/ NIR/UV rays), thermal, acoustic, or magnetic to either dislodge the therapeutic payload or may initiate exacerbating effects on the causative agent “the pathogen” or the infected tissue, so as to cause localized cell death and necrosis to curb the spread of the disease to uninfected tissues (Lim et al., 2015).

12.2.6 Nanobarcoding: Naming and Sorting the GM Crops The barcodes are short stretches of gene sequences or ssDNA, RNA, or complementary DNA that can be immobilized or coated on the surface of a NP or NM (nanobarcodes) to detect (as optical fluorescence, SER, or other signals) preferentially digitally and quantify target genomic ssDNA, mRNA, or complementary DNA (cDNA) in the biological sample, particularly plant material. Geiss et al. (2008) have developed a nanostring nCounter DNA barcoding optical fluorescence based technique to identify and quantify direct mRNA from the sample without the requirement of amplification and formation of cDNA. Thus, there is higher sensitivity due to increased surface area of attachment and reactivity by using NMs, and also there is multiplexing of the platform to identify a large variety of genes simultaneously (Fortina and Surrey, 2008). The basic concept involves the identification of unknown stretches of DNA by hybridization with the known bait DNA molecules attached/adsorbed on to NM for even femtomolar concentrations without any prior amplification (Husale et al., 2009). Moreover, the use of NMs also makes detection systems more versatile to include spectroscopy, microscopy, or both as conjugate system to identify as well as quantify the unknown DNA. A DNA functionalized silica-coated gold NPs based nanobarcode assay involving SERS analysis has been reported to detect transgenic Bt rice in a mixed sample at 0.1 pM level of sensitivity (Chen et al., 2012).

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These platforms do not vary much in their configuration or design from the nanodiagnostic kits or other portable platforms used for identification of pathogens, pests, or other microbes. However, being unique identification markers, these have an additional asset to be most widely and effectively used for identification, authentication, and tracking in agricultural food. The concerns for the GM food have been raised, which calls for the authentication of the agri-based food including grain crops, vegetables, and fruits to be checked and later sorted for the introduced genes. It will serve to be the cheapest and rapid way to track and sort effect of GM crops; for instance, to track the effect of Bacillus thuringiensis (cry Bt endotoxin) gene containing corn, canola, and cowpea on the population of the nontarget insect population comprising the beneficial plant pollinators such as insects belonging to Hymenoptera, Coleoptera, Diptera, and Lepidoptera classes (Nzeduru et al., 2012).

12.2.7 Nanogrowth Enhancers: NMs to Enhance Seed Germination and Plant Growth Engineered NMs are known to possess plant growth altering properties by virtue of their unique physical and surface chemical properties. The application of NMs for seed invigoration and enhancing the growth has been documented by many researchers (Table 12.2). NMs themselves are not expected to have any specific nutritional value, provided, these are not composed of either plant micronutrients like zinc, boron, iron, molybdenum, and many more or are essential rare earth elements required for proper physiological, enzymatic, and biochemical well-being of a growing plant. There are several known as well as unspecified ways by which applied NMs may exhibit a positive growth effect on the test plants (Servin et al., 2015). NMs may be applied to the test plants by many techniques viz., above-ground plant surfaces particularly leaves and aerial stem portion by dusting, electrostatic/aerosol spray, foliar spray, and hydrogel application, while the below-ground root system by root dipping incubation techniques, degradable polymer-encapsulated or embedded NP/NM formulations, and fertigation through dripirrigation systems. Keeping in mind the nanotoxicity issues, cautious and prudent application dosages for particular plant species have to be deduced prior to testing of NMs in open, dynamic test condition or at field scale. Moreover, long-term studies to envisage the impact of applied NMs have to be planned before accomplishing nanorevolution in plant growth nanoformulations.

12.2.8 Bioinspired/Nano-enabled Plants Nature has always been an inspiration for inquisitive and inventive human mind. Biomimetics deals with imitation of the components, models, and systems from the nature to find appropriate solutions for the current and/or possible future human quagmires (Koch et al., 2009). A comprehensive subcategory of biomimetics deals with the construction of synthetic technology systems by imitating the biological principles (Ren and Liang, 2014). Bioinspired or bionic plants are the artificial technological masterpieces that replicate the

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TABLE 12.2 Type of NM

Plant Growth Promoting Effects and Seed Invigoration by Application of Diverse NMs

Concentration Used (in mg L21)

Crop Used in Study

Type of Study

Effect on Growth of Test Plant

References

Raliya and Tarafdar (2013)

METAL/ METAL OXIDE NPS ZnO NPs

10

Cluster bean

Soil study involving foliar spray of ZnO NPs on 14-day old plants

Increased shoot length, root area, dry biomass, grain yield

TiO2 NPs

100 300

Wheat

Field soil study involving foliar application

Increase in yield Jaberzadeh attributing characters et al. (2013) like ear weight and number, biomass, seed number, and yield

Fe2O3 NPs

20 100

Water melon

Nutrient solution study

Increased antioxidase Wang et al. enzyme (Superoxide (2015a,b) dismutase, peroxidase, and catalase) activity, chlorophyll content, and soluble proteins

Mn NPs

0.05 1.0

Mung bean

Pot study involving seed incubation with Mn NPs and planting of germinated seeds in perlite and Hoagland nutrient solution under controlled laboratory conditions

Increased nitrogen assimilation enzyme activity including nitrate reductase activity

Pradhan et al. (2014)

Kole et al. (2013)

CARBON-BASED NMS C60 fullerenes/ Fullerols

0.943, 4.72, 9.43, 10.88, and 47.2 nM

Bitter melon (Mormordica charantia) var. CBM12

Greenhouse study involving seed treatment for 48 hours and planting of germinated seeds in potting mix

Increased biomass, water content, yield attributing characters as fruit length, number and weight, total yield, and anticancer and antidiabetic compound contents

SWCNTs

9, 56, 315, and 1750 (both functionalized, i.e., fSWCNT and nonfunctionalized, i.e., nfSWCNT were used)

Brassica oleracea (cabbage), Daucus carota (carrot), Cucumis sativus (cucumber),

Petri plate seed germination study for 48 hours under controlled conditions

Increased root Canas et al. elongation in (2008) cucumber only and no effect on cabbage and carrot by nfSWCNT (Continued)

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TABLE 12.2 (Continued) Type of NM

Concentration Used (in mg L21)

Crop Used in Study

SWCNTs

56, 315, and 1750

Allium cepa (onion), Lycopersicon esculentum (tomato), and Lactuca sativa (lettuce)

Petri plate seed Increased root Canas et al. germination study for elongation in onion by (2008) 48 hours under nfSWCNT controlled conditions

MWCNTs

5 60 [Pristine MWCNTs (6- to 9-nm diameter and 5-µm long)]

Zea mays L. (maize)

Seedling study performed by growing seeds in nutrient agar gel under controlled ambient conditions

Type of Study

Effect on Growth of Test Plant

20 mg/L MWCNTs increased the growth and water contents of root

References

Tiwari et al. (2014)

biological process(es) and mimic the function(s) of the plant or plant organs. For example, developing a bionic leaf composed of a synthetic polymeric composite adsorbent and a water holding layer coated with green coating that simulates the thermal effects of a transpiring natural leaf transpiration (Yuan et al., 2014) is a great human ingenuity effort or precise mimicking of the key solar spectrum reflection characteristics of plant leaves and transpiration by designing bionic leaf composed of a thin film of polymer, LiCl, and Cr2O3 (Ye et al., 2015, Yang et al., 2010). The know-how gathered by these studies could be utilized for developing efficient silicon solar panels that simulate maximum solar energy harvest, optimized aerodynamic attributes, and ability of restructuring for efficient electricity generation (Za¨hr et al., 2010). The showcasing of the functional internal organs by open and closure of flowers of many plants can be studied for the development of innovative technical lead-through and closure structures, particularly the cable entry systems having high opening to closing ratio to support cable and also protect the switch from dust and water contamination (Masselter et al., 2008). A plant-tendril inspired new generation of robots can be designed having ability to climb smooth nonadhesive surfaces by using the grasping-by-coiling and pulling phase (Vidoni et al., 2015). Similarly, the simulation of stomatal micropore transpiration has been tried to develop a novel low cost, simple structured micropump possessing high and adjustable flow rates (Li et al., 2011). A dichotomy of bionics, plant nanobionics, is a recent approach to engineer new materials by combining/embedding synthetic NPs in plant organelles so as to enhance the processes performed by the plant organelle or the plants (Giacomo et al., 2015). Giraldo et al. (2014) have tried to passively transport SWCNTs to get localized and embedded in the isolated chloroplast lipid bilayer. These nanobionic chloroplasts showed three times higher photosynthesis, highest electron transport rates, and increased the photon harvesting beyond visible light range, i.e., in UV and NIR due to unique optoelectronic properties of SWCNTs. Similarly, another study aimed for the development of a composite multifunctional material comprised biological matrix embedded with MWCNTs to permanently stabilize temperature response of isolated plant cell for enhanced background conductivity

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for developing thermal and distance sensors (Giacomo et al., 2015). These paradigm studies will pave towards the “nanoengineering plant physiological processes” in coming years. Chen et al. (2013) have developed a bionic nanoporous TiO2 nanosheet containing 10 nm TiO2 NPs for better photocatalytic degradation of methylene blue dye in visible range of sunlight. They generated it by calcification of product obtained by using float grass as a biotemplate.

12.3 CONCLUSIONS Genetic transformation and PB are need-of-the-hour technologies catering to rapid and stable food supply for growing world population. The tools, processes, and products of PB require a tweak with the help of nanotechnological acumen. As one of the “key enabling technology,” nanotechnology in agriculture is expected to possess Pandorian potentials to resolve the daunting challenges of agri-sustainability for achieving food security in the present-day cyclic climatic oscillations faced by the agriculturists as a result of global warming. Not just this, these interventions are predicted to curb the paucity of sustainable growth and competitiveness in varied sectors of agri-production, storage, preservation and processing, marketing, and distribution (Parisi et al., 2015). Thus, the crop bioengineering techniques will harness the accuracy, sensitivity, and multiplexing of benefits by using NMs or products derived of nanotechnology.

Acknowledgments The author graciously thanks the Dean, College of Agriculture, for providing the necessary infrastructural and other facilities for carrying out the research work.

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O’Brien, J.A., Lummis, S.C.R., 2011. Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles. BMC Biotechnol. 11, 66. Available from: http://www.biomedcentral.com/1472-6750/11/66. Parisi, C., Vigani, M., Rodrı´guez-Cerezo, E., 2015. Agricultural nanotechnologies: what are the current possibilities? Nano Today 10, 124 127. Available from: https://doi.org/10.1016/j.nantod.2014.09.009. Pasupathy, K., Lin, S., Hu, Q., Luo, H., Ke, P.C., 2008. Direct plant gene delivery with a poly(amidoamine) dendrimer. Biotechnol. J. 3, 1078 1082. Available from: https://doi.org/10.1002/biot.200800021. Pradhan, S., Patra, P., Mitra, S., Dey, K.K., Jain, S., Sarkar, S., et al., 2014. Manganese nanoparticles: impact on non-nodulated plant as a potent enhancer in nitrogen metabolism and toxicity study both in vivo and in vitro. J. Agri. Food. Chem. 62, 8777 8785. Available from: https://doi.org/10.1021/jf502716c. Rafsanjani, M.S.O., Alvari, A., Samim, M., Hejazi, M.A., Abdin, M.Z., 2012. Application of novel nanotechnology strategies in plant biotransformation: a contemporary overview. Recent Trends Biotechnol. 6, 69 79. Rai, M.K., Deshmukh, S.D., Ingle, A.P., Gade, A.K., 2012a. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J. Appl. Microbiol. 112, 841 852. Available from: https://doi.org/10.1111/j.13652672.2012.05253.x. Rai, V., Acharya, S., Dey, N., 2012b. Implications of nanobiosensors in agriculture. J. Biomater. Nanobiotechnol. 3, 315 324. Available from: https://doi.org/10.4236/jbnb.2012.322039. Raliya, R., Tarafdar, J.C., 2013. ZnO nanoparticle biosynthesis and its effect on phosphorous-mobilizing enzyme secretion and gum contents in Clusterbean (Cyamopsis tetragonoloba L.). Agri. Res. 2 (1), 48 57. Ren, L.Q., Liang, Y.H., 2014. Preliminary studies on the basic factors of bionics. Sci. China Tech. Sci 57, 520 530. Available from: https://doi.org/10.1007/s11431-013-5449-1. Rostami, A.A., Shahsavar, A., 2009. Nano-Silver particles eliminate the in-vitro contaminations of olive ‘Mission’ explants. Asian J. Plant Sci. 8, 505 509. Safavi, K., 2012. Evaluation of using nanomaterial in tissue culture media and biological activity. In: 2nd International Conference on Ecolology and Environmental Biological Sciences. EEBS’2012, 13 14 October, 2012, Bali, Indonesia, pp. 5 8. Schinkel, H., Jacobs, P., Schillberg, S., Wehner, M., 2008. Infrared picosecond laser for perforation of single plant cells. Biotechnol. Bioeng. 99 (1), 244 248. Available from: https://doi.org/10.1002/bit. Serag, M.F., Kaji, N., Habuchi, S., Bianco, A., Baba, Y., 2013. Nanobiotechnology meets plant cell biology: carbon nanotubes as organelle targeting nanocarriers. RSC Adv. 3, 4856 4862. Available from: https://doi.org/ 10.1039/c2ra22766e. Shangguan, L., Han, J., Kayesh, E., Sun, X., Zhang, C., Pervaiz, T., et al., 2013. Evaluation of genome sequencing quality in selected plant species using expressed sequence tags. PLoS One 8 (7), e69890. Available from: https://doi.org/10.1371/journal.pone.0069890. Shen, A., Guo, J., Xie, W., Sun, M., Richards, R., Hu, J., 2011. Surface-enhanced Raman spectroscopy in living plant using triplex Au-Ag-C core-shell nanoparticles. J. Raman Spectrosc. 42, 879 884. Available from: https://doi.org/10.1002/jrs.2812. Silva, A.T., Nguyen, A., Ye, C.M., Verchot, J., Moon, J.H., 2010. Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts. BMC Plant Biol. 10, 291. Available from: http://www.biomedcentral.com/1471-2229/10/291. Sokolova, V., Epple, M., 2008. Inorganic nanoparticles as carriers of nucleic acids into cells. Angew. Chem. Int. Ed. 47, 1382 1395. Available from: https://doi.org/10.1002/anie.200703039. Spacciapoli, P., Buxton, D., Rothstein, D., Friden, P., 2001. Antimicrobial activity of silver nitrate against periodontal pathogens. J. Periodontal Res. 36, 108 113. Srinivasan, S., Batra, J., 2014. Four generations of sequencing—is it ready for the clinic yet? Next Gener. Seq. Appl. 1, 107. Available from: https://doi.org/10.4172/jngsa.1000107. Staedler, Y.M., Masson, D., Schonenberger, J., 2013. Plant tissues in 3D via X-ray tomography: simple contrasting methods allow high resolution imaging. PLoS One 8 (9), e75295. Available from: https://doi.org/10.1371/journal.pone.0075295. Steinbock, L.J., Radenovic, A., 2015. The emergence of nanopores in next generation sequencing. Nanotechnol. 26. Available from: https://doi.org/10.1088/0957-4484/26/7/074003074003, 1-5. Sussex, I.M., 2008. The scientific roots of modern plant biotechnology. Plant Cell 20, 1189 1198.

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Swasey, S.M., Leal, L.L., Lopez-Acevedo, O., Pavlovich, J., Gwinn, E.G., 2015. Silver (I) as DNA glue: Ag1-mediated guanine pairing revealed by removing Watson-Crick constraints. Sci. Rep. 5. Article number: 10163. Available from: https://doi.org/10.1038/srep10163. Tiwari, D.K., Dasgupta-Schubert, N., Cendejas, L.M.V., Villegas, J., Montoya, L.C., Borjas Garcia, S.E., 2014. Interfacing carbon nanotubes (CNT) with plants: enhancement of growth, water and ionic nutrient uptake in maize (Zea mays) and implications for nanoagriculture. Appl. Nanosci. 4, 577 591. Available from: https:// doi.org/10.1007/s13204-013-0236-7. Tomizawa, M., Shinozaki, F., Motoyoshi, Y., Sugiyama, T., Yamamoto, S., Sueishi, M., 2013. Sonoporation: gene transfer using ultrasound. World J. Methodol. 3 (4), 39 44. Available from: https://doi.org/10.5662/wjm.v3.i4.39. Torney, F., Trewyn, B.G., Lin, V.S.K., Wang, K., 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotech. 2, 295 300. Vidoni, R., Mimmo, T., Pandolfi, C., 2015. Tendril-based climbing plants to model, simulate and create bioinspired robotic systems. J. Bionic Eng. 12, 250 262. Wang, F.H., Liu, J., Tong, C.Y., Wang, Q.M., Tang, D.Y., Yi, L., et al., 2010. Magnetic nanoparticle as rice transgene vector mediated by electroporation. Chin. J. Anal. Chem. 38 (5), 617 621. Wang, M., Liu, X., Hu, J., Li, J., Huang, J., 2015a. Nano-ferric oxide promotes watermelon growth. J. Biomater. Nanobiotechnol. 6, 160 167. Available from: https://doi.org/10.4236/jbnb.2015.63016. Wang, Y., Yang, Q., Wang, Z., 2015b. The evolution of nanopore sequencing. Front. Genet. 5, 1 20. Available from: http://dx.doi.org/10.3389/fgene.2014.00449. Wang, Q., Chen, J., Zhang, H., Lu, M., Qiu, D., Wen, Y., et al., 2011. Synthesis of water soluble quantum dots for monitoring carrier-DNA nanoparticles in plant cells. J. Nanosci. Nanotechnol 11 (3), 2208 2214. Available from: https://doi.org/10.1166/jnn.2011.3560. Wolfbeis, O.S., 2015. An overview of nanoparticles commonly used in fluorescent bioimaging. Chem. Soc. Rev. 44, 4743 4768. Xia, B., Dong, C., Zhang, W.Y., Lu, Y., Chen, J.H., Shi, J.S., 2013. Highly efficient uptake of ultrafine mesoporous silica nanoparticles with excellent biocompatibility by Liriodendron hybrid suspension cells. Sci. Chin. Life Sci. 56, 82 89. Available from: https://doi.org/10.1007/s11427-012-4422-8. Yah, C.S., Simate, G.S., 2015. Nanoparticles as potential new generation broad spectrum antimicrobial agents. DARU J. Pharm. Sci. 23, 43. Available from: https://doi.org/10.1186/s40199-015-0125-6. Yang, Y., Liu, Z., Hu, B., Man, Y., Wu, W., 2010. Bionic composite material simulating the optical spectra of plant leaves. J. Bionic Eng. 7 (Suppl.), S43 S49. Ye, H., Gao, Y., Li, S., Guo, L., 2015. Bionic leaves imitating the transpiration and solar spectrum reflection characteristics of natural leaves. J. Bionic Eng. 12, 109 116. Yu, K.Q., Zhao, Y.R., Li, X.L., Shao, Y.N., Liu, F., He, Y., 2014. Hyperspectral imaging for mapping of total nitrogen spatial distribution in pepper plant. PLoS One 9 (12), e116205. Available from: https://doi.org/10.1371/ journal.pone.0116205. Yuan, Z., Ye, H., Li, S., 2014. Bionic leaf simulating the thermal effect of natural leaf transpiration. J. Bionic Eng. 11, 90 97. Za¨hr, M., Friedrich, D., Kloth, T.Y., Goldmann, G., Tributsch, H., 2010. Bionic photovoltaic panels bio-inspired by green leaves. J. Bionic Eng. 7, 284 293. Ziemienowicz, A., Shim, Y.S., Matsuoka, A., Eudes, F., Kovalchuk, I., 2012. A novel method of transgene delivery into triticale plants using the Agrobacterium transferred DNA-derived nano-complex. Plant Physiol. 158, 1503 1513.

Further Reading Akin, D., Sturgis, J., Ragheb, K., Sherman, D., Burkholder, K., Robinson, J.P., et al., 2007. Bacteria-mediated delivery of nanoparticles and cargo into cells. Nat. Nanotech. 2, 441 449. Hao, Y.Z., Yang, X., Shi, Y.Z., Song, S., Xing, J., Marowitch, J., et al., 2013. Magnetic gold nanoparticles as a vehicle for fluorescein isothiocyanate and DNA delivery into plant cells. Botany 91, 457 466. Available from: https:// doi.org/10.1139/cjb-2012-0281. Kejzlar, P., Volesky, L., Andrsova, Z., Kroisova, D., 2011. Bionics and nanotechnology. In: Nanocon 2011, held from 21 to 23 September 2011, Brno, Czech Republic, EU.

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13 Techniques in Biotechnology: Essential for Industry Malik G. Mustafa1, Md Gulam M. Khan1, Duy Nguyen1 and Shahid Iqbal2 1

University of Sherbrooke, Sherbrooke, QC, Canada 2University of Agriculture Faisalabad, Faisalabad, Pakistan

13.1 BRIEF HISTORY OF BIOTECHNOLOGY The first use of biotechnology aimed at producing alcohol dates back to Sumerian, Neolithic, and Babylonian cultures (7000 BC). However, there has been a very little progress on the understanding and development of biotechnology for the 86 centuries that followed. For examples, Sumerian, Neolithic, and Babylonian used yeast for fermentation of grapes and grains to produce wine and beer, respectively. This humble beginning was followed by Egyptians who discovered that fermentation carried out by yeast results in the production of CO2 which leavens bread. The early medical uses of biotechnology date back to Assyrians (3500 BC) and Hippocrates (400 BC) who used vinegar to treat chronic middle ear disease. Until the last quarter of 17th century, the history of biotechnology consists of wine, beer, vinegar, leaven bread, and yogurt production and preservation of the milk and some other food items. It was only in 1673 1723 when Leeuwenhoek (having no university connection) who examined water and scraping from the teeth by his simple lenses and published his observation regarding “microscopic animals” in European science amateur. Instead of his important observation, there has been a long standing debate over the spontaneous generation of microorganisms for about a century that hampered the progress of biotechnology. Although Francesco Redi (Italy), Charle Cagniard (France), Theodor Schwann, Friedrich Traugot Ku¨tzing (Germany) resisted the theory of “spontaneous generation of microorganisms,” in mid-19th century Louis Pasteur (France) not only refuted the theory of “fermentation being entirely a chemical process” but also laid the

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foundation of microbiology as a distinct discipline and showed that fermentation was carried out by live yeast. Although Louis Pasteur dominated the 2nd half of the 19th century by significantly contributing in the acceptability of existence and role of microorganisms, the first half of the 20th century was dominated by coordinated interinstitutional and interenterprise work on penicillin discovered by Alexander Fleming (England). Two decades of research on penicillin not only made it the first successful life-saving antibiotic but also laid the foundation of a cascade of life saver antibiotics during the 2nd half of the 20th century. The difficulties encountered during the development of penicillin clearly defined the future needs of antibiotic development in particular and biotechnology in general. However, in the 2nd half of the 20th century, biotechnology enhanced its scope by significantly contributing in the fields of health (antibiotic and pharmaceuticals), chemicals, enzymes, food and feed, paper and pulp, biofuels, and environment, etc. Although biotechnology has benefitted from multidisciplinary techniques (a huge list), the techniques that have been instrumental in expanding the scope of biotechnology include fermentation and biocatalysts/enzyme production which will be discussed comprehensively in this chapter. In addition, relatively newly developed contributions of biotechnology in the field of paper and pulp industry and biofuels would be discussed with special emphasis on the enzymes which significantly contributed in making these industries more economical and environment friendly followed by a very brief description and applications of selected industrial techniques. At the end of this chapter, recently developed/advanced techniques which have the potential to contribute significantly in future in the field of biotechnology including deep sequencing, mass spectrometry, and CRISPR Cas, will also be discussed briefly. For the detailed and state-of-the-art information on the omics approaches on the various applications of industrial biotechnology, the readers are referred to the following book chapters: Chapter 14, Omics Approaches in Industrial Biotechnology and Bioprocess Engineering by Dr. Raja et al.; Chapter 15, Omics Approaches and Applications in Dairy and Food Process Technology by Dr. Dubey et al.; Chapter 16, Omics Approaches in Enzyme Engineering by Dr. Hassan et al.; Chapter 17, Biomedical Engineering: The Recent Trends by Drs. Manju Sharma and Paul Khurana; Chapter 18, Omics approaches in biofuel technologies: toward cost-effective ecofriendly and renewable energy by Dr. Yadav et al.)

13.2 FERMENTATION The process/technique of fermentation has been used and/or known to man for thousands of years. Man’s quest to understand the process of fermentation not only laid the foundation of microbiology but also triggered interest and development of biotechnology. The process of industrial fermentation has following major components which need to be considered vigilantly to make the process economically viable: 1. Fermentation method 2. Inoculum (microorganisms) 3. Substrate

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4. Fermentors 5. Culture conditions 6. Product We will describe the well-established recommendations regarding each component of industrial fermentation in the following sections.

13.2.1 Fermentation Method The process of fermentation might be classified into several types depending on the criteria of classification. For example, regarding the medium of culture for microorganisms, fermentation is classified into solid-state fermentation where substrate is fermented on solid support and H2O2 might only be available through capillary action and submerged fermentation where substrate is submerged or dissolved in solution. Both of these (solid state and submerged) fermentations can be aerobic (in the presence of oxygen) or anaerobic (in the absence of oxygen). In addition, depending on the type of culture of microorganisms, fermentation can be classified to monoseptic (single type of microorganism) and mixed culture (more than one species of microorganisms). Strictly sterile environment is required for monoseptic fermentation whereas mixed culture fermentation is more permissive regarding sterile environment. Regarding the method of inoculum induction and product collection, fermentation can be classified into fed-batch operations (culture medium injected and product collected at defined intervals) or continuous cultures (culture medium injected and product collected continuously). In continuous type of fermentation, the dilution rate of medium must not exceed the growth rate of microorganism under given condition.

13.2.2 Inoculum (Microorganisms) Microorganisms including bacteria, yeast, etc., are used as the agents of fermentation where a substrate in a suitable growth condition is converted to a desired product by the action of microorganisms. Below are the recommendations for inoculum preparation: 1. Inoculum culture must start from fresh frozen culture. Old or overgrown cultures show high lag times and hence result in high cost of production. 2. Use uniform population of inoculum culture. 3. Use defined media for growth and culture of microorganisms instead of complex media. 4. Maintain the growth/culture conditions constant (i.e., pH, temperature, and aerobicity/ anaerobicity) throughout all the production procedure.

13.2.3 Substrate Substrate for the process of fermentation is the substance that is required for the growth of microorganisms and/or synthesis of product.

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1. The substrate must be defined, simple, economical, and readily (easily and continuously) available. 2. The substrate whose fermentation results in product that is easily extractable should be preferred over the substrate that may produce complex mixture of products and byproducts. 3. High concentrations of the substrate can reduce H2O2 activity and hence may result in reduced/limited growth of the fermenting microorganism. 4. It is imperative to understand the effect of substrate on product formation as the substrate can be inhibitory for the product formation.

13.2.4 Fermentors Fermentors are the enclosed containers where the process of fermentation takes place. Below are the recommendations: 1. Fermentors should be easily sterilizable with higher volumetric capacity. 2. Fermentors should have a high surface area for cooling or removal of heat to maintain growth temperature.

13.2.5 Culture Conditions The culture conditions are extremely important for microorganisms’ growth and product formation. In general, there is a direct relation between conditions required for product formation and microorganisms’ growth; however, in some instances the conditions required for growth and product formation can be completely different. That is why it is important: 1. To understand the condition required for growth and product formation; 2. The growth and product formation conditions should be kept constant. Especially, during the process of fermentation the temperature tends to rise, that is why the efficient cooling of the fermentors is required. 3. Similarly, inappropriate pH can completely inhibit the growth and/or product formation, so pH of the growth media must be maintained. 4. The culture conditions (pH, media concentration, and temperature) of inoculum should be similar to the media in fermentors.

13.2.6 Product Industrially required products are obtained by the conversion of substrate, as primary (produced during yeast growth) or as secondary metabolites of microorganism (usually produced during stationary phase). Below is the recommendation: 1. It is of utmost importance to understand the relationship between biomass growth and product formation.

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13.3 BIOCATALYSTS/ENZYMES In a broad term, industrial biocatalysts are produced biologically which could be crude, pure or formulated enzymes or their mixtures, viable or nonviable whole cells or cell fragments. Biocatalysts are essential components of industrial biotechnology and are produced on commercial scale predominantly by genetically modified microorganisms in large fermentor vessels. Industrial biocatalysts offer several advantages over traditional chemical catalysts in terms of catalytic capacity, high specificity for substrates, solubility in organic solvents, and requirements of mild functional environments (temperature, pH, and pressure). In past decade, biocatalysts were subjected to extensive research with an aim of significant improvement for replacing the most applicable enzyme hydrolases with other enzyme classes. Due to some drawbacks of classical strategies, such as immobilization, additives, or process engineering, molecular biology and genetic engineering techniques came forward to ensure large-scale production, long-term stability at various conditions, cost effectiveness and optimized process controls. In this section, we summarize the major achievements made in the past decade in the area of industrial biocatalyst along with the major challenges towards the future. Rational protein design and directed (molecular) evolution are the two major approaches that helped to achieve significant breakthroughs in the past decade. Rational protein design relies on the advanced understanding of molecular modeling of protein structures. Site-directed mutagenesis facilitates altered protein structures thus conferring an augmentation of catalytic properties of protein along with the altered activity or enantioselectivity. With the aid of random mutagenesis along with a high-throughput screening and/or selection step, the in vitro direct molecular evolution techniques proved to be the most efficient and powerful techniques to improve the properties and functional efficiency of biocatalyst proteins. Through unbiased random mutagenesis, a large mutant library of protein coding genes can be developed in this technique. Screening or selection of enzymes with desired properties is done by the cloning and expression of those mutant variants. Mutant libraries can be generated through asexual (nonrecombining) evolution and sexual (recombining) evolution. A huge development has been done in the field of recombinant biotechnology and genetic engineering in the last 15 years. Due to the high error rate of the most widely used asexual error-prone polymerase chain reaction (epPCR) method, Stratagene’s QuikChange method gets more popularity because of the advantage of whole plasmid amplification. A related technique MEGAWHOP does not require restriction digestion and ligation. Recently, isothermal amplification techniques facilitate rolling circle amplification. Recently, more advanced methods have been developed for example, thio-ITCHY (incremental truncation for the creation of hybrid enzymes), Gene Reassembly method, nonhomologous random recombination, and sequence homologyindependent protein recombination to overcome the drawback and disadvantages of classical recombination methods. A completely different strategy has been discovered recently called circular permutation which does not require the introduction of mutation and resulted in higher catalytic efficiency of fungal originated Lipase B than the wild type.

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Recently, more computation approaches have come forward to predict and compare the quality of the mutant library. Recently, it has been proposed that a protein structure indicator, an aminoacid indicator (complemented by codon diversity coefficient), and a chemical diversity indicator could be a useful option for library evaluation in comparison to mutagenesis assistant program (MAP) that is often unable to predict the quality of the mutant library. Furthermore, a plethora of computational approaches has been reported and these approaches facilitate the simulation of mutational process in combination with rationalizing published results. For example, program for estimating diversity in epPCR libraries, library diversity program, program for libraries comprising a random sampling of equally probable variants (GLUE), eShuffle and eSCRATCHY programs, and SIRCH. Due to the complex chemical structure of protein and limited information on sequence function relationship, rational design or directed evolution sometimes results in unconvincing outcome or surprising variants with aberrant properties. To overcome this limitation, recently, focused directed evolution (combinations of directed evolution and rational design) has been proposed. Increased thermostability and enantioselectivity of biocatalyst along with expanded substrate acceptance has been achieved through one of the focused directed evolution approaches called iterative saturation mutagenesis (ISM). In the ISM method, iteratively continued rounds of individual analysis of aminoacid properties followed by saturation mutagenesis offer the selection of best desired and improved biocatalyst. CASTing (combinatorial active site saturation test) and CASTER program have been developed with added advantages. More recently 3D structure or homology model-independent approaches have been developed to achieve significant functional improvement of biocatalysts; for example, ProSAR is an extension of structure activity relationship. Intriguingly, sequence-independent site-directed chimeragenesis and structure-based combinatorial protein engineering approaches rely on semirational attempts along with recombinant mutagenesis using 3D protein structure. Biological selection aims to screen the desired catalysts from mutagenesis library by discarding the uninterested and irrelevant subjects. This selection can be done in vitro or in vivo. In vitro selection of desired mutants in a large library is possible through phage display techniques by cloning the gene of interest in fusion with virion gene encoding coat protein. Protein libraries are frequently displayed on bacteria and yeast. Display of biocatalysts on bacterial surface facilitates quantitative determination of enzyme catalytic activity at the single cell level and free contact to substrate and thus flow cytometric screening is possible. In vivo selection in bacteria can be performed through screening on agar plate containing increasing concentration of antibiotic. Chromogenic product like X-gal or α-naphthyl acetate and Fast Blue/Fast Red are frequently used for esterase activity detection. Recently, higher throughput and quantitative signal determination can be performed through digital imaging. Direct determination of the enantioselectivity of an enzyme is a key requisite for screening improved biocatalysts. Recently, traditional time-consuming assays have been replaced by solid-phase assays with the aid of modern technology such as robot automation and colony-picking technology. Hydrolytic activity of lipases and esterases can be performed through colorimetric and fluorometric assays which replace the traditional pH-stat assay. Major disadvantages of these hydrolytic assays are the false positive outcome and risk of autohydrolysis in different extreme external conditions. Another common screening assay

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is oxidoreductase assay which relies on the formation or depletion of NAD(P)H, although this assay often negatively influences by the purity of cell lysates. Dehydrogenases activity are traditionally measured by the formation of colored compound by the reaction of different dyes (e.g., nitroblue tetrazolium) with NAD(P)H. Oxidative activity can be determined spectrophotometrically by combining enzymatic H2O2 production to an HRP reaction. Oxygenase activity can be performed through several assays like p-nitrophenoxy analog assay (pNA). Hydroxynitrile lyases catalyze the cleavage of cyanohydrins which can be determined spectrophotometrically. Limitations of this method such as poor water solubility can be overcome by the use of emulsifying agents. With the advent of modern technology, a significant breakthrough has been observed in terms of industrial biocatalysts. Still there is a huge scope to improve the properties of biocatalyst, their screening method, activity and stability, and cost effectiveness.

13.4 INDUSTRIAL PRODUCTION OF BIOCATALYSTS/ENZYMES 13.4.1 Enzyme Definition Enzymes are indeed proteins which are originally produced by all living organisms to catalyze biochemical reactions essential for their life, as well as to respond to the changes in their habitat (Reeta et al., 2010; Buchholz et al., 2005). Enzymes can catalyze energy production reactions inside the cells (intracellular) or in the living environment of the organisms (extracellular).

13.4.2 Enzyme Production Methods The main source for enzyme production in industry is microorganisms via their fermentation pathway either through submerged or solid-state culture. Although submerged fermentation is still the major method used in industrial enzyme production, solid-state fermentation has recently been becoming a potential alternative method due to its economic cost and ecofriendly value. Submerged fermentation can be subdivided into four categories: batch cultivation, fedbatch cultivation, continuous cultivation, and perfusion batch cultivation. These submethods differ from each other mainly depending on how the culture medium is supplied and adjusted, and how the raw product is withdrawn during the fermentation. Batch cultivation: microorganism is cultured in a closed bioreactor in which all fermentation conditions (pH, temperature, nutrients) have been already optimized at the beginning of the process. The products will be only collected at the end of the reaction. Fed-batch cultivation: Similar to batch cultivation but the medium is supplied to the bioreactor during the fermentation process (Tsuneo and Shoichi, 1984). Continuous cultivation: Fresh medium is continuously supplied to the bioreactor, and a part of medium containing products is withdrawn simultaneously (Nielsen et al., 2003). Perfusion batch cultivation: This fermentation process is longer than other methods. In this process, fresh medium is continuously supplied and an equal volume of

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spent medium is removed, whereas cells are still being kept throughout the reaction (Wang et al., 2012). In solid-state fermentation, organisms are grown on solid supports, which are more similar to the natural habitat of most of the microorganisms used for enzyme production. This fermentation method requires higher cost for product harvest and purification process, but the overall cost is much more economical.

13.4.3 Purification of Enzyme Purification is the next step following the production of the raw enzyme products. The main purpose of the purification process is to remove living cells from the culture medium and discard unwanted components in the enzyme solution. These contaminants could be DNA fragments, proteins, ions, or cell debris. The required purity level of a certain enzyme is highly dependent on its applications. For example, enzymes used in molecular biology assays and diagnostics usually require highest levels of purity, since these molecular reactions generally are performed in a very small scale. In contrast, enzymes utilized in industrial production, such as textile, paper, and animal feed, often require a large amount of biocatalyst for industrial scale with a less purity requirement. The purification process, applied for both intracellular and extracellular enzymes, can be divided into several critical steps including separation of host cells from culture medium by ultracentrifugation or microfiltration. The primary purified products will be then concentrated by nanofiltration approach. It is noteworthy that, for intracellular enzymes, the purification process is more complicated since it requires extra separation steps to extract and purify protein solution inside the living organism. Concentrated intracellular and extracellular enzymes will be further purified by chromatography. This step is extremely important for enzymes requiring the highest levels of purity. One of the remaining obstacles of the purification process is the loss of enzyme recovery after each step of purification. Recently, a technique called continuous chromatography has been developed to improve enzyme recovery, and thus introduce a more competitive price (Reeta et al., 2010).

13.4.4 Advances in Enzyme Industry Advances in biotechnology, especially in genetic engineering, have seen a rapid development of industrial production of enzyme over the past few decades. Recombinant gene technology and directed evolution have become the two crucial approaches to improve the quantity of current enzymes, as well as to create new enzymes with innovative characteristics (Headon and Walsh, 1994; Kirk et al., 2002). Recombinant gene technology predominantly aims to boost up the production of a desired enzyme at industrial levels. The principle is to clone a coding sequence of the desired enzyme from a natural strain, which generally expresses at low levels, into an optimized strain used for industrial production, which is genetically modified by removing unnecessary native genes. Protein engineering, on the other hand, focuses not only on optimizing the current enzymes, but also on making next-generation enzymes that can perform effectively in extreme reaction conditions such as low/high pH and high temperature (Reeta et al., 2010;

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Kirk et al., 2002). By modifying the encoding gene for the enzyme by mutagenesis, a new biocatalyst will be generated with characteristics towards to a better performance in a desired enzymatic reaction. It is noted that even a minor genetic modification could give rise to the formation of a novel molecule with unexpected functions. It is therefore very important to predict and test those properties by a combination of initiatives in biochemical and biophysical research, before a new enzyme can be generated at industrial scale.

13.4.5 Application of Enzyme It is undeniable that enzymes have been widely applied in almost all aspects of life to meet human needs, especially in food production (chees, wine, bread, etc.), fine chemicals (vitamins, chiral compounds, aminoacids), textiles, hygiene, and leather. Moreover, the use of enzymes in modern applications in molecular biology, pharmacology, paper and pulp industry, and biofuel has been steadily growing (Reeta et al., 2010; Jaeger et al., 2001). Here we present a brief account of contribution of enzymes in the development and progress of paper and pulp industry and biofuels.

13.5 PAPER AND PULP INDUSTRY Recent development of biotechnology initiates a new era for the pulp and paper industry. Besides the advance of our understanding on the mechanism of action of different enzymes related to wood and wood-component degradation along with the continuous upgrading of our knowledge of biotechnology and genetic engineering, nowadays, bulk scale and cost-effective production of crucial enzymes is possible as mentioned in the previous section. Hence, research and developments of microbial biotechnology boost up the pulp and paper industry by facilitating efficient removal of lignin and proper management of pitch problem and thus ensures quality and efficient use of raw material. Plant genomic research contributes to improvement of forest raw material by reducing lignin content through faster trees growth. Intriguingly, the hurdles of environmental damage during processing wood into pulp and paper products have also been overcome. Extensive research and overall progress in bioscience and biotechnology leads the pulp and paper industry to be exclusively dependent on biotechnology. Still there is a huge scope in the field of fiber engineering, nonrenewable energy sources, recycling of forest materials to reduce greenhouse gas emission. In this section, we summarize the current status of biotechnology along with special focus on the enzymes used in different stages, particularly, for pulping, pitch removal, bleaching, and enzymatic deinking. The traditional method for processing pulp and paper requires heavy chemistry, has environmental impacts, and produces a lot of chemical wastage that is unsuitable for further processing and recycling. Traditionally, pulp and paper industry consumed lot of water, energy, and different chemicals. Traditional methods were always being questioned for the environmental issues, energy, and cost efficiency. Moreover, traditional methods always struggled for the improvement of fiber property, refining or recycling of paper, and proper processing of pulp and paper at different stages. Since a few decades,

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extensive studies on several enzymes, those having potential to degrade complex chemical structure of wood components, contribute to further functional development of costeffective and environmental-friendly oxidation and delignification strategies thus improving pulp and paper processes. Cellulase, one of the most extensively studied enzymes, possesses a high degradative potential against the major component of lignocellulosic wooden materials. Combined action of several cellulose enzymes displayed high efficiency hydrolysis of resistant crystalline cellulose. Modern biotechnology has contributed much commercial grade cellulase production in large scale through genetically modified fungal organisms. These multicomponent fungal origin cellulases demonstrate higher specificity and extensive degradation capacity without compromising the quality and strength of pulp. Nowadays more specific process applications are possible through the development of monocomponent fungal origin cellulases. Although xylanases or other hemicellulases are inefficient in lignin degradation, the xylanases indirectly support pulp bleaching. In kraft pulping, endoxylanase supports degradation of unsubstituted backbone of xylans. Until now, only few xylanases, originated from extremophiles, have been qualified for commercial grade application. Mannanases (another type of hemicellulase) also attack oligomeric substrate with the aid of other accessory enzymes. Many xylanases are now being cloned in bacteria for commercial scale bulk production. Endoglucanases are recently used in the pulp and paper industry which specifically attack xyloglucan, an important component in plant cell walls/wood materials that is normally resistant to microbial degradation. Xyloglucan endotransglycosylase has also been emerged as a potential fiber modifier. One fungal derived xyloglucanases recently showed higher potential against tamarind xyloglucan. Lignins, normally incorporated in the carbohydrate polymer matrix of cellulose/hemicellulose, are resistant to hydrolytic attack of many enzymes. Hence, lignin modifying enzymes are extensively studied in the last three decades and several of them have been qualified for industrial delignification. A range of extracellular peroxidises and oxidases produced from fungi showed increased lignin degrading potential when used in different combinations. Laccases and manganese dependent peroxidases have recently showed promising potential in commercial scale lignin degradation because of the nonspecific nature of catalysis. However, it demands further research to understand more detail about lignin modifying enzymes and their mode of action. In the last two decades, the use of enzymes (in combination) is getting an attractive option in the pulp and paper processing at various stages in the industry. For example, use of cellulase and pectinase (or mixtures) in wood (chip) pretreatment facilitates in energy saving in debarking. Application of xylanases, endogluconase, or mixtures in chemical and mechanical pulping offers technical benefit to achieve TMP refining, reduced energy consumption, and increased flexibility of fibers along with decreased consumption of chemical byproducts. Commercial scale use of xylanase, laccase along with different mediators has been proven useful in bleaching of mechanical and chemical pulp through reducing chemical use along with improving higher final brightness. Use of endonuclease and hemicellulases facilitates upgraded paper making in terms of different technical aspects of drainage and paper smoothness quality improvement. Mixed cellulases and amylases are extensively used for enzymatic deinking of recycled paper through increased release of ink particles. The problem of the accumulation of dissolved and colloidal

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substances in the process, has been solved by using endoglucanases and hemicellulose through reduced water usage and facilitating smoother paper machine running. Esterases and along with other hydrolytic enzymes along with chemical additives have been used for biofilm control because of their potential slime degrading capacity. Still there are a lot of challenges and scope of improvement for the application of enzymes in pulp and paper processing specially in the field of penetration of enzymes into the cambial layer and wood chips, preservation of pulp strength and properties, pulp process control, improving efficiency and biodegradability of different hydrolytic enzymes, and cost efficiency. Fiber based industries recently observe a recent transition of increased utilization of biotechnological options for product designing compared to process improvement. Today pulp and paper industries are enjoying the biotechnology based enzyme aided bleaching, deinking, refining, and pitch control. Still there are challenges for the biotech industry to develop more efficient biocatalysts and scope of developing more efficient waste management and renewable energy sources. Production of functionalized, valueadded fibers using different enzymes initiates a new era to develop novel, highperformance paper and paper making materials. Moreover, an integrated biorefinery concepts targeted to develop methods and applications for both large volume and special use products through recycling of wood components and byproducts in the pulp and paper industry confers a huge possibility for biotechnology in the pulp and paper industry.

13.6 BIOFUELS Biofuels are being regarded as a promising alternative source of energy worldwide because of their relatively low impact on climate change and recent success in America and Europe. Developments are underway to increase the contribution of biofuels in energy production but availability of sufficient biomass resources and sustainability of production are the major concerns. Currently, ethanol and vegetable oil methyl esters (VOME) are mostly used for sparkignition and diesel engines, respectively. Both are produced form plant sources: ethanol from sugars and starches, and VOME from vegetable oils. New potential sources are being considered for future production. Biodiesel is presently produced from vegetable oils and fats through esterification process. Current technologies of biodiesel production are mainly based on homogenous catalysis; however, a new technology called Esterfip-H has been recently introduced which utilizes heterogeneous zinc aluminate catalyst. The use of heterogeneous catalysis holds greater potential for future technologies of biodiesel production. Another way of producing biodiesel from vegetable oils and possibly animal fats is through hydrogenation process which transforms triglycerides of oils and fats. Some major developments have been made recently in Europe by improving technologies using the hydrogenation process. The production of ethanol from sugars and starches requires extraction of sugars in case of sugar-crops and conversion of starches of cereal crops to glucose, followed by fermentation carried out by microorganisms. Ethanol is generally consumed in its derivative

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form called ethyl-tertiary-butyl-ether (ETBE) which is produced after reaction with isobutene. Ethanol has a relatively high octane number whereas ETBE has more gasoline-like properties, hence suitable to be used in gasolines line. Since the production of ethanol from sugars and starches requires the use of large quantities of valuable food resources, therefore new technologies using alternative sources are necessary. Notably, lignocellulosic biomass can prove to be an excellent and abundant source of ethanol production. A major component of lignocellulosic biomass is cellulose present in varying proportions depending on the plant material. Although with some limitations, cellulose can be hydrolyzed to extract glucose which can be subsequently fermented and distilled for the purification of ethanol. An important step in this production is pretreatment of biomass in order to make its fractions available to enzymes. Pretreatment methods include acidic hydrolysis and steam explosion. The enzymatic hydrolysis is a key step in production of ethanol from the biomass: a combination of enzymes is used for breakdown of cellulose and production of glucose. The following methods for the fermentation of glucose are similar to those used in conventional methods of ethanol production from sugars and starches. Thermochemical methods have been developed to produce biofuels from lignocellulose biomass using pyrolysis or gasification. The method involving synthesis gas is often referred to as biomass-to-liquid process. Fast pyrolysis is also used to biooil but cannot be directly used as a fuel because it contains several undesirable components. Further developments are underway to convert such biooils into motor fuels by hydrogenation. Synthesis motor fuels are also produced from biomass. In this process, the biomass is decomposed, gasified, and heated-up to eliminate tar or acidic compounds. Entrainedflow gasifiers are best suited for large-scale production. The gasification of biomass can be directly done in the reactor and solid particles are removed by filtration. The biofuels produced by these methods are very high in quality and low in undesirable byproducts. With the availability of above technologies and methods of biofuels production, the development of large-scale production systems such as “biorefineries” is important. Such facilities will not only enhance production but will also allow the usage of diverse resources of biomass. Also, integration of technologies will allow the development of much more efficient production systems. Sustainable resources of biomass production are a major concern in the development of biofuels industry. Although biofuels promise lower impact on environment, they present a competition for food particularly in production through sugars and starches. Further advancements in production through lignocellulose are necessary because they allow the utilization of more abundant resources of biomass. Therefore in order to take advantage of biomass for the production of biofuels, the sustainability and diversity of biomass are important factors to be kept in consideration. Disclaimer: The authors do not necessarily appreciate and/or agree the use of biomass for the production of any sort of biofuels. Although the use of waste biomass is suggested to avoid competition with food and feed, we suggest the use of waste biomass for soil fertilization to reduce use of chemical fertilizers. In addition, we recommend the investment in the research and development of solar, wind, and hydroenergies to make them cost effective and environmentally safe. Similarly, governmental subsidies on electric cars (especially on electric batteries) and/or investment in research and development of

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batteries for electric cars to improve their rapid recharging, and capacity increment would significantly reduce the use of nonrenewable fuels (diesel and petrol) and hence environmental pollution.

13.7 ENVIRONMENTAL BIOTECHNOLOGY The application of modern biotechnology has been extended to solve and manage the problems of environmental and ecosystem risks which is frequently termed as environmental biotechnology. Environment biotechnology uses living organisms (mostly microorganisms) to develop cleaner and green way for the efficient production of industrial products. It also deals with environmental pollution, bioremediation of polluted materials, and biomonitoring waste treatment process. Thus environmental biotechnology is essential for sustaining an environmental-friendly healthy society. Environmental biotechnology mostly relies on prokaryotic microorganisms for the biodegradation of organic waste materials. But fungi, algae, and protozoa are now widely used as an absorbent of heavy metals as well as for the treatment and detoxification of hazardous industrial byproducts.

13.7.1 Application Environmental biotechnology is widely adopted by modern industrial sector for costefficient green production and to reduce the environmental hazards. Some major processes and applications include biomarkers, bioenergy, agriculture, pulp and paper industry, and bioremediation and biotransformation.

13.8 FOOD PROCESS TECHNOLOGY Food process technology deals with the production processes in the food preparing industry which includes a set of physical, chemical, or microbiological techniques used to transform raw ingredients into final food products. Louis Pasteur’s research was the first early attempt to understand microbial connection in the production of wine, alcohol, vinegar, wine and beer, and the souring of milk. After the Second World War, introduction of new technology greatly advanced food processing, and introduced “new” food ingredients. The use of biotechnology and nanotechnology in food processing is increasing. The food industry today adopts a wide range of technologies to: 1. Extend the shelf life of food by using various preservation techniques. 2. Increase versatility of food flavors, colors, aromas, and textures in food without compromising quality. 3. Improve nutritional quality of a food. 4. Facilitate bulk scale and cost-effective production.

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13.8.1 Advances and Applications Applications include preservation by freeze-drying, food packaging by biodegradable and compostable film, use of nanotechnology for the food preparation surfaces, plastic food storage containers and plastic wrap, dairy industry (rehydratable instantized milk powder is a pioneer innovation in the food and milk industry), and biopolymers and bioremediation (used in the degradation of toxins and industrial wastes, removal of oil spills from the sea/river water, cleaning pollutants from river, lake and soils thus reducing environmental risks).

13.9 BIOREFINERY Biorefinery deals with the conversion process of biomass into fuels, power, heat, and value-added chemicals thus maximizing the value derived from the biomass feedstock. Thus biorefinery has been proven to be cost effective, environmental friendly, and increase profitability in bioindustry. The renewable feedstocks utilized in integrated biorefineries include a wide range of components such as: 1. Energy crops, such as switch grass, miscanthus, willow, and poplar. 2. Agricultural, forest, and industrial residues, such as bagasse, Stover, straws, forest thinning, sawdust, and paper mill waste. 3. Algae and other microorganisms. 4. The development of biorefineries offers novel opportunities and prospect to the industrial application of microorganisms. Depending on the feedstock characteristics and desired products, a microbial strain can be genetically modified to adopt an ideal conversion process.

13.9.1 Applications 1. Fiber, pulps for paper, and other materials like plant lignin can be processed through biorefineries into different useful products like sugars, sweeteners, alcohol, and more. 2. Fostering technical and economic viability of integrating and scaling up a range of innovative technologies. 3. Coproduction of bioethanol and probiotic yeast biomass from agricultural feedstock. Biorefineries concepts start a new era in the industry with a huge future potential to optimize the whole economic-environmental-social agrosystem.

13.10 BIOREACTORS Bioreactors are the vessels/containers which provide biological, biochemical, and biomechanical requirements for the optimal growth of the fermenting microorganisms and/or biochemical reactions on the industrial scale for the synthesis of desired products. Efficient bioreactors are capable of maintaining the desired biological activity by controlling the

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temperature, pH, fluid velocity, shear stress, mass and heat transfer, O2, CO2, and nutrient supply, reaction rate, and cell growth. Bioreactors are used in all domains of large-scale industrial biotechnology where a large scale production is required.

13.11 FUTURE TECHNIQUES 13.11.1 CRISPR/Cas9 CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats while Cas9 (a nuclease protein) represents CRISPR-associated genes. Although CRISPR was discovered back in 2002 and proposed to be responsible for adaptive immunity in microbes (for the precise history and contributions, refer to Lander, 2016), Jinek and colleagues showed experimentally for the first time that their reengineered CRISPR/Cas9 system could find the target DNA with the help of guide RNA (expressed from U6 polymerase III promoter) and cleave it (Jinek et al., 2012). The discovery was followed very next year by the “Multiplex genome engineering using CRISPR/Cas systems” (Cong et al., 2013). The level of utility of CRISPR system can be imagined by the fact as of July 2016 both the publications boast of 5000 citations (2200 and 2800 respectively). Since its discovery, it has been used for the genome editing of Homo sapiens, monkeys, mice, nematodes, Ambystoma mexicanum, Drosophila melanogaster, Danio rerio, Saccharomyces cerevisiae (Guo and Li, 2015; Cong et al., 2013; Wang et al., 2013; Jiang et al., 2013; Friedland et al., 2013; Flowers et al., 2014; Gratz et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013). Although there are still issues for the specificity of guide RNA, still CRISPR/Cas9—provided specific genome/ gene knock down and replacement via a very simple system while ensuring specificity and very low cell toxicity. CRISPR can become a technique of choice for industrial biotechnology not only for functional characterization and/or improvement of the genes/enzymes but also de novo discovery of the enzymes/biocatalysts. CRISPR provides promising perspectives for improving the efficiency of currently used biocatalysts/enzymes. The applications of CRISPR/Cas9 or other nucleases would not be limited to gene disruptions and functional understanding rather it has the potential for editing/altering the germ lines of plants (food crops), animals and even humans. Although editing of human germ lines raises ethical issues, it may prove instrumental for the correction of hereditary diseases.

13.11.2 Microbiome and Personalized Medicine The microorganisms (bacteria, archaea, eukarya, and viruses) living in the gut of humans and animals collectively form the microbiome of that organism. Microbiome is the harmless and/or beneficial bacteria present in the gut. The Human Microbiome Project not only aims for the identification and characterization of the collection of humanassociated microorganisms at various anatomic sites including skin, mouth, nose, colon, and vagina. But also aims to determine the mechanisms involved in influencing human health via intra and inter-individual changes in the microbiome. An emerging field related to industrial biotechnology is the development of personalized medicine by understanding

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the (Virgin and Todd, 2011) individual specific interaction between the drugs and the microbiome of the individual.

13.11.3 Sequencing Complete genomic and transcriptomics maps have been very instrumental in our understanding of gene expression and function and the advancements in sequencing the genomes and transcriptomes have played a pivotal role in this regards. Not yet arrived, but the scientific communities around the world are not far from being able to quantify complete genome or transcriptome in 1000 $ only. Next-generation sequencing has been used for sequencing of diverse range of species including, microbes, model organisms, livestock and human genome which has been useful for characterizing inherited disorders and mutation that may drive cancer. Next-generation sequencing would be very instrumental for industrial biotechnology for the development of personalized medicine, de novo discovery of drug targets and clinical trials of drugs (to analyze the effect of drugs on transcriptome).

13.11.4 Mass Spectrometry Although the history of mass spectrometry dates back to the last quarter of the 20th century and has been instrumental in defining the proteome of many model organisms, the quest for enhanced identification and accurate quantification is still undergoing. For examples, it was only in 2013 when Picotti et al. (2013) published first proteome report on S. cerevisiae covering 97% of predicted proteome. Similarly, it was only in 2014 when Minsik-kim et al. (2014) reported 84% of the total human protein (encoded by 17,294 genes) (Picotti et al., 2013). Although progress has been made at the level of identification and relative quantification of the proteomes, there are still issues of reproducibility of the Mass spec results among different research groups that need to be resolved. In addition, absolute quantification through labeled peptides of the proteins is not economical: it costs are comparable to that of antibodies. Instead of all the above mentioned shortcomings, Mass spectrometry is still the technique of choice for drug discovery and environmental pollution detection (industrial biotechnology).

References Buchholz, K., Kasche, V., Bornscheuer, U.T., 2005. Introduction to enzyme technology. Biocatalysts and Enzyme Technology. Wiley-Blackwell, Weinheim3-527-30497-5. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., et al., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339 (6121), 819 823. Available from: https://doi.org/10.1126/science.1231143. DiCarlo, J.E., Norville, J.E., Mali, P., Rios, X., Aach, J., Church, G.M., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41 (7), 4336 4343. Available from: https://doi.org/ 10.1093/nar/gkt135. Flowers, G.P., Timberlake, A.T., McLean, K.C., Monaghan, J.R., Crews, C.M., 2014. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development (Cambridge, England) 141 (10), 2165 2171. Available from: https://doi.org/10.1242/dev.105072.

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Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaiacovo, M.P., Church, G.M., Calarco, J.A., 2013. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10 (8), 741 743. Available from: https://doi. org/10.1038/nmeth.2532. Gratz, S.J., Cummings, A.M., Nguyen, J.N., Hamm, D.C., Donohue, L.K., Harrison, M.M., et al., 2013. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194 (4), 1029 1035. Available from: https://doi.org/10.1534/genetics.113.152710. Guo, X., Li, X.J., 2015. Targeted genome editing in primate embryos. Cell Res. 25 (7), 767 768. Available from: https://doi.org/10.1038/cr.2015.64. Headon, D.R., Walsh, G., 1994. The industrial production of enzymes. Biotechnol. Adv. 12 (4), 635 646. Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., et al., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31 (3), 227 229. Available from: https://doi.org/ 10.1038/nbt.2501. Jaeger, K.E., Eggert, T., Eipper, A., Reetz, M.T., 2001. Directed evolution and the creation of enantioselective biocatalysts. Appl. Microbiol. Biotechnol. 55 (5), 519 530. Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B., Weeks, D.P., 2013. Demonstration of CRISPR/Cas9/sgRNAmediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 41 (20), e188. Available from: https://doi.org/10.1093/nar/gkt780. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E., 2012. A programmable dual-RNAguided DNA endonuclease in adaptive bacterial immunity. Science 337 (6096), 816 821. Available from: https://doi.org/10.1126/science.1225829. Kim, M.-S., Pinto, S.M., Getnet, D., Nirujogi, R.S., Manda, S.S., Chaerkady, R., et al., 2014. A draft map of the human proteome. Nature 509 (7502), 575 581. Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13 (4), 345 351. Lander, E.S., 2016. The heroes of CRISPR. Cell 164 (1 2), 18 28. Available from: https://doi.org/10.1016/j. cell.2015.12.041. Nielsen, J., Villadsen, J., Lide´n, G., 2003. Bioreaction Engineering Principles. Springer978-1-4613-5230-3. Picotti, P., Clement-Ziza, M., Lam, H., Campbell, D.S., Schmidt, A., Deutsch, E.W., et al., 2013. A complete massspectrometric map of the yeast proteome applied to quantitative trait analysis. Nature 494 (7436), 266 270. Available from: https://doi.org/10.1038/nature11835. Reeta, R.S., Anil, K.P., Ashok, P., 2010. The industrial production of enzymes. Industrial Biotechnology Sustainable Growth and Economic Success. John Wiley & Sons978-3-527-31442-3. Tsuneo Y., Shoichi S. (1984) Fed-batch techniques in microbial processes. Adv Biochem Eng/Biotechnol 30:147 194. Virgin, H.W., Todd, J.A., 2011. Metagenomics and personalized medicine. Cell 147 (1), 44 56. Available from: https://doi.org/10.1016/j.cell.2011.09.009. Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., et al., 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153 (4), 910 918. Available from: https://doi.org/10.1016/j.cell.2013.04.025. Wang, L., Hu, H., Yang, J., Wang, F., Kaisermayer, C., Zhou, P., 2012. High yield of human monoclonal antibody produced by stably transfected Drosophila schneider 2 cells in perfusion culture using wave bioreactor. Mol. Biotechnol. 52 (2), 170 179. Available from: https://doi.org/10.1007/s12033-011-9484-5.

Further Reading Parenteau, J., Durand, M., Morin, G., Gagnon, J., Lucier, J.F., Wellinger, R.J., et al., 2011. Introns within ribosomal protein genes regulate the production and function of yeast ribosomes. Cell 147 (2), 320 331. Petibon, C., Parenteau, J., Catala, M., Elela, S.A., 2016. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes. Nucleic Acids Res. 44 (8), 3878 3891. Available from: https://doi.org/10.1093/nar/gkw140. Yu, C.H., Dang, Y., Zhou, Z., Wu, C., Zhao, F., Sachs, M.S., et al., 2015. Codon usage influences the local rate of translation elongation to regulate co-translational protein folding. Mol. Cell 59 (5), 744 754. Available from: https://doi.org/10.1016/j.molcel.2015.07.018.

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14 Omics Approaches in Industrial Biotechnology and Bioprocess Engineering Mustafeez M. Babar1, Hasan Afzaal2, Venkata R. Pothineni3, Najam-us-Sahar S. Zaidi4, Zeeshan Ali1, Muhammad A. Zahid1 and Alvina Gul4 1

Shifa Tameer-e-Millat University, Islamabad, Pakistan 2Riphah International University, Islamabad, Pakistan 3Stanford University, Palo Alto, CA, United States 4National University of Sciences and Technology (NUST), Islamabad, Pakistan

14.1 INTRODUCTION The ground-breaking developments in the field of omics have helped in deciphering the physiological and biochemical makeup of various organisms. In this respect the commercially viable organisms, contributing to various industrial processes, are no exception. The efficient whole genome-sequencing strategies have led to the complete understanding of the functional annotation of various gene products (Golden and Handfield, 2014). The omics tools, both dry and wet-lab based, have helped the bioprocess engineers to gather sufficient information related to the genes, their functions, the genomic structure, the biological/metabolic pathways and their evolutionary history (Belsky et al., 2013). Additionally, in order to fully understand the microbial biological processes, they have to be quantified by functional characterization studies of the genes, coding regions, proteins, and the metabolic products. The advancements in these fields have led to the development of a new set of technical expertise which are referred to as genomics, transcriptomics, proteomics, metabolomics, and interactomics (Low et al., 2013). All these approaches are highly efficient and follow a high-throughput mechanism. Moreover, all the biological

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00014-0

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players are considered while studying any biological process using these approaches. Hence, there is no stand-alone data point but each player is affected by multiple entities of similar or different kinds. Moreover, these experimental approaches, coupled with various computational and statistical tools, help in the generation of large amount of highly representative data (Berger et al., 2013). Based on the rationale, not a single high-throughput omics approach can provide a complete picture of the metabolic process. Therefore, multiple approaches have to be applied and manipulated to achieve the industrial targets. The different omics techniques that are exploited for industrial bioprocess development have been summarized in Fig. 14.1. The employment of these omics approaches is now considered the cornerstone of any industrially viable procedure. The industrial processes rely on the production and availability of highly efficient microbial strains for the production of maximum amount of a

Next-generation sequencing

Mutagenesis

Reverse genetics

Cell line modifications

Synthetic biology and bioinformatics tools

FIGURE 14.1 Various omics tools developed for understanding and manipulating the microbial biome for efficient processing.

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bioproducts (Keasling, 2012). The omics approaches can not only help in the complete understanding of a biological process but also contributes to the generation of such microbial entities. In general, the fields of genomics and transcriptomics help in understanding the genetic (DNA and RNA) elements of a metabolic process. Similarly, proteomics provide information about the protein makeup of a biological process. Metabolomics is the study of the metabolites and how they are produced and regulated. Interactomics deals with the study of the interaction of all these players. Many recent developments have helped in improving the overall outcomes of these processes. However, strategies still need to be employed for maneuvering these techniques and gaining a complete control of various biological and metabolic procedures (Nielsen et al., 2014). The experimental procedures in conjunction with the computational and statistical models need to be optimized for improving the overall efficiency of the omics approaches for the industry. The current chapter provides a review of the importance of omics tools in the industrial biotechnology processes. The experimental platforms employed in omics technology have then been summarized. Towards the end of the chapter, various issues hindering the adaptability of these tools by many of the industrial setups have been presented.

14.2 THE OMICS REVOLUTION: IMPLICATIONS FOR INDUSTRIAL BIOTECHNOLOGY The discovery of the molecular players involved in a biological process has led to a deeper understanding of the biochemical and metabolic process. This has, ultimately, resulted in the development of improved means to modify these organisms for achieving maximum biomedical benefits, directly or indirectly. The recent developments in medical, pharmaceutical, chemical, nutrition, and agricultural industries all owe their progress to the omics-based approaches. In general, the generation of small amounts of biopharmaceuticals is cost effectively attained in the industry (Harris et al., 2015). However, for the production of larger volumes, economically feasible alternatives have to be designed, optimized, and developed. In order to achieve these, over the past several decades, microbes that have been produced have a greater capability to produce better products, qualitatively and quantitatively (Kim et al., 2015). Efficient metabolic engineering techniques have, recently, been developed to carry out these processes (Snyder, 2015). A number of genetic elements, coding as well as regulatory regions, have been studied and manipulated to achieve maximum commercial benefits (Erickson and Winters, 2012). Fig. 14.2 summarizes various omics tools that are exploited for improvement in microbe-based industrial processes. Using a combination of dry and wet-lab experimentation, massive data have been generated over the past years that have facilitated the design and development process of new metabolic pathways. The omics approaches have, hence, been successful in providing a knowledge base for the genetic manipulation processes. Genomics, for instance, has helped in deciphering the genetic code of the organisms (Bush and Moore, 2012; Barrick and Lenski, 2013). Similarly, the transcriptomics approaches have led to the identification

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Transcriptomics

Genomics Study and exploitation of genetic and genomic data

RNA-based approaches

Phenotype

Proteomics

Metabolomics

Expression control and product design

Product synthesis and metabolic control

FIGURE 14.2

The use of omics tools for the study of biological processes of microbes.

of the key region necessary for coding of a protein (McGettigan, 2013). The structural and functional characterization of the protein product is studied in proteomics (Masselon, 2014). The metabolic process resulting in the production of regulatory players in the form of metabolites or signaling molecules is investigated in metabolomics (Ballereau et al., 2013; Tachibana, 2014). The interaction of two or more of these fields is dealt with by the interactomics approach (Finkelstein, 2015; Feng et al., 2015). The progress in these various fields of omics has provided a basis for the development of a thorough knowledge base of biological processes. Though the huge amount of data generated by these individual fields was earlier considered separately, a number of research studies have now integrated the information obtained from various tools and employed it for developing industrially feasible organisms (Meng et al., 2014). In addition to these high-throughput experimental procedures, the innovations in the field of computational modeling and simulation has helped to decipher the structural and functional basis of biological processes (Segata et al., 2013; Kholodenko et al., 2012). This data has, therefore, helped in understanding, predicting, and designing the cellular pathways of microbes under various biological and environmental perturbations (Nemergut et al., 2013). The system- and, even, organism-level manipulation of microbes can be attained by combining the high-throughput wet-lab experimental procedures with the statistical and computational approaches. Hence, the genomic, transcriptomic, proteomic, and metabolic data points can be utilized for the development of an adequate biological and metabolic process. This would ultimately facilitate the generation of newer commercially feasible strains that can efficiently produce the required biopharmaceutical agent.

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14.3 OMICS TOOLS IN INDUSTRIAL BIOTECHNOLOGY AND BIOPROCESS ENGINEERING 14.3.1 Next-Generation Sequencing Among the many biopharmaceuticals, currently being produced in the industry, a number of products rely on the determination of the exact sequence of the target organism. Vaccines, DNA/RNA-based inhibitors, and enzymes are a few examples of these products. Apart from knowing the basic genoproteomic makeup of the organisms, the technique can help in optimizing the conditions, owing to genetic composition of the organisms. In light of the immense importance of knowing the exact sequence of the DNA molecules and the mutations observed in them, newer techniques and methods must be employed for ultrafast sequencing of the samples. Next-generation sequencing (NGS), commonly referred to as high-throughput sequencing, refers to the set of techniques employed for reading millions of nucleotides in a very rapid fashion (Marx, 2013). It involves the sequencing of huge data using specific fragment libraries which possess an improved efficiency in comparison to the conventional-sequencing techniques. A number of genome-sequencing platforms have been developed for the purpose including Roche 454 sequencer, Illumina sequencer, and SOLiD sequencer. Roche introduced the first NGS system known as Roche 454 FLX sequencer (Mardis, 2008). The instrument works on the principle of pyrosequencing—a polymerase-based approach that produces luminescence owing to the cleavage of oxyluciferin by the luciferase enzyme (Metzker, 2010). The methodology employs reading the samples using the sample sequences hybridized to agarose beads. Using the principles of in vitro amplification and hybridization, this sequencing method can read hundreds of thousands of nucleotides, hybridized to up to 454 beads, within a matter of hours. Using specialized detectors, the light emitted after the incorporation of a particular nucleotide during the sequencing process is quantified and it helps in the correlation to the specific nucleotide. This instrument can help in an average sequence read of around 250 bp per sample, i.e., around 100 million nucleotides in a 7-hour operation of the machine. Illumina, another highly efficient system, sequences by de novo synthesis and reads the nucleotide incorporation using a luminescence reader. The incorporation of nucleotides at the 30 end is followed by the computerized imaging of the incorporated nucleotides. The process generally continues for around 4 days and millions of sequences can be read during one read. SOLiD or Sequencing by Oligo Ligation and Detection is another tool employed for the NGS process (Morozova and Marra, 2008). The sequencing methodology employs the use of ligase enzyme to read up to 34 Gb of sequence data within 45 days. The SOLiD system processes a large number of samples simultaneously. However, an added advantage of the method is that there is an additional 20 -base encoding which double checks the incorporation of the hybridized nucleotide. In case any of the NGS method is employed, it can be used to a number of processes in the industrial biotechnology field. It can not only be employed for the mutation discovery process but also for the metagenomics characterization in the R&D section of the industry. The coding and noncoding regions can also be identified by initially sequencing the samples followed by the application of the bioinformatics techniques. Additionally, the proteinprotein interactions and the proteinligand interactions can also be observed.

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Moreover, the confirmation of site-directed mutagenesis carried out in many procedures for improving the overall outcomes of any industrial process can also be verified, optimized, and validated using the same technique. The NGS-based approach is quite a new technique but its incorporation in the main stream industrial biotechnology can definitely help in improving the quality of the product and the yield of the bioprocess.

14.3.2 Mutagenesis The mutation of the gene sequences can result in the generation of new proteins. Moreover, mutation in the regulatory regions can cause a significant alteration in the kinetics of the gene expression. These mutations can either be beneficial or harmful for any biological process. Industrial biotechnology, based mainly on the exploitation of biological processes for extracting beneficial products, relies highly on the use of intelligently designed biosystems. Introducing specific mutations or deleting the unwanted regions can help in significantly improving the outcomes of any bioprocess. Site-directed mutagenesis (SDM) is one such approach employed for the introduction of mutations within the genetic sequence. A number of methods can be employed for this purpose (Cobb et al., 2013). Performing polymerase chain reaction (PCR) with modified primers can help in introducing new sequences at the terminal ends of an amplicon (Carey et al., 2013). Though commonly employed, this method can only effectively introduce a limited number of nucleotides (,100 bp) in the target. Another technique employed for the purpose is the primer-extension mechanism which uses nested primers to introduce a mutation in the target region. Using a dual-step PCR reaction, the first set of primers anneal containing a small mismatch or the “desired mutation” in the sequence. In the second step, the newly introduced sequences anneal and hybridize to form a new mutated product. The method is more efficient and can introduce/delete around 100 nucleotides with higher efficiency. Inverse PCR is another approach used for mutating plasmids. Much similar to the previously described methods, this technique uses a number of primers for the amplification of the plasmid. The primer-binding regions can be exploited to contain the mutagenic regions. In order to introduce new sequence, flanking regions can be associated with the primers. In comparison, for deleting any region, a space can be left between the two primers. This method is much similar to the primer-extension method and can easily carry out indels (insertiondeletion mutations) of up to 100 bp (Ratan et al., 2015; Ajawatanawong and Baldauf, 2013). In addition to the SDM with their capability to mutate small regions, larger regions of the genome can also be modified using various techniques including the vector-based technologies and restriction enzymes. Adeno-associated vectors are of prime importance in this regard. These viral systems can aid the protein engineering process and be helpful in the generation of useful biocatalysts for industrial applications. These viral vectors can successfully transfer the genomic material to various tissues and tissue systems. The required mutations are then introduced into the biological system aiding the regulation of the enzyme process or the bioproduct. In addition, among the newer more specific techniques are the nuclease-based mutagenic process. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are the restriction enzymes that can be developed

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to cleave specific regions of the genetic material (Gaj et al., 2013). ZFNs utilize the zinc finger DNA-binding domain for identification of the region while the cleavage domain acts to produce the sequence specific cuts (Ochiai and Yamamoto, 2015). Similarly, TALENs are made by a combination of a DNA-binding domain and a DNA-cleavage domain (Pu et al., 2015). They bind specifically to the desired regions and introduce double-stranded breaks. Both the tools are highly effective in genome editing. One of the recent, yet highly accepted, technique is the CRISPR/Cas system. Clustered regularly interspaced short palindromic repeats or commonly referred to as CRISP, is also a DNA-editing tool (Ran et al., 2013a; Hsu et al., 2014). It utilizes the RNA transcript for the recognition of specific regions and then cleaves the DNA to produce the double-stranded breaks. Hence, the availability of newer tools and techniques for introducing specific mutations within the cells and organisms to tailor-make specific gene products at a desirable pace is achievable. The site directed, as well as in certain cases the random mutagenesis, has led to the development of a number of platforms that have positively contributed to the development and progress in the field of white biotechnology.

14.3.3 Reverse Genetics The era of genomics and proteomics has led to a better understanding of the molecular basis of organisms and as to how can these systems can be manipulated for exploiting maximum monetary and biological benefit. The generally adopted practice for the manipulation of the genetic machinery follows the classical screening of following a phenotype presentation to its genotype origin. Once established, the genetic elements are manipulated using various molecular biology techniques. This scheme has helped in establishing the functions of many genes and has contributed to the understanding of many essential biological processes. The contemporary method starts with the study of the gene sequence and relates it to the ultimate gene product and function (Civelek and Lusis, 2014). This technique helps in specifically modifying a gene, its expression level, and then characterizing the effect of the changes produced as a result of the introduced mutations. The approach is, hence, a cornerstone to the development of any modern industrial process involving protein engineering. Both classical and reverse genetics combine to achieve the adequate bioprocess goals. They generally exploit common tools and techniques for the purpose. For instance, among the various tools employed for understanding the function of an introduced mutation, SDMs are introduced. Using this technique, the genes or the regulatory regions of a gene are modified, resulting in the generation of new proteins. Similarly, the mutation can help in producing null alleles, the genes that do not play any biological function (Segal and Meckler, 2013). Gene knockout strategies are also employed in certain lower multicellular organisms that can help in studying the biological function of these genes. Similarly, in certain organisms, gene sequences are also “knocked in” to introduce conditional alleles in the biological system (Sabri et al., 2013). These conditional alleles are mainly helpful in the selection/screening procedures. Another technique referred to as Targeting Induced Local Lesions in Genomes (TILLING) is another efficient method of mutagenesis using chemicals as the essential mutagens (Choudhary and Swamy, 2016). Among these chemical agents,

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ethyl methanesulfonate is a very potent agent for identifying mutations in a specific gene. Gene silencing is another method employed in reverse genetics. RNA interference or RNAi identifies specific regions of the transcript, binds to and generates a knockout effect without producing any change in the original DNA of interest (Wilson and Doudna, 2013). This technique contributes to the silencing of the gene expression and identify any potential mutated phenotype. The process relies on the cellular biological systems like the Dicer protein or the RNA-induced silencing complex (RISC) (Hutva´gner and Zamore, 2002). A simpler alternative utilizes the Morpholino technology (Schulte-Merker and Stainier, 2014; Subbotina et al., 2016). This technique uses specific antisense RNA molecules that bind to the target region without requiring any cellular component for their activation. Both the techniques have been proved to be effective during in vitro and in vivo experimentation. Another widely adopted method of gene manipulation is based on the overexpression of proteins. The generation of the transgenic organisms expressing higher levels of proteins has especially been very effective in industrial biotechnology and bioprocess engineering. This is generally carried out by modifying the regulatory domain or by introducing a mutation in specific amino acid residues through ubiquitation, phosphorylation, or related mechanisms. An optimized process on this pattern can help in gaining desirable quantities of the desired gene of interest. The reverse genetics approach, owing its basis to the omics revolution, can hence help in the optimization and selection of appropriate biological agent in the industry. Many enzymes and proteins can be engineered using these sophisticated, yet easily adaptable, tools of reverse genetics. Similarly, the introduction of mutations in the regulatory regions can facilitate in controlling the rate and the output of the process.

14.3.4 Cell Line Development The omics era has revolutionized industrial biotechnology. Progress in the field of genomics, transcriptomics, proteomics, and metabolomics has all led to optimizing and improving the upstream and downstream processes. Among the various industrial biotechnology products, the production of enzymes and biopharmaceuticals relies on the selection of adequate production platforms. Cell lines, especially of mammalian origin, are widely used for the production of these products. These cell lines, both in their natural forms and in the variant forms, facilitate the industrial processes. Chinese Hamster Ovary or CHO cells are generally the most reliable source of protein expression (Farrell et al., 2014). They, like a number of other mammalian cell lines, provide the correct protein folding and posttranslational modifications (Young, 2013). Hence, availability of the adequate cell system is essential for the production of high-quality products. Omics approaches are also facilitating in producing efficient production platforms. Cell line processing, both cell line development and cell engineering to maximize the output, has rapidly developed over the past several decades. Earlier, for instance, growth supplementation with fetal bovine serum (FBS) was considered to be an essential component of any cell culturing procedure. Owing to the risks of infection and high costs, the cell cultures have now been modified to grow even in the absence of FBS. Using the genomics

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approaches, cell lines have been developed that are highly resistant to signals that inhibit their expression capacity. Cyclins, protooncogenes and certain growth factor genes have been introduced into the cells to achieve superior production capability. The modifications in the production capacity of the cell lines is sometimes associated with a decrease in the quality of the product. Recombinant cell lines are, hence, being designed and developed that have a highly regulated posttranslational protein modification assembly. Recombinant cell lines are the modified forms of the cell lines. Each cell acts as an individual clone. Selecting the appropriate clone is the most important part of the cell line development process. Cells show clonal variability and, hence, the most appropriate clone should be selected for performing a particular bioprocess. A cell, for instance, might be efficient in producing certain specific form of a protein under specific process conditions. Therefore depending upon the need of the end-user, the production platform is characterized, developed, and utilized (Vaxelaire et al., 2015). Cell lines are developed in a manner that they respond adequately to certain controlling factors such as temperature, gaseous content, and/or certain chemical agents (Lai et al., 2013). These factors are used as inducing/controlling agents for the expression process. Many industries develop their own platforms in order to select the most desirable features. In case, a recombinant cell line is being developed, the gene of interest is first expressed in a transient expression system (Kim et al., 2012). This step helps in identifying the potential of that gene to be expressed under varying conditions. Once it has been proven that the gene can successfully incorporate into a cell and is expressed, the gene is introduced into cells that have been selected separately (Ram et al., 2016). The gene then gets integrated into the host-cell genome at a random location. Specific clones of cells with desirable characteristics are then selected using suitable screening techniques (Young, 2013). Significant improvements have been made in mammalian cell culture technology after the advent of omics approaches. The genomic tools have been employed to understand the genome of the cell lines, the key players involved in the expression of proteins and their regulation through molecular techniques. Similarly, the proteomics techniques have been employed to study the structure of proteins, their folding process, and the posttranslational modifications. Furthermore, the tools of metabolomics and fluxomics can help in deciphering the metabolic machinery of the cell systems.

14.3.5 Synthetic Biology The field of synthetic biology incorporates a number of omics approaches in order to improve the quality of life. In industrial biotechnology, these tools are employed to develop adequate means for the production of biological products. The main focus of many of the industrial biotechnology processes is the generation of products from renewable resources using microbes. The processes should, hence, be highly efficient in terms of output and cost. Hence, “hyper-productive” microbial strains should be developed (Costanza et al., 2012). Although classical genetics helped in achieving these goals through laborious mutation-screening repetitive cycles, the advent of metabolic engineering, based on systems biology tools, has yet to optimize the production of reliable, highly efficient

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microbial strains. The process makes use of the manipulation of the chemical and metabolic networks within the biological system. Synthetic biology and metabolic engineering mathematically model the biological networks and identify the ratios of reactants leading to the production of a particular product and determining the points of the pathways that might be exploited for increasing the yield of a process (Wang and Wu, 2015). Afterwards, using the genetic engineering techniques, the production can be maximized for the industrial benefits. These tools are generally aimed at one or more of the four principle ways: overexpression of a “favorable” gene, blocking of a “competing” gene, introducing a foreign “highly efficient” gene or the “enzyme” engineering. By adapting any of these mechanisms, the metabolic networks can be controlled and manipulated for the benefit of industrial bioprocesses. The basic aim of synthetic biology is, hence, to optimize and standardize the biological system providing a greater controlling capacity. Information systems coupled with the progress in genomics have helped in deciphering the mystery of biological processes. Computer-aided design of genes and proteins is now easily achievable. De novo gene design and synthesis is now being carried out throughout the world. These genes can then be introduced into the target cells to get integrated within the genome for the generation of the final bioproduct. Conversely, in order to edit the genome, the coding, or the regulatory part, a number of techniques are available. CRISPR/Cas system is one such remarkable innovation that has shortened down the gene editing time from weeks to days (Ran et al., 2013b). Similarly, the recent developments in NGS have led to providing the synthetic biologists a broad work space for employing the tools for devising efficient means to increase the quality and quantity of the product. Moreover, efficient sequencing methods aid the optimization and screening of the designed biological organisms. Apart from the genetic modification, proteins can also be modified in a manner to improve their efficiency. Intracellularly produced fusion proteins with higher stability are designed using the same procedures. Similarly, the conjugated proteins composed of a combination of proteins and/or peptides are also helpful in improving the activity and the efficiency of the system. Controlling the posttranslational modifications is also achievable and is being utilized for improving the outcomes of many industrial processes. Similarly, introduction of secretory signals helps in getting the expressed protein secreted from the cell, hence, making the cell a renewable source of production of bioproducts. Moreover, after the design of a protein molecule, it can computationally modeled to study various structural features. The proteins can then be modified or synthesized. These modeling techniques can also be employed for studying the interaction of various biological players and or small ligands/inhibitors. The molecular simulation techniques can, hence, be used for studying important biomolecular interactions during the process of replication, transcription, translation, and regulation. Similarly, the fluxomics approaches can be helpful in studying the enzyme kinetics and the rate of metabolic processes by physically measuring the output and rate of a reaction to predict the biological mechanisms. These tools of systems biology can hence facilitate the intelligent design of the bioprocess and the product. They can aid the production process as well as the up- and downstream bioprocessing. The rich interdisciplinary methodology of systems biology facilitates the production of ideal biopharmaceuticals by aiding their conception through syntheses to their ultimate usage.

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14.3.6 Data Depository and Bioinformatics Tools The progress in industrial biotechnology and metabolic engineering can be attributed to the availability of abundant data for the genoproteomic analysis and manipulation. Full length genome-sequencing projects have led to the provision of data about the genetic basis of biological processes. The information provided not only helps in designing and optimizing appropriate metabolic product but also provides insight about various other biological and biochemical processes. Based on these approaches, the metabolic models can be constructed for designing and devising appropriate products and processes. Similarly, the bioinformatics tools of genomic and proteomic analysis also help in the metabolic engineering process. The integration of wet-lab experiments with the computational methods has now become a cornerstone of industrial processes and basic and clinical research. Among the data repositories, the most important source of genomic data is the National Institute of Health’s National Center for Biotechnology Information (NCBI). It has the richest databank of gene sequences from around the world (Benson et al., 1999). Most of the other bioinformatics tools utilize the data obtained from NCBI databank. Similarly, the European Molecular Biology Laboratory has a separate unit known as European Bioinformatics Institute that has a large number of bioinformatics tools ranging from data repository to structure analysis to ontological information. Additionally, there are a large number of tools that address only certain specific needs. For instance, the GenMAPP is a tool used to interpret genetic data in terms of metabolic pathways and biological processes (Dahlquist et al., 2002). It is generally employed for interpreting the microarray data. The genes can be mapped with respect to biological pathways in order to interpret the upregulated and downregulated elements. Hence, the role of various genes within the biological process can be determined and manipulated for biological regulation. Similarly, a large number of tools help in the determination of gene-function prediction using codon sequence, amino acid sequence, protein structure (secondary and/or tertiary), interacting molecules, and expression profiles of the genes. Among these tools PSI-BLAST, STRING, SMART, PROSITE, Pfam, and PSORT are worth-mentioning. PSI-BLAST or positionspecific iterative basic local alignment search tool is based upon the homology search method and provides more specific information about the protein functionalization (Altschul et al., 1997). STRING is another tool employed for predicting the functional relationship between genes and proteins (Jensen et al., 2009). It relies on interpreting the genomic signatures. In STRING, in case a query gene is located near to a particular gene with identified function in a number of genomes, the results would indicate that the query gene performs a similar function to that of the earlier identified gene. Hence, this tool is based upon the phylogenetic analysis of the gene sequences. Similarly, SMART and PROSITE are the databases that rely on the sequence homology models and provide a functional attribute to the sequences (Letunic et al., 2012; Hofmann et al., 1999). Pfam is another database that is based upon the Hidden Markov Models (HMM) and provide statistical representations with regards to multiple sequence alignments (Eddy, 1996). The sequence information can also be used for analysis and prediction of the subcellular localization of genes. The server makes use of the protein features like the signaling sequences/regions for predicting these features. Apart from these bioinformatics tools available for genomic and

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proteomic studies in metabolic engineering, a number of computational platforms are available for determining the metabolic products and the rate of reactions. In brief, the introduction of multidisciplinary scientific process has contributed to the development of a number of biological and industrial processes. The field of genomics, for instance, has helped in understanding the genetic basis of metabolic processes. This information has, in turn, been used for optimizing and developing reliable solutions in industrial biotechnology. Similarly, the field of proteomics has helped in understanding the protein structure and function. It has also provided a basis for protein engineering. The fields of metabolomics and fluxomics have been aiding the understanding of the bioprocess outcomes and their rate. Hence, the tools of bioinformatics have provided significant contribution to all the omics approaches for understanding and manipulating life processes.

14.4 COMBINED OMICS APPROACHES The multifaceted omics approaches have helped developing highly effective industrial bioprocesses. All the sections of the “biomics” including genomics, transcriptomics, proteomics, metabolomics, and fluxomics have aided the industrial biotechnology in a manner that has made it the basis of the production of not only biopharmaceuticals but also the source of development of many other products as well (Demain and Vaishnav, 2009). The study of the genome and the whole genome sequences have eased the study of complex cellular and metabolic networks. This information has aided the optimization of the development of microbial strains of industrial importance. Moreover, these interaction patterns are not only limited to the genes but, using the data repositories and bioinformatics tools, are extended to other nongene players as well. These include the noncoding regions of the genomes, the proteins, and the metabolites involved in the biochemical processes. The data obtained from these omics approaches have, hence, helped in understanding the cellular mechanisms and aided the metabolic and protein engineering steps during the microbial strain development. The stain development process has, for instance, been fully supported by the whole genome-sequencing projects. Over the past several years, a number of groups have reported the reconstruction of the whole genome using the omics technology. The commercially and clinically viable organisms with major changes in their genomes include Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae (Huang et al., 2012; van Dijl and Hecker, 2013; Stovicek et al., 2015). Fig. 14.3 summarizes the nature of interaction between various omics techniques for process and product development. Apart from the introduction of major changes in the microbial genome for optimizing the bioprocess and product development, a number of tools are exploited for studying the microbial capability to produce these metabolites. Among the many techniques, the flux analysis by means of computational biology methods and then their validation by in vitro and in vivo analysis has become one of the cornerstones for the industrial biotechnology processes. Metabolic flux analysis, commonly referred to as MFA, is one such experimental technique which is employed for studying the generation and consumption of the metabolic products of a bioprocess (Dalman et al., 2015). The procedure involves the use of isotopic forms of carbon (13C) in association with various spectroscopic or

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Transcriptome:

Proteome:

High-throughput data: mRNA profile

High-throughput data: protein profile

Equipment: microarray

Equipment: 2D Gel, MS/MS

C D

E

B

A

Fluxlome: High-throughput: metabolite profile Equipment: GC–MS/ NMR

F

Genome:

Metabolome:

High-throughput data: DNA sequence data Equipment: DNA sequencer

High-throughput: metabolite profile Equipment: GC–MS/NMR

FIGURE 14.3 The interdisciplinary approaches of omics for deciphering the metabolic processes.

chromatographic techniques (like NMR and GC-MS) (Crown and Antoniewicz, 2013). Using certain computer generated algorithms, the experimental procedure can be helpful in determining both the intracellular and extracellular flux of the substrate and the metabolite. Similarly, a number of studies have been presented in which a combined, integrated approach has been employed for studying the relationship between various genetic and protein players that influence the metabolic processes. These approaches have been employed for studying the transcriptomic and metabolic profiles of various organisms by using DNA, RNA, or proteins as the basic data points. These approaches have also been employed for analyzing the bioprocesses of biological and industrial importance in various bacteria, fungi, and plants. Various pathways, for instance tricarboxylic acid (TCA) cycle, glycolysis, lysine production process, and NADH regeneration, are used as markers for the study of these processes. These integrative approaches have helped in deciphering important regulatory mechanisms. Incorporation of various genes for increasing the yield of the bioprocesses followed by their metabolic profiling has helped in understanding the role of various genes and gene products in the metabolic processing. Similarly, the effects of the environmental factors can also be studied in conjunction with the biological and metabolic processes. A number of studies have, for instance, compared the role of aerobic and anaerobic

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TABLE 14.1 Applications of Omics Techniques Employed for Human Betterment Omics Techniques

Applications

Genomics

Diagnosis of genetic disorders Predicting the predisposition to genetic diseases Rational drug development Drug repurposing for the “genomic few” Gene therapy

Transcriptomics

Understanding of gene regulatory networks Development of animal models including genetically engineered mice (GEM) Improvement of tolerance to a/biotic stress factors in case/food crops

Proteomics

Understanding the hostpathogen interactions Discovery of biomarkers for human diseases Understanding of variable response to drug therapies

Metabolomics

Global analysis of metabolic functions in human tissues Analysis of metabolic loads on various cellular entities

Fluxomics

Understanding of metabolic networks involved in various diseases including infectious and noncontagious conditions Analysis of metabolic processes for improvement of process yields

growth conditions during the metabolic processes. The energy spilling and secondary metabolite pathways can be studied and correlated with the bioprocess engineering networks. The biological and environmental perturbations of the biological system can, hence, be studied by a variety of different tools including microarray techniques, MFA, and crystallographic analysis. These highly sensitive methods can be employed for identifying and manipulating the metabolic pathways of the microbial strains for attaining maximum yields of the metabolic product. Table 14.1 provides a summary of the omics techniques employed for human betterment. The proper use of the “omics” tools can only be substantiated in the presence of the adequate information about their applications. Integrating various approaches of these techniques has to be based upon statistical grounds of their usage. A number of research groups employ these methodologies for the qualitative and quantitative improvement of the bioprocesses. Efforts should be made in order to correlate the data obtained from genomics, transcriptomics, proteomics, and metabolomics studies and to use them adequately for product and process engineering. Statistical methodology and computer algorithms can be employed for the study of various players within the cellular system. Multivariate statistical tools for studying the relative gene expression within a biological system have been developed that make use of the abundant gene and protein data aiding the modeling

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of appropriate biological and metabolic processes (Khatri et al., 2012). These tools are not only used for the postprocessing studies but can be employed for the prediction and the development of the most adequate growth conditions. Co-inertia Analysis or CIA, for instance, is based on the statistical inference of the gene and protein data points (Gomez-Cabrero et al., 2014). This systematic analysis utilizes these information obtained from different samples and project them in the form of various graphical representations. The outcomes of such analysis can, hence, help in the study, analysis, and manipulation of various biological processes. Owing to their importance, the integrative omics approaches are now considered to be one of the basic necessities for the designing, optimization, development, and validation of an efficient industrial bioprocess.

14.5 CHALLENGES IN OMICS-FOR-INDUSTRY Over the last two decades, the omics tools have helped in understanding the genomic and proteomic basis of many essential biological processes. The availability of advanced tools for high-throughput screening methods has significantly contributed to the establishment and linking of various essential biological players. Myriad complex cellular and molecular pathways have been deciphered which have not only provided a lot of information about these pathways but also provided means to manipulate them. The integration of various fields have helped in providing the ultimate solution in many instances. However, a number of concerns have been put forward by the scientific, industrial, and regulatory communities. Many research groups in the scientific community claim that the benefits of the omics tools have been over hyped for both financial and scientific interests. Many of the groups, due to certain technical reasons, are against the widespread commercialization of the omics techniques. Their chief concerns include the lack of reproducibility and specificity. Both the problems are quite related to one another and are interdependable. The generation of a large amount of data and the “selection” of the results to support the proposed hypothesis lead to these problems. Commonly referred to as fishing, the hand-picking of the favorable results and presenting them causes a significant decrease in the sensitivity of the outcomes. Similarly, none of the omics approaches can alone provide a solution to all the scientific questions. Inappropriate selection of interpretation and validation tools adversely affects the process dependence. Many of the biological players are not very stable. Proteome, for instance, is a very sensitive player and is prone to significant degradation over time. The results of proteomic studies are sometimes not easily reproducible which hints at the failure of any experimental procedure. While devising and optimizing strategies for the industrial processes, such unreliable procedures are generally not adapted (Gomez-Cabrero et al., 2014). Moreover, the role of various confounding variables has to be considered while optimizing these strategies for the genetic manipulation of commercially viable organisms. Many of the experimental techniques do not produce the same results when they are being studied under various environmental and experimental conditions. Hence, the sensitivity of the

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methods has to be considered while studying the overall dynamics of a process. The outcomes of any biological process should only be presented once they have been verified and validated under various experimental conditions (Machado et al., 2015). The academiaindustrial gap is sometimes one of the basic reasons for the failure of the omics approaches to be adapted by the biotechnology industry in general. The success of genetic modification in an academic setup is generally quite different from that considered to be a successful by an industrial setup where, in general, a high-commercial outcome is the main driving force. Moreover, due to financial constraints, many industrial units do not put in adequate amount of funds for the establishment of appropriate omics facilities. They rely highly on a contract organization for providing them the data obtained after experimental manipulation of a commercially viable microorganism. The data from various sources might not be of equal credibility as that obtained from an in-house facility. Additionally, the discussion of development of a super-producer of any metabolite is quite controversial. On these grounds, the ethical concerns raised by the regulatory authorities, academia, and industries defy the adaptability of the integrative omics approaches by many industrial facilities. These challenges, hence, need to be addressed while devising an adequate “omics-forindustry” policy. The massive amount of data obtained from various omics sources should, hence, be validated. The reproducibility of data can only be achieved once there are adequate facilities for practicing omics at the industry. Similarly, the study of integration of various techniques under varying environmental and biological conditions has to be performed before these manipulated organisms are introduced in the industrial setup. In future, it would only be through the efficient integration of various omics tools in close relation with the statistical and computational techniques that appropriate methods are devised for metabolic engineering of microbes.

14.6 CONCLUSION AND FUTURE PERSPECTIVES The data suggest that the advent of omics has led to a significant improvement in all the fields of biomedical sciences. However, due to certain technical, regulatory, and financial constraints, these tools have not been employed in many of the industrial setups. The tools of omics technology, alone and in combination, have been employed for understanding the bioprocesses and bioproducts in a number of studies. They help in the analysis of complex biological processes at different levels of biomolecular complexity, thereby, contributing to the overall understanding of these biological systems. Though many advances in this field validate their efficiency in various systems, yet efforts need to be made to improve the sensitivity, specificity, cost effectiveness and, the overall, efficiency of these techniques. Devising efficient methods to integrate various techniques of genomics, transcriptomics, proteomics, and metabolomics can aid the analysis of the metabolic pathways and the use of the data for extrapolating various experimental conditions in silico before their wet-lab testing. The development of these technological processes can, hence, contribute to the understanding of the genomic and proteomic players that can be manipulated for quantifiable improvement of the industrial processes by optimizing the metabolic engineering pathways.

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C H A P T E R

15 Omics Approaches and Applications in Dairy and Food Processing Technology Rekha Chawla1, Jaspreet S. Arora1, Rajesh K. Dubey2 and Chandra S. Mukhopadhyay1 1

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India 2 Punjab Agricultural University, Ludhiana, India

15.1 INTRODUCTION The word “omics” formally refers to study related to genome, proteome, or metabolomics, and aims at characterization of large family of cellular molecules and exploring the role, relationship of one molecule with another, their interactive effect, and actions of various molecules of an organism. However, in context of food, the constituent’s entities (particularly proteins) play a pivotal role and affect various properties of food (includes milk as well; the major component of food). Therefore, identifying the changes of their state and conditions of transition in their structure is of particular importance to dairy and food industry. Among various classes of studying behavior, proteomics plays a major role and studying the same gives an idea to understand the cellular changes taking place within the molecule. Various different definitions have been given by various researchers. However, in simple language, proteomics can be defined as “the qualitative and quantitative comparison of proteomes under different conditions to understand cellular mechanisms underlying biological processes” (Anderson and Anderson, 1996). Proteomics involves separation, identification, and post-translational characterization of constituent proteins. It uses different high-performance separation techniques such as two-dimensional gel electrophoresis, one-dimensional and multidimensional chromatography, combined with high-resolution mass spectrometry, and has the power to monitor the

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protein composition of foods and their changes occurring during the production process. At the same time, it has the enormous potential in clarifying biochemical and physiological mechanisms of complex diseases at a molecular level (Dunn, 2000).

15.1.1 Historical Perspective In a broad philosophical perspective, the concept and origin of omics (and omes) is perhaps as long as human history. It is a further knowledge classification process, as in other subjects such as chemistry, biology, physics, and mathematics, which is further fused with various other knowledge-seeking disciplines. However, in biology, it further elaborates the studying genome (genomics), proteins (proteomics), lipidomics (cellular lipids), metabolism (metabolomics), food omics, transcriptomics, and many more, but in the context of this chapter, a few relevant categories have been taken up and their effect has been described in detail. As far as the term “proteome” is concerned, it was first coined by Wilkins et al. (1995) to describe the protein complement to the genome. It involves systematic separation, identification, and posttranslational characterization of constituent proteins from a common source (i.e., cell type, secretion, or subcellular compartment). Proteomic approaches can be conveniently classified into six groups: expression proteomics, protein protein interactions, functional proteomics, structural proteomics, proteome mining, and posttranslational modifications (Bindexin 2013) and can be well described diagrammatically (Fig. 15.1). The techniques have also been used to characterize foods from vegetable and Signal transduction

Medical microbiology

Protein expression profiling

Drug discovery Target identification/ validation

Disease mechanism

Glycosylation Posttranslational modification

Proteome mining

Phosphorylation Proteolysis

Differential display

PROTEOMICS Yeast two-hybrid

Yeast genomics Affinity purified Protein complexes

Functional proteomics

Protein-protein interaction

Structural proteomics

Mouse knockouts

Co-precipitation

Phage display Organelle composition

Subproteome isolation

Protein complexes

FIGURE 15.1 Classification of proteomics approaches. Source: Courtesy: Carbonaro, M., 2004. Proteomics: present and future in food quality evaluation. Trends Food Sci. Technol. 15, 209 216.

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animal sources (Alomirah et al., 2000) including eggs (Raikos et al., 2006) and meat (Di Lucia et al., 2005).

15.1.2 Biotechnological Developments in Dairy and Food Processing The major chunk of developments in dairy industry can be attributed to fermented dairy products, wherein modified cultures have been used to get the maximum beneficial results. Among top rated products, cheese stands at first, being consumed at a large scale by majority of population. A brief description of the same is enclosed herein. 15.1.2.1 Cheese The making of cheese as a means of preserving the most important constituents of milk in highly concentrated form is in vogue all over the world. It provides a palatable milk product of high food value, which can be kept fresh for a long time. Cheese is an excellent source of milk protein and is rich in calcium and vitamins. It is a nourishing and easily digestible food. In India, earlier cheese was not a much popular food item, owing to the use of animal rennet in preparation. Most cheese varieties were made by coagulating milk with selective proteinases, like rennet, which is an enzyme preparation extracted from fourth stomach (vells) of young calves. Addition of rennet to milk breaks down the casein micelle stabilizing protein, called kappa casein, at a specific bond (i.e., Phe105-Met106) and destroys its ability to stabilize the casein micelle. Thus, it brings about an enzymatic modification of casein. Consequently, pure-kappa casein is formed, whose aggregates in the presence of tonic calcium at a temperature of about 20 C forms a gel. However, in the past few decades due to the short supply of calf vells, production of calf rennet has not been sufficient to meet the demand of growing cheese industry all over the world and led to the search of rennet substitutes suitable for cheese making. The suitability of any rennet substitute depends on several factors, such as availability of the source, ease of production, purity, absence of antibiotics, and thermostability, along with fact that milk-clotting activity (MCA) of rennet substitute should not be much dependent on pH and the ratio of MCA to proteolytic activity (PA) should be high. 15.1.2.1.1 MICROBIAL RENNET AND RECOMBINANT CHYMOSIN

Several proteinases from animals, plants, and microbial sources possess the ability to coagulate milk under suitable conditions, but most of them are too proteolytic relative to their clotting ability, which causes development of defects in flavor and texture as well as reduction in yield of cheese. However, a few microbial sources produce rennet substitutes which meet the suitability requirements and have reached the stage of commercial production (Table 15.1). Like chymosin, these substitutes are acid (aspartyl) proteinase and similar in molecular and catalytic properties. The specificity of rennet substitutes from fungal sources such as Mucor miehei, Mucor pusillus and Endothia parasitica is quite different. The acid proteinases of M. miehei and M. pusillus, like chymosin, preferentially hydrolyze the same Phe105-Met106 bond of K-casein, while that of E. parasitica preferentially cleaves the Ser104-Phe105 bond.

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TABLE 15.1 Commercially Available Microbial Rennet Sources of enzyme

Brand name

Mucor miehei,

Hannilase, Rennilase, Fromase, Miki, Marzyme, Modilase

Mucor pusillus (Lindt)

Noury, Meito, Emporase

Endothia parasitica

Suparen, Sure curd

Bacillus subtilis

Mikrozyme

Aspergillus niger

Chymogen

Escherichia coli

Chymax

Kluyveromyces lactis

Maxiren

Although microbial rennet is relatively cheap, these have attracted the attention of industrial enzymologists and biotechnologists. The gene for prochymosin has been cloned in Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. The enzymatic properties of the recombinant enzymes are indistinguishable from those of calf chymosin. The uses of recombinant chymosin have been assessed on many cheese varieties, and always come up with very satisfactory results. Three recombinant chymosins are now marketed commercially: Maxiren secreted by K. marxianus var. lactis and produced by Gist Brocades; Chymogen secreted by A. niger and produced by Chr. Hansen; and chymax secreted by E. coli and developed by Pfizer. The genes for Maxiren and chymogen were isolated from calf abomasums, while that used for chymax was synthesized. Microbial chymosins have taken market share from both calf rennet and especially fungal rennet and now represent 35% of total market. The gene for Rhizomucor miehei proteinase has been cloned and expressed in Aspergillus oryzae. It is claimed that this new rennet (Mazyme GM) is free of other proteinases or peptidases activities that are present in fungal rennet and may reduce cheese yield. Cloning of the gene for R. miehei proteinase has created the possibility for site-directed mutagenesis of the enzyme.

15.1.3 Bio Yogurt Intestinal region of almost all warm blooded animals contains microflora which supports the function of intestinal environment. Amidst a large variety of flora, selected groups of bacteria actively dominate the enteric environment inhibiting several undesirable microbial/biochemical functions. The two important bacterial systems are Lactobacillus acidophilus and Bifidobacterium bifidum. The bacterium B. bifidum plays a significant role in human infants’ enteric system, especially in breast-fed individuals. On weaning, due to non-availability of specific growth factor (bifidus factor), this bacterium disappears in a nonhuman milk environment. Such a disappearance can encourage growth of undesirable flora leading to problems of ill health. Similar to bifidobacteria,

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Lb. acidophilus plays an active role in adult human subjects. Growth of these two flora creates conditions unfavorable to undesirable flora, which promotes the general status of health of host, human system. Hence, the function of these floras is referred to as probiotic. Bio or BA yogurt is made with different live cultures, called bifidus and acidophilus. It is often claimed that these particular bacteria aid digestion by supplementing the body’s natural flora. When these bacteria are used as the starter for our own yogurt, they will reproduce and make bio yogurt. Bio yogurt is not as sharp as ordinary yogurt, and has a milder, creamier flavor. All yogurts are easier to digest than plain milk, because of the action of the bacteria, and they are a good source of calcium and phosphorus, which are essential for strong teeth and bones. Yogurt is also said to be good for maintaining the general health of the skin and the digestive system, and when soluble fiber added to them, it may help to lower the level of cholesterol in the blood. Apart from this, approaches in gene transfer and cloning technologies provided numerous opportunities for the application of genetic approaches to strain development program as per the requirement of dairy processing industry. Also, modified and tailor-made enzymes can be used for speeding up the slow processes like cheese ripening to harvest more benefits in a short span of time. Not only this, proteomics deals with proteins from recombinant technologies, thus indicating its potent use in biotechnology industry (Liu, 2000), whereas in food industries, enzymes, amino acids, vitamins, organic acids, PUFA (poly unsaturated fatty acids), and complex carbohydrates used in various food formulations are currently produced using Genetically Modified Organisms.

15.2 OMICS: FROM FARM TO FORK Animal biotechnology is not a new field and has been practiced in one form or another since the beginning of the domestication of animals. The older but still effective tools of animal breeding, genetics, and nutrition have played an important role in the selection, propagation, and management of desirable and economically important characteristics in livestock. With biotechnology, modern livestock production is possible for development of improved feedstuffs, feed ingredients, vaccines, biologicals, enzymes, high-quality genetics, genetic markers, assisted reproduction, etc. Recently, several new “omics” (such as genomics, transcriptomics, proteomics, and metabolomics) technologies came into existence that are now readily available to scientists or industry for its application in livestock production. The biggest advantage with these technologies is that they offer a holistic instead of a reductionist view of the biological phenomena. The microarray technology for microRNA is available today for bovine, pigs, and chickens. When combined with appropriate bioinformatics tools, they have been of great help in understanding livestock genomics. Among the livestock species, large-scale SNP (single nucleotide polymorphism) arrays are available today only for bovines. Epigenomics (the study of the non-DNA hereditable factors affecting the phenotype) has been used for large-scale studies, but data have not been generated using this technology in livestock. Systems biology has emerged to investigate “interrelationships of all of the elements in a functioning system in order to understand how the system works.” A systems biology approach is only possible by combining a single or multiple “omics”

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technique(s) along with bioinformatics for a broad purpose such as to study the whole system, organism, or comparison between organisms (Metzker, 2010). To harness the breakthroughs from “omics” techniques, efficient animal breeding and reproduction of these rare genetic individuals is required. For decades, assisted reproductive technologies (ART), such as artificial insemination (AI), superovulation, embryo transfer (ET), and in vitro embryo production (IVEP), have contributed to animal breeding programs allowing faster transmission of desirable traits in livestock populations in a shorter period of time compared to classical approaches. The use of transgenic technologies along with ARTs to introduce single or multiple genes into existing genomes of livestock has played an increasingly larger role in the genetic development of our production livestock. Addition of appropriate stem cell technologies to the genetic “toolbox” has further increased our capabilities to enhance, cure, and modify livestock genomes and physiology (Mundim et al., 2009).

15.3 PROTEOMICS: GENERAL STRATEGIES AND ANALYTICAL METHODS The study of the whole set of proteins encoded by a genome at a certain time and under certain conditions involves different steps that depend on various technologies. A typical proteomics workflow consists of: 1. 2. 3. 4.

protein extraction, protein or peptide separation and quantification, protein identification, and data analysis and interpretation.

15.3.1 Protein Extraction Protein extraction itself can be a difficult task as many foods are of plant origin that largely consists of fibrous cell wall material. In addition, the watery content of the plant vacuole results in very low protein yields compared to bacteria or animal tissues. Several methods have been reported for protein extraction from plant materials that takes into account the presence of interfering compounds such as phenolic compounds, carbohydrates, proteolytic, oxidative enzymes, and pigments. For animal and bacterial tissues, various protein solubilization buffers, chaotropic agents, detergents, reducing agents, buffers, and ampholytes are used for higher protein yields (Morzel et al., 2004). Due to the complex chemical nature of proteins and their broad dynamic range (ratio between the smallest and largest protein levels), each technique generally focuses on a particular set of proteins. To overcome the problem of dynamic range, prefractionation techniques such as organelle fractionation prior to two-dimensional electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) are currently employed. Typically, these prefractionation techniques involve differential density ultracentrifugation (Ho et al., 2006).

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15.3.2 Protein Separation Proteins can be separated from each other using either gel-based or gel-free approaches. These approaches are complementary because they focus on specific subsets of proteins that are only partially overlapping. They differ in the way how proteins or peptides are isolated, separated, and detected. 15.3.2.1 Gel-Based Proteomic Approach The gel-based proteomics approach depends on 2-DE for the separation of proteins based on two properties—isoelectric point (pI) and molecular weight (Mw). This technique was introduced in the 1970s by O’Farrell et al. (1977). Briefly, isoelectric focusing (IEF) separates proteins by their differences in electric charge taking advantage of the amphoteric character of proteins (the fact that proteins charge depends on the pH of the environment). To accomplish the separation, an electric current is applied to an immobilized pH gradient (IPG) strip (acrylamide gel matrix copolymerized with a pH gradient). When a protein is in a pH region below its pI, it will be positively charged and migrates towards the cathode and vice versa. When proteins reach their isoelectric point (pH at which the protein has no net charge), they stop migrating and are said to be “focused.” After completion of the IEF, the IPG strip with the separated proteins is used for the second dimensional separation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE separates proteins based on the molecular weight. Before this second dimension can be carried out, proteins on the IPG strip need to be equilibrated for elimination of the secondary and tertiary structures of proteins and reduction of the disulfide bridges between cysteines. The electrophoretic mobility of proteins is now solely dependent on molecular weight. The acrylamide separating gel is composed of different particle sizes; thus, smaller molecules will move faster compared to large molecules which will be slowed down. Once proteins are separated, they are visualized for quantitative analysis with staining strategies such as colloidal coomassie blue (Neuhoff et al., 1990), silver (Blum et al., 1987), radiolabeling (Patton, 2002), and fluorescence (Chevalier et al., 2004). The stains discussed above present certain limitations in terms of the detection limit (e.g., colloidal coomassie blue), dynamic range, and reproducibility (e.g., silver). One of the limitations of comparative 2-DE is the high gel-to-gel variation that makes the analysis difficult in terms of distinguishing biological variation from experimental variation. To overcome this issue, a two-dimensional difference in-gel electrophoresis (DIGE) technology was developed (Unlu¨ et al., 1997). In DIGE, the samples are labeled prior to the electrophoretic separation with spectrally resolvable dyes (Cy2, Cy3, and Cy5). Subsequently, the samples are mixed prior to IEF and resolved on the same 2-DE gel (Fig. 15.2) (Unlu¨ et al., 1997). DIGE increases the confidence in terms of detection and quantification of differences in protein abundance and also reduces the number of gels needed to run in an experiment. The primary advantage of multiplexing samples is that an internal standard (representing an average of all samples in an experiment) can be included to normalize protein abundance across multiple gels. Thus, each gel will contain an image with a highly similar spot pattern, improving the confidence of inter-gel spot matching and quantification. Basic or hydrophobic proteins are still difficult to separate under gel-based 2-DE approaches in

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15. OMICS APPROACHES AND APPLICATIONS IN DAIRY AND FOOD PROCESSING TECHNOLOGY Protein digestion

Separation of extracted proteins Gel-based Molecular mass

Molecular mass

Isoelectric point

1D 2D SDS-PAGE Gel free

Excised spots/bands or collected HPLC fractions are digested with a protease to generate peptides

Hand-made microcolumns packed with materials with different affinities or

Reverse phase lon exchange t (min)

Shot gun Whole proteome is digested with protease(s)

Peptide identification Protein inference Search against protein databases m/z

nano-HPLS or micro-HPLC

HPLC fraction

or

Bioinformatics

MS Intact peptides m/z values

1D band

HPLC

No previous separation of proteins

Mass Spectrometry analysis

Separation

2D spot

or collected

Reverse phase lon exchange Size exclusion

Peptide purification concentration and/or

Relative abundance

Tissue collection and subcellular fraction preparation

Individually or a combination of these approaches

MS/MS Aminoacid inference from peptide fragments Relative abundance

278

Sequenced organism Unsequenced organism Homology driven protein Specific protein identification databases

Pathway analysis

m/z

FIGURE 15.2 Schematic representation of the bottom-up proteomic workflow. HPLC, high-performance liquid chromatography. Source: Courtesy: Soares, R., Franco, C., Pires, E., Venstosa, M., Palhinhas, R., Koci, K., et al., 2012. Mass spectrometry and animal science: protein identification strategies and particularities of farm animal species, J. Proteomics, 75(14), 201, 4190 4206.

spite of the introduction of IPG strips up to pH 14. Substitution of the reducing agent dithiothreitol for tributylphosphine or hydroethyldisulphide (commercially known as Destreak) partially overcomes the problem of a lack of resolution of basic proteins during the IEF run (Olsson et al., 2002). Gel-based proteomics is the most powerful option for non-model organisms (e.g., most plant-based foods in study of isoforms and posttranslational modifications). Some of the limitations of this approach are the unequal resolving power of 2-DE (e.g., bias towards high abundant proteins, hydrophobic, or very acidic proteins is not resolved, comigration of proteins resulting in spots containing multiple proteins) and the limited dynamic range covered besides the difficulty for automation. 15.3.2.2 Gel-Free Proteomic Approach Gel-free approaches in most of the cases use a bottom-up strategy, meaning that proteins are first proteolyzed, and the resulting mixture of peptides is then separated based on hydrophobicity via reverse-phase chromatography. Subsequently, the eluted peptides are introduced into a mass spectrometer. All the tandem mass spectra gathered are then used to search databases and reconstruct the original proteins. This approach is successful for relatively simple protein mixtures from sequenced species. The problem of resolution of proteins from complex samples was overcome by the introduction of MudPit (multidimensional protein identification technology) (Washburn et al., 2001). The application of MudPit aided the separation of membrane proteins as well as the ability to detect low abundant proteins (Roe and Griffin, 2006). However, the limitation of this approach is the lack of provision of quantitative information, which has been overcome by the use of stable isotope labeling and dilution strategies for the relative quantification of proteins (Roe and Griffin, 2006). Gel-free approaches have the disadvantage that qualitative and quantitative information on protein isoforms and differential posttranslational modifications are lost (Carpentier et al., 2008). Besides that, cross-species identification for poorly sequenced genomes, e.g., most plant-based foods, is not possible. Cross-species

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identification depends on the identification of proteins by comparing peptides of the proteins of interest to orthologous proteins from other species that are well characterized.

15.3.3 Protein Identification 15.3.3.1 Mass Spectrometry Proteins need to be digested before being introduced in a mass spectrometer. In most of the cases the unknown protein of interest is cleaved into smaller peptides by using a trypsin enzyme which specifically cleaves proteins on the carboxy-terminal side of arginine and lysine residues (Steen and Mann, 2004). A mass spectrometer consists of an ion source (e.g., ESI or MALDI) to produce ions from the sample, one or more mass analyzers (e.g., quadrupole, time of flight (TOF), ion trap) to separate the ions based on their mass to charge (m/z) ratios, a detector to register the number of ions coming from the last analyzer, and a computer to process the data and produce the mass spectra. In addition, an inlet device is necessary to introduce the sample into the ion source (Lane, 2005). 15.3.3.1.1 IONIZATION TECHNIQUES

Matrix-assisted laser desorption ionization (MALDI) (Karas and Hillenkamp, 1988) and electrospray ionization (ESI), (Fenn et al., 1989) are the two ionization techniques of mass spectrometry that has revolutionized the proteomics platform making possible the highthroughput identification of proteins. For ESI, the sample is dissolved in a solvent mixture (e.g., acetonitrile water) and then injected into a capillary held at a potential of 3 4 kV. As a result, a very fine spray of solvent droplets containing ions of the forms (M 1 nH)n 1 (where M is the peptide molecule, nH is the number of protons attached to the molecule, and n 1 is the net charge of the ions) is formed. Multiple charged gas-phase ions are subsequently formed during the desorption process due to the evaporation of the solvent, which will then enter the mass analyzer. MALDI relies on a laser which is fired at a sample plate containing a dried mixture of matrix (α-cyano-4-hydroxycinammic acid) and sample to ionize the latter. The matrix absorbs radiation from the laser resulting in excitation of the matrix molecules. As a result, a dense plume containing both the matrix and the analyte molecules is produced. The analyte molecules interact with protons from the matrix to form mainly single charged ions (Steen and Mann, 2004) that enter the mass analyzer. The formed ions are separated in a mass analyzer according to their m/z ratio. 15.3.3.2 Mass Analyzers There are different mass analyzers, each having their strengths and weaknesses. TOF uses an electric field to accelerate the ions at the same potential, and the time needed to reach the detector is measured. For particles with the same charge, their kinetic energy is the same, and therefore their velocity is solely dependent on their mass; ions with smaller m/z values will reach the detector first (Wollnik, 1993). Quadrupole (Q) uses oscillating electrical fields to selectively stabilize or destabilize ions passing through a radio frequency quadrupole field (Lane, 2005). Ion traps, or more specifically quadrupole ion traps,

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trap ions in a dynamic electric field and sequentially eject them into the detector according to their m/z values (Steen and Mann, 2004).

15.3.4 Comprehensive Data Analysis Proteomics, as a high-throughput platform, generates a tremendous amount of data from which high-quality data have to be filtered. Gel-based or gel-free approaches require proper data analysis to avoid misleading conclusions, but first of all a good experimental design should be emphasized (Chich et al., 2007). The experimental design should consider the sources of variability (e.g., technical and biological) selecting the appropriate number of replicates needed to characterize treatment differences (Horgan, 2007).

15.4 PROTEOMICS OF MILK AND MILK PRODUCTS In dairy industry, studying variation at proteomic level of milk proteins reveals a new and an interesting picture. Milk in legal terms (FSSAI, 2015) may be defined as the normal mammary secretion derived from complete milking of healthy milch animal without either addition thereto or extraction there from unless otherwise provided in FSSAI regulations. It shall be free from colostrum. Or in general, it may be defined as the secretion of the mammary glands of mammals and has primary natural function regarding nutrition of the young ones. The constituent portion of milk protein (active component) responsible for the enhanced and specific functionalities is of immense importance and is termed as bioactive peptides. These bioactive peptides are proteins synthesized in the cell in the form of large prepropeptides (either directly or upon enzymatic hydrolysis in vitro or in vivo), which are then cleaved and modified to give active products (Sharma et al., 2011). However, these peptides are inactive within the sequence of the parent protein molecule and can be liberated by (1) gastrointestinal digestion of milk, (2) fermentation of milk with proteolytic starter cultures, or (3) hydrolysis by proteolytic enzymes (Hannu and Anne, 2006) and can be screened by various proteomic techniques. In a 2-DE proteomic method, applied to bovine whey, after been fractionized to acidic, basic, and nonbound components coupled with anion and cation exchange chromatography, a large number of newer unidentified bioactive peptides from group of osteopontin have been found (Fong et al., 2008), whereas some other researchers used this technique to evaluate milk from different species so as to find a substitute to human milk (D’Auria et al., 2005).

15.4.1 Proteomics of Milk Proteins The main nutritional and functional properties of milk are attributed to the protein composition and amount of it present in any dairy product. Thus, proteomics of milk proteins, whey proteins, and milk fat globule proteomics, and changes therein during lactation, processing, and other heat treatments during storage has remain an interest of researchers and number of studies have been conducted on the same. A number of proteomic techniques have been applied to the study of milk and milk products, allowing the separation

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Milk protein PTM analysis (phosphorylation) Whole milk

Milk fraction separation

Fat layer MFG: Low abundant proteins

Centrifugation

Milk protein fractionation

ICP-MS : Ciavardeli, 2010

Combinatorial ligand library (ProteoMinor): Cunsolo, 2011 D’Amato, 2009

1-DE, 2-DE or LC-MS: Scrensen, 2003; Holland, 2011

Cysteine tagging: Holland, 2006

LC tandem MS: Cunsolo, 2009; Mateos, 2009; Mateos, 2010

Milk protein MS-quantitation

Milk protein PTM analysis (glycosylation)

Skimmed milk fraction:

Amine-eactive isobaric tags (TRAQ): Reinhardt, 2006

casein whey proteins and peptides

Label –free: Boehmer, 2010

Ultracentrifugation

PNGaseF: Tao, 2008; Nwosu, 2012 Lectins: Cebo, 2010 LC tandem MS: Tao, 2008; Nwosu, 2012 Glycobotting; Takimori, 2011

Stable isotope dilution: Affolter, 2010

Gel-free milk proteomics

MALDI-TOF linear mode: Ham, 2012 MALDI-TOF/TOF: Takimori, 2011 2D-LC ORBITRAP: Affolter, 2010 1-DE LC-MS/MS: LC-FTICR: Tao, 2008 D’Amato, 2009; Pisanu, 2011 IRMPD: Tao, 2008 nLC-ESI-CID: Arena, 2010 2-DE-MS: Addeo,1995; CE-MS: Muller,2008; Somma, 2008; Lecoeur, AlonsoFauste, 2011; Anderson, 1982; Bianchi, 2009; 2010 Cunsolo, 2011; D’Amato, 2009; nLC-qTOF-MS/MS: Nwosu, 2012 D’Auria, 2005; Holland, 2005; Holland, Milk protein bioinformatics 2006; Pisanu, 2011; Roncada 2002 Sequence analysis: Khaidi, 2011

Gel-based milk proteomics

Soluble fraction: whey proteins and peptides

Insoluble fraction: casein and peptides

Differential in gel electrophoresis (DIGE): Gene Ontology anlysis(GO): D’Alessandro, 2011; Lbeagha-Awemu, 2010 Addis,2011 Ingenuity Pathway analysis (IPA): D’Alessandro, 2011; Addis, 2011

FIGURE 15.3 Milk prefractionation steps and proteomic experimental strategies. PTM, posttranslational modification; MFG, milk fat globules. Source: Courtesy: Roncada, P., Piras, C., Soqqiu, A., Turk, R., Urbbani A., Bonizzi L., 2012. Farm animal milk proteomics. J. Proteomics 75, 4259 4274.

of major proteins, including caseins (αs1-, αs2-, β- and κ-casein) and whey proteins (β-lactoglobulin, α-lactalbumin, and bovine serum albumin). Outline of milk fractionation and strategies is illustrated in Fig. 15.3. Use of proteomics led to the identification of 151 proteins in milk, removing major fractions such as casein and whey proteins along with immunoglobulin A (IgA), lactoferrin, half of which were not previously identified (Palmer et al., 2006). Similarly, applying 2-DE, Vanderghem et al. (2008) used number of detergents for the extraction of milk fat globule membrane and found that inclusion of 4% CHAPS resulted in the removal of the highest amount of skim milk proteins. A comparison of different milk obtained from different species has been made possible with the use of two-dimensional milk patterns combined with IEF in first dimension and SDS-PAGE or Urea-PAGE in second dimension (Kim and Jimenez-Flores, 1994; Goldfarb, 1999). Immunological detection with polyclonal antibodies following analysis by 2-DE had been commonly applied to caprine and ovine milks and allowed determination of the heterogeneity of caseins and whey proteins (Chianese, et al., 1992; Lopez-Galvez et al., 1995; Chianese et al., 1996) The findings of 2-DE on proteinase derived from Pseudomonas species on milk proteins and hydrolysis of different proteins by Penicillium caseicolum and Penicillium roqueforti have been compared (Trieu-Cuot and Gripon, 1981).

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Studying proteolysis using proteomics in fermented products like cheese is of great interest as the textural characteristics of such products are highly dependent on extent of proteolysis. Apart from this, various flavor generation compounds are due to degradation of these proteins and their peptides made thereof after the fermentation process and possess health promoting features as well. As every product has some significant observation to assess its quality, monitoring rate of proteolysis in cheese carries an immense importance. The different types of proteolytic enzymes active within cheese and their specificity have been determined through identification of peptides released in cheese (Singh et al., 1994, Gagnaire et al., 2004). A detailed study on Emmental cheese carried out by Gagnaire et al., 2004, and the method adopted was based on prefractionation of the cheese aqueous phase at the end of ripening, by size exclusion chromatography (to reduce sample complexity and to facilitate detection of the least abundant components); protein separation by 2D electrophoresis and identification by MALDI-TOF MS and/or de novo sequencing by nanoscale LC-ESI-MS/MS. Study revealed constant release of GroEL from Streptococcus thermophilus and Lb. helveticus, which is an indicator of constant stressing conditions as well as cell lysis conditions. Thus, results revealed that different peptidases arose from St. thermophilus and Lb. helveticus, suggested that streptococci are involved in peptide degradation in addition to the proteolytic activity of lactobacilli. Similarly, proteomics helped to study Lactic acid bacteria (LAB) and its ability to adapt to different environmental stresses when used under various fermentations conditions to be used in food preparations (Champomier-Verges et al., 2002). Though a number of products have been targeted to improve quality of products using proteomics, cheese, among all, has received ample attention being consumed at larger scale and relished in many developing countries. Products like yogurt have also been monitored with respect to their change in milk protein profile due to action of Lb. delbrueckii subsp. bulgaricus and St. thermophilus using MALDI-MS (Fedele et al., 1999). Proteomics has also been shown to provide a powerful tool to characterize milk and microbial proteins as shown in Table 15.2.

15.5 PROTEOMICS OF FOOD TECHNOLOGY The relevance of high-throughput proteomic approaches is to increase insight and understanding of how food processing is affected by the physiology of the product and, through this, to enable the optimization of the overall food production process. Proteomics can also be used for validation and control of industrial processes of food products. The use of proteomics in food technology is presented, especially for characterization and standardization of raw materials, process development, detection of batch-to-batch variations, and quality control of the final product. Further attention is paid to the aspects of food safety, especially regarding biological and microbial safety and the use of genetically modified foods.

15.5.1 Postharvest Processing Proteomics can also be used for validation and control of industrial processes of food products. As we know that protein constitute a good amount in almost every food, these

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TABLE 15.2 Main Applications of Proteomics Concerning the Study of Milk Proteins and Dairy-Related LAB Proteomes Proteins studied

Main applications

Whole milk proteins and caseins

High-resolution protein patterns, 2D maps of milk proteins 2-DE from different mammal species. Study modifications of caseins during lactation. Determination of casein hydrolysis.

Caseins/whey proteins

Determination of heterogeneity due to genetic variants and posttranslational modifications in different ruminant species. Localization of some glycosylation and phosphorylation sites. Quantitative determination of casein content in milk from transgenic cattle. Determination of lactosylation of whey proteins.

2-DE

Identification of phosphorylation sites and localization of oligosaccharide sequence in glyco- and phosphopeptides.

HPLC 1 ESI-MS

Minor milk proteins

Detection and identification of low-abundance proteins in milk and colostrum. Determination of biomarkers. Immunogenicity of minor proteins.

2-DE 1 microsequencing

LAB protein expression

2D maps. Determination of Mw and pI, protein identification. Comparison of different LAB species or strains. Differential expression in different growth media. Stress response and adaptation.

2-DE

Identification of casein hydrolysis products using different microbial and gastric enzymes. Determination of whey protein truncated forms. Monitoring of the appearance of casein degradation products in cheese and yoghurt. Specificity of microbial proteinases. Selectivity of enzymes in cheese. Study of proteolysis throughout cheese ripening. Identification of proteins liberated by LAB throughout ripening.

2-DE

Milk peptides

Complex dairy matrices: Cheese/ yoghurt proteins

Proteomic techniques

2-DE 1 immunodetection 2-DE 1 MALDI-TOF MS 2-DE 1 nano-ESI-TOF MS/MS

MALDI-PSD MS

2-DE 1 MALDI-TOF MS

2-DE 1 MALDI-TOF MS 2-DE 1 Q-TOF MS/MS

2-DE 1 MALDI-TOF MS 2-DE 1 Q-TOF MS/MS HPLC 1 ESI-MS HPLC 1 MALDI-TOF MS Nano-HPLC 1 ESI-MS/MS

HPLC, High-performance liquid chromatography. Courtesy: Manso, M.A., Leonil, J., Jan, G., Gagnaire, V., 2005. Application of proteomics to the characterization of milk and dairy products. Int. Dairy J. 15, 845 855.

techniques offer a new very promising approach to identify protein in food matrix and can offer to study the protein protein interactions in raw and processed foods, as well as interactions between proteins and other food components (Carbonaro, 2004). It can direct to the detection of markers for either specific food processing technologies or for quality of processed food (Van der Werf et al., 2001). In a research study, the 2-DE provided a convenient way to identify the different proteins produced in tomato fruit under heat stress conditions (Iwahashi and Hosoda, 2000). In food processing industry, proteomes of certain food (wheat, wine, and fish) can be used to identify the origin of a particular food or its quality during the food processing (Carbonaro, et al., 2003; Iwahashi and Hosoda, 2000; Quaranta et al., 2001).

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15.5.2 Cereal and Other Crops Cereals are the most important staple foods for mankind worldwide, and its outer coat represents the main constituent of animal feed. In other words, cereals are at the core of our life: The cultivation of cereals dates back to 10,000 years ago, which marks the start of civilization. The availability of cereals and quality of cereal products is key to the social stability of communities. Compositionally, cereals consist of 12% 14% water, 65% 75% carbohydrates, 2% 6% lipids, and 7% 12% protein. Cereals are quite similar to millets in gross composition being low in protein and high in carbohydrates. Though cereals are known for their carbohydrates reserve food content, apart from this, these are good sources of proteins as well. These storage proteins account for about 50% of the total protein in mature cereal grains and have important impacts on their nutritional quality for humans and livestock and on their functional properties in food processing (Shewry et al., 2002). Therefore studying proteomics of cereals and related crops through the application of gel and non-gel approaches offers a new way to characterize the protein component of foods, which not only allows the type of protein expressed but also provides the information regarding composition of protein in different tissues. Processing conditions like severity of heat treatment, protein protein interactions, and many other factors affect a lot to protein composition of the fresh produce, which lead to the modifications in their primary, secondary, or tertiary structure depending upon the severity. These modifications include side-chain oxidation, cross-link formation, and backbone cleavage, and critically influence key food properties such as shelf-life, nutritional value, and digestibility and health effects (Kerwin and Remmele, 2007). Modifications in proteins, as discussed, resulted from the processing techniques and heat or chemical treatment applied to foods for processing led to the formation of a series of xenobiotics, including lysinoalanine and ornithoalanine, cross-linked modifications which have been implicated in nutritional damage and adverse health effects (Fay and Brevard, 2005; Gliguem and Birlouez-Aragon, 2005; Rerat et al., 2002; Silvestre et al., 2006). Likewise, a range of modified products resulted from oxidation of tryptophan and tyrosine can be used as oxidative markers (Asquith et al., 1971; Davies et al., 1999; Dean et al., 1997; Guedes et al., 2009; Simat and Steinhart, 1998; Zegota et al., 2005). These changes significantly affect the quality of protein derived from the crops, notably cereals and grains. Implications of these can be seen as oxidation of proteins, which influences the formation of off-flavors and the nutritive value (Heinio et al., 2002). Therefore, studying proteomics can be used as a tool to investigate the cereals quality and also helps in detection of maintaining internal quality, so as to control the quality of finished product made thereof. Not only in assessing quality, being a powerful tool in the separation and analysis of complex protein mixtures, it can also provide valuable information on digestion patterns of food proteins too, and, therefore, on their bioavailability. Despite having knowledge on wide usage of proteomics techniques, industries are facing problems due to lack of information on complete genome sequence of many plant species. Though the situation is now improving rapidly, the genome of important plants such as rice that are important for human and animal nutrition are now either sequenced or their sequencing is the topic of ongoing projects (Kim et al., 2007). A significant importance has been given to rice, being the staple food of the various countries or can be said as more than half of world’s population (Sasaki and burr, 2000) and construction of a rice

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proteome database based around 23 reference 2-DE maps ranging from the cataloging of its individual proteins to the functional characterization of some of its component proteins, including major proteins involved in growth and stress response was done by Komatsu and Tanaka (2004) and resulted in an addition of 5092 proteins in the database. Proteomics also provided useful information concerning the relationship between the protein quality components and the quality of bread rice. Role of 15 proteins in disease resistance and quality determination during grain filling of rice plant was examined by Kim et al. (2009) and proteomics approach to study how the plant cops under stressful conditions and to various other environmental factors was evaluated by Kang et al. (2010). Apart from rice, proteomics of various other crops like maize, barley, and wheat (Agrawal and Rakwal, 2006), and corn (Ricroch et al., 2011) were also studied. A comprehensive characterization of allergens present in wheat was studied by Akagawa et al. (2007), using 2-DE approach coupled with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS), resulting in identification of nine subunits of low-molecular weight glutenins as being the most predominant IgE-binding proteins. Also, proteomics was utilized for the identification of stress-induced proteins in wheat lines and has special role in food science (Horvath-Szanics et al., 2006). The approach also provided very useful information about protein components linked to bread wheat quality and particularly to kernel hardness. Therefore, the proteome of wheat can be used to predict the quality of bread produced from it and further to simplify the data handling multivariate component analysis was applied (Gottlieb et al., 2002). Not only the cereals crops but also the legumes play a pivotal role in one’s life. Increased cultivation of legumes is essential for the regeneration of nutrient-deficient soils and for providing needed protein, minerals, and vitamins to humans and livestock (Anon, 2015). Proteomic approach was applied to seven different legumes, which when applied resulted in 54% increase in average protein score and an average 50% increase in average matched peptide (Lei et al., 2011). Knowledge of these protein sequences also helped to identify difference in quality of storage protein globulins, in soy crop, for difference in protein content greater than 45% of dry seed weight than standard soybean cultivar (Krishnan and Nelson, 2011). The same approach was used to verify health claims associated with soy (Erickson, 2005). Apart from this, specific applications of proteomics in analysis of food quality are reported in Table 15.3.

15.6 PROTEOMICS IN ASSESSING 15.6.1 Quality of Foods Quality of various fermented products can be assessed by monitoring the growth of microorganisms which in turn can be assessed using proteomics, either by carrying out a comprehensive systematic chromatographic study in the form of 2-DE maps to investigate the protein expressed by a microorganism in a particular physiological state or by studying the protein patterns of particular strain under different conditions. Studies under the differential method revealed that composition of protein varies with change in strain and

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TABLE 15.3 Various Applications of Proteomics in Relevance to the Food Quality Application study

References

Determination of wheat quality

Gottlieb et al. (2002)

Analysis of wheat kernel amphiphilic proteins

Amiour et al. (2002)

Glutenin subunit mapping

Cozzolino et al. (2001)

Metabolic pathways in rice

Koller et al. (2002)

Tomato protein expression under heat stress

Iwahashi and Hosoda (2000)

Identification of hazelnut 11S allergens and markers of sesame seed allergens

Beyer et al. (2002)

Map of commercial bovine milk

Galvani et al. (2001)

Collection of bioactive peptides of β-casein

Righetti et al. (1997)

Reference map of fat globule membrane proteins

Quaranta et al. (2001)

Bioavailability of milk proteins

Carbonaro et al. (2003)

Courtesy: Carbonaro, M., 2004. Proteomics: present and future in food quality evaluation. Trends Food Sci. Technol. 15, 209 216.

media undertaken (Guimont et al., 2002). As we know, microorganisms are important entity to many fermentation industries and are used in various food processes. However, their complete proteomics knowledge can help to detect and prevent the contamination by these agents. Undesirable protein profiles can also be checked using a detailed knowledge of proteomics of foodstuff (Kanemaki et al., 2003). Hence its applications can be used for providing a valuable tool for the evaluation of safety, body distribution, and metabolism of food ingredients (Kvasnicka, 2003), detection and control of food spoilage, and the presence of pathogenic microorganisms (Washburn and Yates, 2000). In complex fermentations, by employing varying microorganisms and complex substrates, the quality of the proteome or metabolome culture can be used to predict the quality of the fermented end-product. Therefore, evaluating and eliminating bad culture (contaminated) can result in lots of savings in term of controlling wastage using these technologies (Carbonaro, 2004). The allergy from various food components is another alarming situation and the cases in the same arena are increasing day by day. Studies have been reported to check either the genes for allergic disease or proteins (Beyer et al., 2002 Toda and Ono, 2002, Yu et al., 2003). Hence using proteomics specific gene can be traced and eliminated with regard to the occurrence of allergens.

15.7 TRANSCRIPTOMICS IN FOOD SAFETY A transcriptome is the full range of messenger RNA, or mRNA, molecules expressed by an organism in a particular cell or tissue type. In contrast with the genome, which is characterized by its stability, the transcriptome actively changes. In fact, an organism’s

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transcriptome varies depending on many factors, including stages of development and environmental conditions. Transcriptomics is the study of that transcriptome in a global fashion, most often using high-throughput technologies, such as microarrays, which has become a powerful tool to better understand the process of disease and other complex biological processes such as food spoilage and biofilm formation. Microarrays have improved our understanding of food microbes particularly transcriptomics of food microbes during growth in stressful environments and various foods such as milk, cheese, and yogurt. Recently, it was demonstrated that administration of Lb. plantarum can elicit immunomodulatory responses in gene-expression studies from the duodenum of humans (Van Baarlen et al., 2009). Transcriptomics has revealed an important and valuable insight into pathogenicity, adaptation, and survival of pathogens in food. It has been used to investigate intracellular expression of genes in Listeria monocytogenes (Joseph et al., 2006) during infection and host adaptation, and those genes activated by the transcriptional regulars PrfA, VirR, and the sigma B regulon (Hain et al. 2008). Microarray technology has also expanded to include tiling arrays, which are designed to cover the complete genome rather than covering just the annotated ORFeome of a single genome (Mockler et al., 2005). These arrays have facilitated a deeper view of transcription responses in food microbes such as Bacillus subtilis, L. monocytogenes, and E. coli O157:H7. ChIP (chromatin immunoprecipitation), the technique that was originally applied to eukaryotes is now used in prokaryotes in association with microarrays (termed ChIP-chip) for studying protein DNA interactions (Fig. 15.4). It was first applied in bacteria in 2002 and has since been utilized to study transcription factors in Bacillus subtilis, E. coli, and Helicobacter pylori (Wade et al., 2007). Next-generation sequencing (NGS) technologies are fast emerging as valuable tools in transcriptomics as well as understanding regulatory processes. Recently, NGS technologies have been applied to sequencing of the transcriptome referred to as RNA-seq or RNA deep sequencing (Wang et al., 2009). To date, NGS has mostly been utilized to sequence the transcriptome of eukaryotes, including eukaryotic microorganisms. This is due to the presence or absence of poly-A tail in eukaryotic and prokaryotic mRNA, respectively, which is utilized in the synthesis of cDNA. However, recently there have been some publications on sequenced transcriptome via RNA-seq in food pathogens (Oliver et al., 2009). Direct RNA sequencing for transcriptomics has the advantage over DNA microarrays, as transcripts are only detected on DNA microarrays if there is a corresponding probe on the array and DNA microarrays often do not cover the complete transcriptome but rather just annotated open reading frames. The main outcome of cDNA derived from RNA (depleted of 16S and 23S rRNA) in Salmonella typhi from RNA-seq using Illumina sequencing technology was the correction of the original genome annotation, the identification of transcriptionally active prophage genes, 40 new noncoding RNA sequences, and members of the OmpR regulon (Perkins et al., 2009). The OmpR regulon in Sa. typhi regulates transcription of numerous genes, including those associated with Vi polysaccharide synthesis, two component regulatory systems, and outer membrane porins (Fernandez-Mora et al., 2004). In the case of Vibrio cholerae sequencing of noncoding or small RNA (sRNA) using 454 sequencing technology, 20 known V. cholerae sRNAs, 500 new putative intergenic sRNAs, and 127 putative antisense sRNAs from 407,039 sequence reads were identified. Additionally, a novel sRNA regulator of carbon metabolism was discovered (Liu et al., 2009). II. INDUSTRIAL AND ENVIRONMENTAL TECHNOLOGIES

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FIGURE 15.4 DNA microarray for geneexpression profiling. (A) DNA microarray; (B) array tube. Source: Courtesy: Lancova, K., Dip, R., Antignac, J.P., Le Bizec, B., Elliott, C., Naegeli, H., 2011. Detection of hazardous food contaminants by transcriptomics fingerprinting TrAC. Trends Anal. Chem. 30(2), 181 191.

Similarly, Illumina sequencing technology was also used to sequence the stationary phase stress response transcriptome of Listeria (Oliver et al., 2009) for the comparison of the transcriptomes of an L. monocytogenes strain with an isogenic mutant of the sigma B regulon, which was identified through microarray transcriptome studies as an important regulator of genes involved in virulence, stress response, transcriptional regulation, and carbohydrate metabolism and transport. Using RNA-seq, Oliver et al. (2009) demonstrated that 83% of all L. monocytogenes genes were transcribed in stationary phase and identified 96 genes with significantly higher transcript levels in the parent strain compared with the isogenic mutant indicating sigma B control of these genes. Additionally, RNA-seq also led to the identification of 67 (including 7 novel) noncoding RNA molecules (ncRNAs) transcribed in stationary phase L. monocytogenes and 65 putative sigma B promoters upstream of 82 of the 96 sigma B regulated genes (Oliver et al., 2009). This latter study demonstrates the key role of RNA-seq in our knowledge enrichment, which can ultimately help the scientific community better understand the pathogenicity of L. monocytogenes. This technology will no doubt be applied to additional food microbes. DNA microarrays are still the method of choice due to the high cost of RNA-seq, but in the near future direct sequencing of RNA transcript will be common with advanced technologies, improved bioinformatics software, and lower costs. Indeed, single molecule sequencing such as the Helicos system and other methods under development will also be applicable to RNA sequencing (Ozsolak et al., 2009). Direct RNA sequencing will bypass the need for cloning and/or amplification and reverse transcription of RNA to generate cDNA. Exclusion of these experimental steps will reduce time and cost and also avoid experimental bias during the amplification of the original RNA template.

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An advance in microbial genomics and bioinformatics has led to greater insights into the emergence and spread of food-borne pathogens in outbreak scenarios. One such development is ArrayTrack, an FDA-developed bioinformatics tool for microbial genomics research on molecular characterization of bacterial food-borne pathogens using microarrays. It has been extended to manage and analyze genomics data from bacterial pathogens of human, animal, and food origin. It utilizes the bioinformatics data from public databases such as NCBI, Swiss-Prot, KEGG Pathway, and Gene Ontology for facilitating pathogen detection and characterization. Its platform has been extended to support microbial data with the addition of the Microbial Library and three functional tools (the flag concordance heat map, flag-based hierarchical clustering analysis, and mixed scatter plot). The software can be used as a one-step solution for analyses enabling detection and characterization of bacterial food-borne pathogens. Application of it to different microarray platforms demonstrates its utility for improving the capabilities of the FDA and other government agencies in rapid identification of food-borne bacteria and their genetic traits (e.g., antimicrobial resistance, virulence, etc.) during investigations of threat of bacterial pathogens in accidental or deliberate outbreak scenarios. ArrayTrack is free to use and available to public, private, and academic researchers at http://www.fda.gov/ArrayTrack (Fang et al., 2010).

15.8 FUTURE PROSPECTS It is clear that scientists have made significant progress in the fields of genetic map construction, QTL (quantitative trait loci) mapping, genetical genomics, transcriptome analysis, and proteome analysis; however, additional efforts are needed to further develop omics resources and approaches to fully and effectively use them in animal genetic improvements and biological research. In particular, the following areas of omics research should be emphasized.

15.8.1 Transcriptomics, Proteomics, and Metabolomics One can expect a future where all the “omics” technologies, together with yet-to-be developed “omics” tools, will be analyzed simultaneously in the same sample to provide all levels of information, from the functional (e.g., proteomics, metabolomics) to the mechanistic (e.g., genomics, CHIP-ChIP, miRNA) to the hereditable (e.g., SNP, epigenomics) understanding of the system. Many technical and computational issues will need to be addressed before such a scenario can be reached. The identification and characterization of genes implicated in economically important traits will substantiate genetic progress as the genome sequences for major livestock species are now available. The refinement of ART (AI, ET, IVEP) for all important livestock species will be necessary to realize the full benefits from superior animals. The use of transgenic technologies to introduce single or multiple genes into existing genomes of livestock will play an increasingly larger role in the genetic development of our livestock production in the future. Networks between technologies need to be fully understood to effectively utilize them for improving livestock

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production. Systems biology approach can greatly improve stem cell biology, study and discovery of QTL, effective production of transgenic animals, and efficient ART. In the future, livestock production will rely even more heavily on existing and emerging biotechnological advances to produce our food. However, improvements are still needed in product composition and production efficiency, especially in growth, disease resistance, and reproduction. The attainment of such improvements will depend heavily on our ability to quantify desirable traits, to identify markers linked to gene(s) responsible for those traits, to select or redesign populations of superior individuals, and to propagate those animals efficiently, practically, and economically. The realization of these potential improvements of our livestock production will demand investments in technological expertise, education, and animal resources. Societies and especially countries that possess or are willing to make such investments will be on the leading edge of development of genetically superior livestock for food production in the 21st century.

15.8.2 Integrating Omics Future directions will also include the integration of different omics in dairy and food processing technology. The trends in biological investigation are shifting from individual omics towards integrated omics and system biology. The integration of molecular profiling technologies into animal developmental biology has just begun, and many exciting developments can be anticipated in the near future. Therefore, with high-throughput data acquisition by genomic projects, it is possible and necessary to better integrate multiomics technologies and system approaches that will generate many intriguing insight into omics of dairy and food process technology.

15.9 CHALLENGES AND OPPORTUNITIES IN FOOD OMICS Genomics, transcriptomics, and proteomics have revolutionized our approaches to detection, prevention, and treatment of food-borne pathogens. With development and accessibility of good sequencing platforms in the near future, it is expected that the price for sequencing any genome is not going to be the limiting factor to carry out proteomicsbased applied research to foods. Microbial genome sequencing in particular has evolved from a research tool that can be used to characterize food-borne pathogen isolates as part of routine surveillance systems. Genome sequencing efforts will not only improve outbreak detection and source tracking but will also create large amounts of food-borne pathogen genome sequence data, which will be available for data mining as well as better source attribution and provide new insights into food-borne pathogen biology and transmission. The NGS technology in particular for RNA profiling will lead to discovery of more novel tags that are differentially expressed. While practical uses and application of metagenomics, transcriptomics, and proteomics data and associated tools are less prominent, these tools have also started to yield practical food safety solutions.

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Currently obesity is a major concern. Obesity is the consequence of several interrelated factors such as genetic, environmental, and behavioral factors. One of the challenges of the 21st century is the reduction of obesity, which has increased threefold in Europe since the 1980s. Efforts are being undertaken by the food industry to come up with less caloric products but that provide satiety. Aroma induces satiation, thus, there are great possibilities for managing weight by controlling aroma profiles in foods (Ruijschop and Burgering, 2007). Understanding metabolic pathways (metabolomics) related to aroma biosynthesis for instance can lead to proper manipulation and design for achieving a target aroma profile that induces satiation. More volumetric portions but less caloric foods might also have a positive effect in obesity reduction. Proteins have different functional properties related to texture. Thus, this is another challenging area of application for proteomics.

15.10 CONCLUSION Food quality of human is governed by many factors and in the era of awareness, consumer has expectations of high-sensory quality products, increased functional and nutritional properties, guaranteed safety with less processing, fewer additives used, and fewer technological interventions. At the same time, human food is a complex and a composite entity, and thus its processing and safety is of paramount importance. Proteomics technology using different high-performance separation techniques such as 2D gel electrophoresis, one-dimensional and multidimensional chromatography, combined with highresolution mass spectrometry, has the power to monitor the protein composition of foods and their changes during the production process. Proteomics can also be used to track the state of the raw material, fine-tune processing steps, and predict shelf life. In addition to this, the combo pack utilization of omics technology can lead to the tailored desired quality right from growing these food crops to the table of the consumer. Hence, there is no doubt that omics approaches are on the verge of potentially making major impacts in the areas like food safety, managing food quality, and providing data bank (e.g., The Pathogen-annotated Tracking Resource Network (PATRN) system: A web-based resource to aid food safety, regulatory science, and investigations) (Gopinath et al., 2013) of foodborne pathogens and diseases, to prevent various outbreaks at a first instance, thereby helping in a better way to human mankind. However, there are a number of areas where use of these approaches is still in its infancy and requires scientific interventions.

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Koller, A., Washburn, M.P., Lange, B.M., Andon, N.L., Deciu, C., Haynes, P.A., et al., 2002. Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad. Sci. USA. 99, 11564 11566. Komatsu, S., Tanaka, N., 2004. Rice proteome analysis: A step toward functional analysis of the rice genome. Proteomics 5, 938 949. Krishnan, H.B., Nelson, R.L., 2011. Proteomic analysis of high protein soybean (Glycine max) accessions demonstrates the contribution of novel glycinin subunits. J. Agric. Food Chem. 59, 2432 2439. Kvasnicka, F., 2003. Proteomics: general strategies and application to nutritionally relevant proteins. J. Chromatogr. B 787, 77 89. Lancova, K., Dip, R., Antignac, J.P., Le Bizec, B., Elliott, C., Naegeli, H., 2011. Detection of hazardous food contaminants by transcriptomics fingerprinting TrAC. Trends Anal. Chem. 30 (2), 181 191. Lane, C.S., 2005. Review Mass spectrometry-based proteomics in the life sciences. Cell. Mol. Life Sci. 62, 848 869. Lei, Z., Dai, X., Watson, B.S., Zhao, P.X., Sumner, L.W., 2011. A legume specific protein database (LegProt) improves the number of identified peptides, confidence scores and overall protein identification success rates for legume proteomics. Photochemistry 72, 1020 1027. Liu, J.M., Livny, J., Lawrence, M.S., Kimball, M.D., Waldor, M.K., Camilli, A., 2009. Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing. Nucleic Acid. Res. 37, e46. Liu, T.Y., 2000. Natural and biotech-derived therapeutic proteins: what is the future? Electrophoresis 21, 1914 1917. Lopez-Galvez, G., Juarez, M., Ramos, M., 1995. Two dimensional electrophoresis and immunoblotting for the study of ovine whey protein polymorphism. J. Dairy Res. 62, 311 320. Manso, M.A., Leonil, J., Jan, G., Gagnaire, V., 2005. Application of proteomics to the characterization of milk and dairy products. Int. Dairy J. 15, 845 855. Metzker, M.L., 2010. Sequencing technologies-the next generation. Nat. Rev. Genet. 11, 31 46. Mockler, T.C., Chan, S., Sundaresan, A., Chen, H., Jacobsen, S.E., Ecker, J.R., 2005. Applications of DNA tiling arrays for whole-genome analysis. Genomics 85, 1 15. Morzel, M., Chambon, C., Hamelin, M., Lhoutellier, S., Sayd, T., Monin, G., 2004. Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures. Meat Sci. 67, 689 696. Mundim, T.C., Ramos, A.F., Sartori, R., Dode, M.A., Melo, E.O., Gomes, L.F., et al., 2009. Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation. Genet. Mol. Res. 8, 1398 1407. Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehrhardt, W., Taube, D., 1990. Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels and their solution. Electrophoresis 11, 101 117. O’Farrell, P.Z., Goodman, H.M., O’Farrell, P.H., 1977. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133 1142. Oliver, H.F., Orsi, R.H., Ponnala, L., Keich, U., Wang, W., et al., 2009. Deep RNA sequencing of L. Monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed non coding RNAs. BMC Genomics 10, 641. Olsson, I., Larsson, K., Palmgren, R., Bjellqvist, B., 2002. Organic disulfides as a means to generate streak free two dimensional maps with narrow range basic immobilized pH gradient strips as first product dimension. Proteomics 2, 1630 1632. Ozsolak, F., Platt, A.R., Jones, D.R., Reifenberger, J.G., Sass, L.E., et al., 2009. Direct RNA sequencing. Nature 461, 814 818. Palmer, D.J., Kelly, V.C., Smit, A.M., Kuy, S., Knight, C.G., Cooper, G.J., 2006. Human colostrum: identification of minor proteins in the aqueous phase by proteomics. Proteomics 6, 2208 2216. Patton, W.F., 2002. Detection technologies in proteome analysis. J. Chromatogr. B 771, 3 31. Perkins, T.T., Kingsley, R.A., Fookes, M.C., Gardner, P.P., James, K.D., et al., 2009. A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi. PLoS Genet. 5, 1000569. Quaranta, S., Giuffrida, M.G., Cavaletto, M., Giunta, C., Godovac- Zimmermann, J., Canas, B., et al., 2001. Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins. Electrophoresis 22, 1810 1818. Raikos, V., Hansen, R., Campbell, L., Euston, S.R., 2006. Separation and identification of hen egg protein isoforms using SDS-PAGE and 2 D gel electrophoresis with MALDI-ToF mass spectrometry. Food Chem. 9, 702 710.

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C H A P T E R

16 Omics Approaches in Enzyme Discovery and Engineering Yousef I. Hassan1, Daria Trofimova2, Pavel Samuleev3, Mohammad F. Miah2 and Ting Zhou1 1

Guelph Research and Development Centre, Guelph, ON, Canada 2Queen’s University, Kingston, ON, Canada 3Royal Military College of Canada, Kingston, ON, Canada

16.1 INTRODUCTION All forms of life on Earth, spanning from viruses to mankind, perform thousands of chemical reactions all the time to sustain life. Most of these reactions have slow rates or do not occur under normal conditions (temperature, pressure, etc.) without being catalyzed. With a few exceptions (e.g., ribozymes), the role of biological catalysts is performed by enzymes. Enzyme-catalyzed processes such as fermentations have been used by humans for thousands of years in the production of bread, cheese, as well as various fermented beverages. Since the first enzyme urease was crystallized in 1926 by James B. Sumner from jack beans, scientists in the 19th and 20th centuries began to study enzymatic actions in a systematic manner paying more attention for functional details. Today, more than 5000 different enzymes are known and characterized, and many of them have been crystallized to determine their structure by X-ray crystallography. In this chapter, we introduce the reader to pioneering approaches that aid in the discovery of novel enzymes with unique functions or re-designing enzymes already in use for better yields and/or unique usage conditions. Finally, we list examples of enzymes that were refined lately using protein bioengineering, molecular modeling, and targeted mutations for commercial-scale applications within different industries.

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16.2 NOVEL ENZYMES DISCOVERY FOR INDUSTRIAL APPLICATIONS Nature is full of living organisms that show high degree of adaption to their environment. Extreme temperatures (high or low), pressure, and moisture form no barriers in front of these organisms to grow and expand. Through evolution, these organisms have accustomed to the surrounding unfavorable conditions to reproduce and even thrive. The biggest tools that help them to overcome the harsh environmental factors are outstanding enzymatic systems that remain active under such conditions. Identifying such organisms and isolating the responsible enzymatic systems for later industrial applications is considered a very attractive prospective nowadays. Fig. 16.1 outlines the general process of isolating novel enzymes with unique functions. The first phase usually focuses on finding a living organism that performs the required function; in other words, it contains the desirable enzyme. Different isolates, strains, and living sources can be screened in order to test the availability of such enzyme. Early attempts to obtain active, highly expressed, or thermostable enzymes involved screening different sources of fungi and bacteria. For instance, in order to identify enzymes that can tolerate high temperatures, researchers investigated thermophilic microorganisms collected from hot springs or deep sea hydrothermal vents (Haki and Rakshit, 2003). These thermostable enzymes have huge industrial potential. One of the industrial enzymes that is favored by the industry for such characteristics is α-amylase. This enzyme is used in the liquefaction process of starch where the reaction occurs at about 105 C for several minutes

Determine what are the desired characteristics of the requested enzyme (tolerance for high temperatures/pH....)

Screen different organisms/isolates for the desired functions/characteristics in a standardized assays

Phase 1

Characterization of the new enzymatic process (time, temperature, pH, reproducibility, efficiency, purity of cultures, growth conditions....)

Mechanistic purification and enzyme/gene identification

Phase 2

Gene cloning, expression, purification, enzyme 3D structure elucidation, and optimization with site-directed mutagenesis

Phase 3

FIGURE 16.1 A general outline for phases and steps involved in novel enzyme(s) isolation and optimization by protein engineering for the industrial use.

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followed by another hour or more at 90 C. Termamyl (commercial name) is an example of a thermostable α-amylase obtained from the thermophilic bacteria, Bacillus licheniformis and Geobacillus stearothermophilus (Kandra et al., 2006). Later, the thermostable ortholog was heterologously expressed in faster growing and easier-to-handle hosts such as Escherichia coli (Nevalainen and Peterson, 2014; Elena et al., 2014). Artificial selection methods can be utilized in this phase too. In the case of microorganisms, growth patterns on media containing high levels of the tested substrate are usually monitored. The incorporation of such substrates into growth media is more likely to keep the selective pressure on the involved enzymatic systems hence induce their expression at higher levels. Furthermore, monitoring the conversion of substrates to final products is another way to sort out different isolates and strains. Mass spectrometry analysis (LC-MS/MS and GC-MS) is a very valuable technique in this regard. The only drawback of this analytical method is its high costs and labor demands, which in reality can hinder/slow the screening of large sets of clones/strains/isolates. In most cases, alternative sorting/screening methods can be optimized such as robust colorimetric assays that can track the appearance/disappearance of certain by-products and intermediates within the reaction or the reduced toxicity of some compounds. For example, a MTT-based bioassay (colorimetric assay for assessing cell viability) was developed and optimized for detecting and tracking enzymatic and nonenzymatic zearalenone and deoxynivalenol detoxifications (Cetin and Bullerman, 2005a,b, 2006; Hassan and Bullerman, 2008). Such colorimetric assays can facilitate screening large sets of factors/ conditions in a reasonable time frame. The second phase, after the identification of a source organism that contains the desired function, is to isolate and purify the target enzyme(s) as well as amplify and clone its encoding gene(s). There is more than one approach to tackle such tasks related to this phase; spanning conventional biochemical methods to state-of-the-art molecular techniques. Usually, the most suitable methods are considered after thorough discussions of the scientific approach, the availability of research tools, the amount of expected troubleshooting, and the level of technical expertise required. For example, researchers might take advantage of the ability of enzymes to recognize and bind specific substrate (s) to isolate and purify them in affinity-based techniques. These include antibody affinity chromatography or phage-display technologies (Fig. 16.2). A phage-display technology can be effectively used for small enzymes (up to 1200 amino acids) with multiple rounds of substrate affinity enrichments. After the final round of selection, cDNA targets can be amplified by polymerase chain reaction (PCR) and used to generate a new library that can be enriched once again for its substrate-binding/affinity (Sheehan and Marasco, 2015; Eldridge and Weiss, 2015). Proteomic techniques such as the comparisons of proteomic profile of microorganism before and after challenging with a particular substrate, which induces the expression of substrate-related transcripts, using 2D/ differential-gel electrophoresis can sometimes identify unique proteins that are likely to be involved in the microorganism’s response elicited by the substrate. Furthermore, control strains (that lack any activity) can also be included in these comparisons. The second approach is usually preferred as it helps to eliminate/reduce stress-response transcripts that are not directly involved in the metabolic pathways under investigation. Upregulated/unique protein spots identified on the 2D gels can be excised and later sent

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FIGURE 16.2 The phage-display approach connects the genetic material of source organism (encoded within the phage) with peptides/proteins expressed and displayed at the outer coat of the same phages. This powerful technique simplifies the process of identifying novel functional enzymes.

to mass spectrometry facilities for identification (Friedman et al., 2009). Reverse translation is used to design DNA primers or labeled probes, which can be used to mine for cDNA constructs and colonies that possibly encode the genes/enzymes carrying the desired structural/functional domains (Renzone et al., 2005). One of the major hurdles in the 2D/differential-gels approach is the magnitude of possible changes and the day-today variations. Experimentally speaking, it is possible to detect large numbers of proteomic changes, which makes sorting and tracking such changes a daunting task. To overcome such hurdles, protein fractionation protocols (coupled with enzyme-activity tracking) can be adopted before proceeding with differential-gel electrophoresis (Brewis and Brennan, 2010). Another feasible approach is to extract total mRNA or genomic DNA of the desired strains and establish cDNA and genomic DNA libraries that encode for the preferred enzymes. By amplifying these libraries and introducing them in exogenous hosts such as E. coli for heterologous protein expression, clones harboring unique cDNA/DNA fragments can be screened and identified for their enzymatic activity (Aharoni et al., 2005; Bhatnagar et al., 1989).

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Entire genome sequencing and de novo genome assemblies have become feasible approaches recently (Hassan et al., 2015). Deep sequencing can determine all open reading frames (ORF) that encode for proteins/enzymes/polypeptides within the obtained strains/genome in a matter of days. Sequencing microorganism’s entire genome was once a very costly task, which required joint efforts of several laboratories and collaborators to be accomplished (Bertelli and Greub, 2013; Soon et al., 2013). With the recent advancements in next-generation sequencing technologies, such tasks are more affordable nowadays and the sequencing of multiple bacterial genomes can be simultaneously accomplished in a fraction of the pervious costs. The annotation and assembly of the acquired sequences into genome contigs is another challenge for this approach but it can be accomplished with an acceptable degree of accuracy especially if more than one sequencing platform is used (a combination of Illumina and PacBio for example) to complete the draft genome. Recent pan-genomics tools do make comparisons between different isolates/strains more robust (Fig. 16.3) and less cumbersome (Sun et al., 2015; Xiao et al., 2015), yet and in order to narrow down the number of genetic clusters of interest to a tangible range with some confidence, the biochemical/mechanistic nature of the investigated enzymes/reactions should be established earlier in the process to complement the genomic data.

FIGURE 16.3 Capable tools and databases with easy access web interfaces are available for the comparative genomics of bacteria: (left panel). The Integrated Microbial Genomes (http://img.jgi.doe.gov/) is powerful portal with free microbial genomic data. The IMG genomic annotation proceeds with several functional references such as Gene Ontology (GO), Pfam, Clusters of Orthologous Groups (COG), KEGG, TIGRfam, and MetaCyc; (right panel). The Pathosystems Resource Integration Center (https://www.patricbrc.org/portal/portal/patric/Home) is another dominant database (but with a more pathogens-oriented focus) that includes sequence typing data, genomes, transcriptomes, protein structures, and interactions. Bacterial genomes are annotated using the RAST server when deposited in PATRIC.

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16.3 MOLECULAR ENGINEERING OF AVAILABLE INDUSTRIAL ENZYMES Quite often the useful enzymes are expressed in minute amounts and mixed up with many other proteins, thus the purification costs of such enzymes in the original endogenous organisms are rather high. At the end of the 1980s, new genetic technologies, heterologous expression, and protein bioengineering techniques became available, which boosted enzyme production and optimization dramatically. These technologies made cloning, tagging, purifying, and functionally characterising enzymes far easier. A simple PCR cloning strategy using primers deduced from identified ORF can help in subcloning such frames into suitable prokaryotic expression vectors (usually with 6xHis or GST tags). Subsequent expression within a suitable host will yield enzyme(s) that can be tested for activity and specificity later. If the genetic origin of the desired enzymes is prokaryotic, then no major concerns over host-selection should exist, as these hosts are most likely to share the same posttranslational modifications/mechanisms. Otherwise, such as the cases of expressing mammalian genes in prokaryotic hosts, close attention should be paid to such modifications in addition to the host’s codon bias. In the past decade, the production of inexpensive enzymes became feasible. Final yields of recombinant enzymes produced using biofermenters/bioreactors have increased tremendously, by a factor up to 100. This has led to much reduced prices, typically lower by a factor of 10, as compared to enzymes produced by conventional means (Buchholz et al., 2012). Lipolase by Novozymes was the first industrial lipase used in detergents. It was originally isolated from Thermomyces lanuginosus with a low level of protein expression. Only by using heterologous expression within Aspergillus oryzae, the production of this lipase became economically possible. Presently, the most common hosts of expression for industrial enzymes belong to the genus Bacillus (Gram-positive bacteria) or Aspergillus or Trichoderma (filamentous fungi). Furthermore, most enzymes produced commercially are engineered to be secreted outside the cell (extracellular), which simplifies the purification process drastically. The most powerful tool for improving enzymes, known currently, is protein engineering and re-designing. Protein engineering is the process of constructing proteins (enzymes) with altered properties or structures. Protein engineering employs a combination of tool sets that allow any amino acid within the protein sequence to be replaced by one of the 19 naturally occurring amino acids in order to change enzyme’s properties, stability, conversion efficiency, or expression level. Two main approaches are used in protein engineering. One is the random generation of mutations within the target gene by PCR methods and then screening the resulting mutants for enhanced properties. Mutants with improved properties can undergo additional rounds of mutagenesis until they fulfill the criteria of the final desired product. This method is named directed evolution (Packer and Liu, 2015; Lane and Seelig, 2014; Kurtzman et al., 2001). An alternative approach is to introduce specific mutations based on the known structural and functional properties of the enzyme. At the molecular level, it is noteworthy to mention that enzymes may differ in their amino acid sequence or source organism but still catalyze identical chemical reaction. Such enzymes are called isozymes. The kinetics

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Crystal structure of alkaline cellulase K showing the enzyme’s active pocket where the substrate binds (different color). The catalytic pocket is formed by the 265H, 269W, 296Y, 299E, 333H, 373E, 485E, 519W, 524K, and 526E amino acids residues.

FIGURE 16.4 The catalytic domain of the alkaline cellulase K enzyme showing the binding site and some important residues involved in its function.

and regulatory properties of isozymes may differ substantially. The later method requires the presence of a solved 3D crystal structure (Fig. 16.4) of the targeted enzyme (Shirai et al., 2001), and the knowledge of substrate-binding sites/active pocket of the enzyme, folding properties of different amino acids, and site-directed mutagenesis (Foley and Burkart, 2007; Yuan et al., 2005) protocol optimization. Usually, the amino acid sequence of the engineered enzyme is aligned against prokaryotic, archeal, and eukaryotic orthologs and homologs to highlight consensus regions. Structural features (outer and inner positions, distance from the active cavity, etc.) of these consensus regions are closely scrutinized. Drastic mutations that affect such regions are usually avoided as such conserved domains are most likely to be critical for enzyme activity; hence their conservation throughout evolution in diverse life domains. In real practice, a combination of both of the above approaches (random directed evolution and targeted site-directed mutagenesis) might be the most fruitful in some cases. Finally, enzymes are rather expensive and susceptible catalysts. However, their attachment to a solid support allows multiple reuses of the enzymes, increases their stability, and prevents contamination of the final product(s) by the catalysts (Ding et al., 2015). Immobilization can be achieved through the physical or chemical attachment of enzyme molecules (or whole-cells) to a solid support. There are several techniques to immobilize enzymes such as covalent binding, ionic binding, physical adsorption, and entrapment of enzymes into polymer matrices or membranes (Sulaiman et al., 2015; Barbosa et al., 2015; Es et al., 2015). Some examples are cited later.

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16.4 INDUSTRIAL APPLICATIONS OF ENZYMES AND EXAMPLES OF BIOENGINEERED ENZYMES CURRENTLY IN COMMON USE Modern manufacturers operating within the food, biofuel, textile, grain processing, and many other industries cannot succeed without the extensive use of enzymes. Enzymes perform processes faster, more efficiently, and help reduce chemical wastes. Almost 65% of the industrial enzymes belong to the class of hydrolytic enzymes (Fig. 16.5). These enzymes accelerate the hydrolysis reactions of different types of molecules including proteins (proteases), carbohydrates (carboxylases), and fats, lipids, and oils (lipases and phospholipases). α-Amylases (hydrolytic enzymes) are the most utilized enzymes in industry due to the fact that their target substrate, starch, is one of the most abundant biopolymers on Earth. Some applications of α-amylases include starch degradation in corn sirup production; degradation of starch-containing stains in laundry and dishwashing; clarification of fruit juices; controlling the viscosity of oil-well fluids; removal of the protective layer of starch in textile manufacturing; and many more. The leading producers of industrial enzymes worldwide are: Novozymes (Bagsværd, Denmark), Danisco including Genencor enzymes (Copenhagen, Denmark), DSM (Heerlen, the Netherlands), National Enzyme Company (Forsyth, MO, United States), AB Enzymes (Darmstadt, Germany), and Amano Pharmaceuticals (Nagoya, Japan). An example of Novozymes sales is shown in Fig. 16.6 to give an idea on which industry uses enzymes the most.

16.4.1 Enzymes in the Food Industry Enzymes have been used in the processing of food since c.7000 BCE. The first known usages of natural enzymes (i.e., bacteria, fungi, or yeasts) were in the production of bread, yogurt, and fermented beverages. Today, enzymes are still essential for the food industry. For example, a major application of enzymes in the dairy industry is to coagulate milk, which is the first step in the production of cheese. Enzymes from both microbial and animal sources are used for this process. In the past, calf rennet was the sole source of enzymes for coagulation. The rennet contains two useful enzymes, chymosin and pepsin.

FIGURE 16.5

Distribution of enzymes (based on their classification) used in current industrial applications reveals interesting facts about the extensive use of hydrolytic enzymes.

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FIGURE 16.6 Enzyme sales in 2012 for a leading enzyme and biotechnology company, Novozymes, reflect the growing industries and sectors that utilize these biocatalysts in their production lines.

Today, inexpensive microbial enzymes are used in the process instead of the standard animal rennet. Recombinant DNA techniques have made it possible to clone the actual gene of calf chymosin into selected bacteria, yeasts, and fungi (Vallejo et al., 2008; Kapralek et al., 1998). Another example of an enzymatic application in dairy production is the lactase (β-galactosidase) enzyme, which is used commonly to hydrolyze lactose, the sugar of milk, and to increase milk-digestibility/acceptance among lactose-intolerant people. Lactase is also used to improve the solubility or sweetness of various dairy products (Horner et al., 2011; Jarvis and Miller, 2002; Pivarnik et al., 1995). Another large market for enzymes is in the baking industry. For example, amylases are added to the dough to produce high-quality bread. Furthermore, α-amylases can increase the shelf life of bread by preserving its freshness. The significant antistaling effect of α-amylase is connected to starch modifications that take place near temperatures where most of the starch start to gelatinize. The resulting modified starch granules remain more flexible during storage, giving breads produced with α-amylases, a softer and more elastic crumbs compared with breads produced with just distilled monoglycerides emulsifiers (Palacios et al., 2004; Leon et al., 2002; Kulp and Ponte, 1981). Bakeries utilize in addition to α-amylases other enzymes such as hemicellulases, xylanases, lipases, and oxidoreductases that can improve the quality of the final products directly or indirectly through improving the strength of formed gluten networks (Saarinen et al., 2012; Dornez et al., 2011). Enzymes are also used in the juice industry. The plant material, such as fruits, is treated with enzyme mixtures to breakdown cell walls and gives higher juice yield, improve color and aroma of extracts, and clear juices from indigestible particles (Bogra et al., 2013). Some enzymes used within the food industry can be called “universal” as they have multiple applications within multiple industries. A good example of such enzymes is transglutaminase (EC 2.3.2.13). This enzyme catalyzes the formation of an isopeptide bond between the amino group in a lysine residue and the carboxyamide group in a glutamine residue. This reaction can form a crosslink between two amino acids within the same or different molecules of protein (Kieliszek and Misiewicz, 2014; Porta et al., 2011). Such crosslinking of proteins leads to the formation of high-molecular proteinaceous biopolymers that influence the rheological properties (i.e., hydration, gelation, emulsification, and foaming) of final products (Wilcox and Swaisgood, 2002).

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Historically, transglutaminase has long been used in Japan for the processing of special ground fish paste known as surimi (Banlue et al., 2010). The first microbial transglutaminase was purified in 1989 from Streptoverticillium sp. (Mariniello and Porta, 2005) and was commercialized since then. Transglutaminase is heavily exploited on the industrial scale to alter whey, casein, soy, meat, and fish proteins. The enzyme is simply known as the “meat glue.” It can produce restructured meat by binding meat pieces together. The binding process involves the use of both caseinate and transglutaminase simultaneously (Ma et al., 2012). The caseinate, when treated with transglutaminase, acts as a glue to hold food components together. The above restructuring of minced meat can effectively be used to produce large variety of consumer-appealing commodities. The manufacturing of processed sausages with improved textural qualities is one typical application of transglutaminase. The internal structure of sausages, when strengthened by transglutaminase, can be made more resistant to high temperatures and freeze/thaw cycles due to the artificially formed crosslinking bonds (Ahhmed et al., 2007). Ham is another example. In this case, transglutaminase improves sliceability, a very important feature to ham manufacturers. In a similar fashion, the polymerization of milk proteins with transglutaminase results in the formation of protein films that can improve the rheological properties of dairy products (Rossa et al., 2011). Here the enzyme helps to keep yogurt in a homogeneous state, preventing unsightly separation. In the baking industry, transglutaminase is extensively used to increase the quality of flour, as well as the texture of bread (Moore et al., 2006) and cooked pasta (Kuraishi et al., 1997). Lastly, by utilizing the unique properties of transglutaminase, uncommon textures and odd combinations of different food delicacies such as noodles made from shrimp or peanut butter pasta can be created without the addition of flower or eggs (Marques et al., 2010; Buchert et al., 2010). Due to the paramount importance of transglutaminases, they were targeted by molecular engineering for better processing qualities. For example, Streptomyces transglutaminase is extensively used in food processing. The enzyme is naturally synthesized as zymogen (proenzyme), which is processed later to produce the active form through N-terminal pro-peptide cleavage and removal. When the α-helix (37G-42S) within the propeptide was substituted with three glycines and three alanines, respectively, the mutants exhibited higher specific activity and the efficiency of propeptide cleavage was enhanced (Chen et al., 2013). Further mutations were constructed by introducing linker peptides within the C-terminus of the propeptide. Mutants with GS (GGGGS) and PT (PTPPTTPT) linker peptides exhibited 1.28- and 1.5-fold higher specific activities than the wild-type enzyme, respectively (Chen et al., 2013). By using nuclear magnetic resonance, Shimba et al. (2002) have shown that amino acids residues exhibiting relatively high flexibility within transglutaminases are localized to the N-terminal region. Furthermore, this terminus was shown to influence substrate-binding. Several mutants with higher activity were obtained by targeting this terminus through site-directed mutagenesis (del1-2, del1-3, and S2R) (Shimba et al., 2002). Lately, the expression conditions of a more readily soluble recombinant transglutaminase from Zea mays were optimized in Pichia pastoris. The coding sequence was optimized according to the codon bias of P. pastoris and successfully transformed into the P. pastoris GS115 strain by electroporation. During expression, a final concentration of 0.5% methanol

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was used and the transglutaminase enzyme was successfully purified by affinity binding yielding 4.4 mg/L enzyme and a specific activity close to 0.889 U/mg (Li et al., 2013). Similarly, Date et al. (2004) used a chimeric proregion consisting of Streptomyces mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of S. mobaraensis transglutaminase in Corynebacterium glutamicum. The chimeric proregion was shown to have an increased secretion rate compared to the native proregion (Date et al., 2004). As a final point, the extensive use of enzymes within the food industry yields a large number of processed foods that contain residual enzymes. At that point, enzymes used during processing become incorporated into an inseparable part of that particular product. This is the main reason why regulations about safe enzyme-usage are pivotal (Pico et al., 1999). In Canada, enzymes implemented in food processing are regulated through the food additives act. Health Canada is responsible for conducting a premarket safety assessment of any enzyme to be used by the industry and for approving their incorporation in such processes. Among the factors that influence the safety assessment is the source organism. In general, edible plants and animals have a history of safe usage in food industry as sources of enzymes. Any microorganism that will be used for food-enzymes production must be well characterized. The final enzyme preparation should not contain any pathogens, toxins, or antibiotics. Soil microorganisms, which humans are commonly exposed to through environment and diet, fall within the accepted group of microorganisms that can be used as natural sources of industrial enzymes (Ladics, 2008; Woodcock et al., 2007). Another primary consideration is having the enzyme formulation itself, in addition to the commercialized final product, to pass toxicity tests before the final clearance (Ku et al., 2007; Olempska-Beer et al., 2006).

16.4.2 Enzymes in the Animal Feed Industry Animals digest food with the help of enzymes. However, the available levels of endogenous enzymes are not sufficient to process the entire feed intake with high-conversion and productivity rates. For instance, swine with their naturally secreted enzymes will only be able to utilize about one-fifth of their administrated daily ration (Bezerra et al., 2013). The largest component of animal feed is grains such as wheat, barley, rye, corn, and sorghum. These cereals contain many antinutritional factors, which restrict their value to animals. Overcoming the above problems is possible by using enzymes as feed additives. Examples of enzymes used in the animal feed are xylanases, β-glucanases, phytases, proteases, and amylases. Some factors that govern the successful use of enzymes in animal industry are the speed that these enzymes act upon the substrate/feed. In general, interactions between enzymes and substrates should be as quick as possible and during the feed passage through the animal’s digestive system (hours range). Another factor is the enzyme’s stability at pH values associated with the gastrointestinal tracts (de Vries et al., 2014; Wu et al., 2014). Historically, enzymes were first used in animal feed around the mid-1980s. β-Glucanase was added to barley-based rations for poultry. Since that time, the poultry industry became the biggest user of feed-enzymes (Cowieson and Masey O’Neill, 2013). Poultry and swine do not have the endogenous enzymes needed to breakdown fiber and adding fiber-degrading

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enzymes is very beneficial in this case. Numerous studies have shown that the growth performance of chicks was enhanced by adding xylanase to their diet (Pettersson and Aman, 1989; Preston et al., 2000; Wang et al., 2005). The fiber of cereals mainly consists of polysaccharides such as arabinoxylan in wheat and rye, and β-glucan in barley and oats. Some of the enzymes that can target and degrade these polysaccharides are xylanases (EC 3.2.1.8) in wheat-based diets and β-glucanases (EC 3.2.1.6) in barley-based ones. Phytic acid is a phosphorus-containing compound found in abundance in grains and seeds. Nonruminant animals cannot utilize this compound as they lack the required enzymes involved in its metabolism. Low absorbability of phytate leads to its intact passage through the gastrointestinal tracts of animals’ causing elevated levels of phosphorus in manure. Excess phosphorus excretion leads later to a problematic environmental-pollution, mostly noticed after rain and water runoff from agricultural lands. Another disadvantage of high levels of phytic acid in the diet is its ability to chelate metals (such as zinc, iron, calcium, and magnesium), making them to be poorly absorbed and causing symptoms of deficiency and low productivity in the affected animals. Due to the low activity levels of endogenous phytases in both poultry and swine particularly, supplementing their diet with phytases obtained from external sources is considered a widely acceptable practice within the modern animal industry (Maller et al., 2013). The exogenous phytase accelerates the hydrolysis of orthophosphate from phytic acid, producing inositol phosphate intermediates, and myo-inositol that can be readily absorbed. Phytase as an enzyme exists in two forms, either as 3-phytase (EC 3.1.3.8), where the phosphate group at position C3 is the first to be hydrolyzed, or as 6-phytase (EC 3.1.3.26), where the hydrolysis reaction of the phosphate group takes place at the C6 position first. The main source of commercial phytases is fungal, although bacterial and yeast enzymes have been isolated and purified recently (Luo et al., 2007). Currently, a phytase obtained from Aspergillus species accounts for the largest source of phytases on the market (Shi et al., 2009; Casey and Walsh, 2004; Troesch et al., 2013). Site-directed mutagenesis optimization of Bacillus amyloliquefaciens DSM 1061 phytase was performed to increase its activity (Xu et al., 2015). Mutations targeting its surface (D148E, S197E, and N156E) or nearby its active site (D52E) were selected. Analysis of the generated enzyme variants showed that mutants D148E and S197E remarkably had increased activities by 35% and 13% over a wide temperature range of 40 75 C in comparison with the wild-type enzyme. In a different study, the ability of inner-coat oxalate decarboxylase (OxdD) to drive the display of an endogenous and active phytase at the spore surface was tested. The recombinant OxdD-Phytase fusion was demonstrated to successfully translocate to Bacillus subtilis spore outer-coat layer in a functional format. This study showed the potential of using spores as enzyme-delivery vehicles that can aid in overcoming the harsh conditions connected with feed preparation/processing and gastrointestinal tracts (Potot et al., 2010).

16.4.3 Corn and Cellulose Processing One of the high-volume agricultural processes that involve converting raw materials to final products is the wet milling of grains. A good example of such a process is the wet milling of corn. The final products of this process are: (1) corn starch (converted into corn

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FIGURE 16.7 Steps involved in starch and cellulose conversions to glucose, fructose, or ethanol with the aid of different enzymes.

sirup or ethanol in a later stage), (2) corn gluten meal (for use in animal feed), and (3) corn oil. The wet milling consists of a series of steps, some of which utilize enzymes. Important enzymatic steps are the hydrolysis of starch by α-amylase, hydrolysis of dextrin with glucoamylase and isomerization of glucose by glucose isomerase (Fig. 16.7). Because of the large magnitude of these processes, these enzymes are produced in greater quantities than any other enzymes within the industry. Starch is composed of long chains of glucosyl residues linked by α-(1 4) glycosidic bonds, with branches of amylopectin. In general, α-amylases attack the interior bonds of starch chains faster than those near chain termini, and a mixture of products with intermediate numbers of glucosyl residues, called dextrin, is formed in preference to glucose or maltose. This step is called liquefaction. The slurry of starch granules is mixed with α-amylases (commonly obtained from B. licheniformis or G. stearothermophilus), and the mixture is held at 105 C and pH 5.6 6.5 for several minutes. This dissolves the granules and exposes starch chains to the enzyme. The final dextrin mixture is often held for another hour or more at about 90 C with fresh α-amylase supplementation. The usage of α-amylases in the liquefaction step helps first in shortening the length of starch chains so they do not form gels at lower temperatures and second, exposes more chain-ends in preparation for the glucoamylase enzyme action. α-Amylases in the above reactions should survive some of the most extreme temperatures that have ever been reported for any enzyme with an astonishing stability and functionality. For that reason, they are often targeted by protein engineering for stability improvement. For example, the oxidative stability of α-amylase (obtained from Thermotoga maritima) was improved substantially by mutating the following methionine residues at positions 43, 44, 55, and 62 to oxidative-resistant alanine residues (Ozturk et al., 2013). A 50% residual activity (in the presence of 100 mM H2O2) was observed in the M55A variant.

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Furthermore, the stability and catalytic efficiency of an α-amylase (from B. subtilis) was improved under acidic conditions by mutating four basic histidine (His) residues H222, H275, H293, and H310 in the catalytic domain and replacing them with acidic aspartic acids (Asp). Of note, 45% 92% of initial activity was retained (at pH 4.5 and 25 C for 24 h) after mutation. The introduced changes around the enzyme catalytic domain increased hydrogen bonds and salt bridges interactions as revealed by the conducted structural modeling hence significantly improved protein’s stability at lower pHs (Yang et al., 2013). Next, glucoamylase hydrolyzes the dextrin mixture to glucose. This process is called saccharification. Glucoamylase attacks the nonreducing ends of malto-oligosaccharide chains to produce glucose. Glucoamylase is a very slow enzyme but this is compensated by its low cost. After this step, the glucose solution can further be used to make ethanol or high fructose sirup. Glucose isomerase (EC 5.3.1.5) converts glucose to fructose by isomerization. The glucose solution is passed through a column containing glucose isomerase immobilized on porous solid particles. Because this enzyme is expensive, it must be immobilized so that it remains within the column and can be used for multiple times. There are two common ways to immobilize glucose isomerase on a solid support: electrostatically on charged carriers (such as granular DEAE-cellulose or silica-based material) or covalently crosslinked with whole or macerated Streptomyces cells by glutaraldehyde. Currently, glucose isomerase is derived from Streptomyces bacteria. This is, by a large margin, the biggest use of an immobilized enzyme in the world (Bhosale et al., 1996). Glucose can be obtained not only from starch, but also from cellulose. Stalks, leaves, and husks of corn plants, wood chips, and sawdust are all used as sources of cellulose (Kawaguchi et al., 2016). Such biomass contains a lot of lignocellulosic fibers. It is more challenging to isolate glucose from these fibers than from starch-based materials. However, it is not impossible. Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin. Cellulose consists of long chains of glucose molecules, the same as starch, but connected by a different type of chemical bonds, β-(1 4) glycosidic, that is more resistant to hydrolysis. The structure of cellulose makes it difficult to degrade unless it is pretreated prior to the enzymatic hydrolysis. Cellic CTec3 from Novozymes is an example of a state-of-the-art cellulase and hemicellulase complex that allows the conversion of lignocellulosic materials to glucose. A novel concept of expressing cell wall degrading (CWD) enzymes such as xylanases in plant feedstocks was introduced recently (Shen et al., 2012). This approach if successful can reduce the amount of enzymes required for feedstock pretreatments and hydrolysis during bioprocessing to release soluble sugars. In planta expression of xylanases reduces biomass yield and plant fertility and in order to overcome this obstacle, a thermostable xylanase with a thermostable self-splicing bacterial intein to control the xylanase activity was engineered. Intein-modified variants were selected that have ,10% wild-type enzymatic activity but recover .60% enzymatic activity upon intein self-splicing at temperatures .59 C (Shen et al., 2012). The greenhouse-grown maize expressing the bioengineered enzyme showed normal seeds and fertility while processing the dried maize stover by temperature-regulated activation and hydrolysis in a cocktail of commercial CWD enzymes produced .90% theoretical glucose and .63% theoretical xylose yields (Shen et al., 2012).

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16.4.4 Enzymes in Surfactants and Detergents Laundry detergents need to remove all sorts of soils and stains from different kinds of fabric. In general, water-soluble components of soil are easy to remove. Water-insoluble stains can be removed by using surfactants; however, the results are often unsatisfactory. Surfactants remove stains by physically detaching them from the fabric. Lipids, proteins, and carbohydrates can be decomposed with the help of enzymes into smaller molecules, which are generally more soluble and easier to remove by surfactants. Yet for an absolute removal of stains, the combined effect of enzymes, surfactants, and mechanical agitation is required. Proteases and amylases can degrade protein and starch particles, respectively. Fats and oils can be removed with the help of lipase enzymes even at temperatures where the fatty material is still in a solid form. Smaller particles of dirt are usually difficult to remove from garments because they are trapped into the fabric. The removal of such particles can be improved with the use of cellulases. Such enzymes remove cellulose fibrils from the surface of the fabric, making it smoother. As dirt adheres to fabric surface by a glue of proteinaceous, starchy, or fatty material, enzymes may assist in removing the dirt even though it does not attack the dirt directly. Enzymes are extremely beneficial additives to laundry detergents; however, they are not easy to introduce into a detergent mixture. Surfactants, extreme wash temperatures of 4 90 C, pH 7 11, ionic strength, bleach, the presence of other enzymes (especially proteases), and mechanical handling all have deteriorating effects on the enzyme(s) performance and stability during the washing process as well as prolonged storage. Here, enzyme-engineering plays a pivotal role for overcoming these obstacles and finding empirical solutions. The first enzyme-containing detergent was presented to the market back in 1913 by Ro¨hm and Haas (Germany). The enzymes cocktail was extracted from porcine pancreas and was mixed with Burnus detergent. This product did not work well. The extremely poor performance of the mixture was due to the high alkalinity of surfactants in the mix. A more successful attempt to use enzymes in laundry detergent was made in 1963 by Novo (Bagsværd, Denmark). A bacterial protease, subtilisin A from Bacillus species, was developed and marketed as Alcalase. This enzyme appeared to be more alkali-tolerant and has effectively been used in detergents since then. In the early 1970s, the first carboxylase enzyme, amylase, was developed and launched in the market by Novozymes. In those years, all enzymes were isolated from different kinds of bacteria and fungi. However, at the end of 1980s, advances in genetic technologies, heterologous protein expression, and engineering techniques have dramatically boosted enzyme production and optimization. For example, protein expression has been greatly improved by the use of heterologous expression systems. In such systems, a target non-native gene (or parts of a gene) can be expressed in a different host organism, which is more convenient to manipulate. In addition, the production of low-cost enzymes became feasible as well as the gene modification of enzymes to achieve specific properties such as chemical or thermal stability. Lipolase (one of the lipases) by Novozymes was the first commercially available lipase for detergents in 1988. It was originally isolated from T. lanuginosus with a low level of protein expression. However, large quantities of this lipase were achieved by using the heterologous expression system, A. oryzae (Yaver et al., 2000; Prathumpai et al., 2004).

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At present, three types of enzymes are exploited in detergents: proteases, carboxylases, and lipases (Aehle, 2007). Proteases are used extensively to remove blood or food stains (Kotb, 2015). Most commercial detergents contain subtilisins (EC 3.4.21.62). These enzymes are serine endo-peptidases. They all have very broad substrate specificity and mainly vary in their pH 6 12 and temperature of 10 80 C tolerance, bleach sensitivity, and Ca21 demand (for the catalytic process) (Vojcic et al., 2015). The inclusion of bleach in the detergent mixture can negatively impact the stability of proteases during long-term storage. The inactivation of these proteases in the presence of bleach is attributed to the oxidation of some amino acids (specifically, a methionine residue that is situated next to a serine within the active site of the enzyme). Engineering these proteins by the replacement of the aforementioned methionine residue within the active site of subtilisin A has improved their long-term storage in the presence of bleach (Saeki et al., 2007). Two types of carboxylases are utilized in modern detergents: α-amylases and cellulases. α-Amylases (EC 3.2.2.1) catalyze the endo-hydrolysis of 1,4-α-D-glycosidic linkages in polysaccharides. Generally speaking, if the soil starch material is in the amorphous form, it will be difficult to remove from garment and cloth surfaces without boilwashing (heat). Furthermore, gelatinized starch may form a film on the fabric that can result in an increased accumulation of particulate soil after washing. Such contaminations are much harder to remove than just the starch alone. As a result, white laundry items turn gray after repeated wash cycles. The presence of α-amylase makes boil-washing unnecessary and enhances performance of detergents at lower wash-temperatures. The incorporation of α-amylases in laundry detergents maintains or even contributes to increase whitening of dingy fabrics and prevent the graying of white ones (Ee and Misset, 1997). Another type of carboxylases used in laundry detergent is cellulases. Such enzymes (EC 3.2.1.4) accelerate the endo-hydrolysis of 1,4-β-D-glycosidic linkages in cellulose, lichenin, and cereal β-D-glucans. These enzymes have different mechanisms of action: they do not degrade the soil itself but rather prevent trapping it into the cotton yarn. The enzyme hydrolyzes and removes exposed bonds in the cellulose fibrils and pills. This in turn decreases the trapped soil within the yarn as well as softens and colorbrightens the fabric in general. Lipases (EC 3.1.1.3) hydrolyze triglycerides such as fat, lipids, and oils. Triglycerides are extremely hydrophobic molecules, which are very difficult to remove from laundry at low temperatures (Jiewei et al., 2014). The products of triglycerides hydrolysis are free fatty acids, mono- and diglycerides, and glycerol with noticeably lower hydrophobicity. Lipase under the brand name of Lipolase Ultra by Novozymes is a protein-engineered variant of the enzyme with enhanced performance at temperatures lower than 20 C. A negatively charged aspartic acid in the enzyme’s active site was replaced with leucine (neutral and hydrophobic). The neutrally charged leucine reduces the repellent electrostatic forces between soil particles and the enzyme, making the affinity to a lipid contact zone on the fabric surface tighter. The catalytic activity of lipases depends on water, reaching the maximum activity at low water content. This makes lipases even more active as presoakers (in undiluted form) for the treatment of tough fatty stains. The lower water content on the fabric in this situation is believed to be responsible for the high lipase activity and therefore stain-removal performance. Surprisingly lipases are

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also active during the drying step, this means that decomposition of any residual fatty stuff will occur while garments are drying. Another usage for enzymes in household detergents is automatic dishwashing machines. Basically, enzymes offered on the market for this application are the same as for laundry detergents. Due to differences in performance/stability, some proteases and amylases are preferred over others (Crutzen and Douglass, 1999). To be well suited for the use in automatic dishwashing machines, the selected enzymes must exhibit an optimum activity at alkaline pH, efficacy, and stability at broad range of temperatures (20 70 C), and have a broad range of substrate specificity (Crutzen and Douglass, 1999).

16.4.5 Enzymes in Organic Bio-Synthesis In the last two decades, biocatalysis has been established as a powerful synthetic tool. Enzymes are highly attractive as catalysts for organic bio-synthesis due to their ability to accelerate reaction rates, together with unique stereo-, regio-, and chemo-selectivity, and mild reaction conditions. Due to the chiral nature of enzymes and their unique stereochemical properties, most of the focus regarding their usage was invested in preparing enantiomerically pure compounds, which are rather hard to produce using conventional chemical reactions. At the industrial scale, certain classes of enzymes are used more often than others. Most of the enzymes that have been used as biocatalysts in the industry are either hydrolases (65%) or oxidoreductases (25%) (Faber, 1997). Lipases, which are hydrolases, are considered versatile and flexible biocatalysts for any organic bio-synthesis due to their high compatibility with organic solvents. Such flexibility ranks them on the top of most frequently used enzymes in this arena. A classic example for their use is the production of (S,R)-2,3p-methoxyphenylglycyclic acid (MPGA), an intermediate for diltiazem and other calciumchannels blockers. In this process, the precursor methyl-p-methoxyphenyl glycidate is hydrolyzed in a stereo-specific fashion to produce MPGA by a lipase catalyst. Oxidoreductases catalyze oxidation and reduction reactions; as a result these enzymes are very attractive for industrial uses. However, they often need expensive cofactors such as nicotinamide adenine dinucleotide (NAD/NADH) and flavin adenine dinucleotide (FAD/FADH). In fact, NADs are required by about 80% of oxidoreductases. Fortunately, several NAD(H) regeneration systems have been developed, the most widely used being the formate dehydrogenase (FDH) system (Chenault and Whitesides, 1987). An example of a pharmaceutical synthesis reaction involving an oxidoreductase is the synthesis of 3,4-dihydroxylphenyl alanine (DOPA). DOPA is a chemical used in the treatment of Parkinson’s disease. The industrial synthesis of DOPA is catalyzed by polyphenol oxidase (EC 1.14.18.1). The monohydroxy compound is oxidized by the regio-specific addition of a hydroxyl group (Faber, 1997). Another example is the use of leucine dehydrogenase coupled with FDH for the reductive amination of tri-methylpyruvate to L-tert-leucine, a building block for various pharmaceutically-active compounds (Kragl et al., 1996). The whole process is carried out in a membrane reactor in which the cofactor NAD is regenerated by FDH. This process has recently reached the ton-scale production levels.

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Lyases are enzymes that promote breaking of various chemical bonds by means other than hydrolysis. The production of acrylamide with nitrile hydratase enzyme is one of the most impressive industrial applications of lyases. Acrylamide is an important building block for the production of polymers and copolymers. Conventionally, acrylamide is produced with the assistance of copper as a cofactor. This cofactor, however, is poisonous and difficult to dispose safely. In addition, the chemical reaction is carried out at 100 C making it a very resource-consuming one. In 1985, a new enzymatic method of acrylamide synthesis through the addition of water to acrylonitrile was devised by Nitto Chemical Industries (Osaka, Japan). Nitrile hydratase (EC 4.2.1.84) catalyzes the hydration of nitriles to amides (Yamada and Kobayashi, 1996). Immobilized cells of Rhodococcus or Pseudomonas chlororaphis overexpressing nitrile hydratase catalyze the addition of water to such substrates (Nishiyama et al., 1991; Hashimoto et al., 1994). Based on the optimized enzymatic reaction, the production of acrylamide is estimated to total more than 400,000 ton per year worldwide. Furthermore, the process is conducted at much lower temperatures, 10 C, consuming a fraction of the initial energy and generating fewer toxic by-products with a reduced possibility of polluting water with heavy metals.

16.4.6 Other Promising Applications for Enzymes Within the Textile and Carbon Capture Industries Enzymes have been used in textile processing for more than 2000 years. First men applied microorganisms to help the retting of bast fibers. Today, enzymes are utilized in many processes of textile production such as starch de-sizing, scouring and bleaching of cotton, aging of denim, degumming of silk, and wool treatment (Singh and Mukhopadhyay, 2012; Rodriguez Couto, 2009). Many of these processes when carried out by conventional methods use harsh or toxic chemicals at high temperatures, and require massive amounts of water and energy, making the textile industry rather a resourceconsuming and environmentally polluting one. The aforementioned reasons make the use of enzyme technologies a “green” alternative or at least a beneficial addition to traditional methods. Since the early 1990s, the use of enzymes in the textile industry has increased dramatically by virtue of their environment friendliness, reduced water and energy usage, and the specificity of reactions catalyzed by enzymatic agents (Demarche et al., 2012). One of the examples where an enzymatic process has become very important is the aging of denim. The well-worn appearance of denim articles is obtained by pumice stone washing in which dyed denim is faded by the abrasive action of pumice stones on the garment surface. At present, in addition to pumice stones, a number of cellulases are used for this step (Phitsuwan et al., 2013). Denim is a sturdy cotton textile. The main component of cotton fiber is cellulose, a polysaccharide consisting of a linear chain β-(1-4) linked D-glucose units. Enzymes that catalyze the hydrolysis of such polymer are called cellulases (e.g., endoglucanases EC 3.2.1.4, cellobiohydrolases EC 3.2.1.91, and cellobiases EC 3.2.1.21). Cellulases accelerate endohydrolysis of 1,4-β-D-glycosidic linkages in cellulose. Denim garments are often treated with surface dyes like indigo, sulfur, and vat-based dyes, which only color the surface of the yarn. The cellulase binds to the exposed fibril on the yarn’s surface and hydrolyzes it,

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leaving the interior part of the cotton fiber intact (Ulson de Souza et al., 2013; Montazer and Maryan, 2010). As a result of the enzymatic hydrolysis and mechanical action, dyes are released from the surface and light, uncolored areas become visible, as desired. An endoglucanase obtained from Trichoderma reesei was found to be supreme, both in removing color from denim and producing the stone-washed effect with a low degree of fiber hydrolysis (Miettinen-Oinonen and Suominen, 2002; Heikinheimo et al., 2000; Csiszar et al., 2001). Because a small dose of enzyme can replace several kilograms of stones, the use of fewer stones results in less garment damage and less wear on machines (Phitsuwan et al., 2013). About 80% of all aged denim products worldwide are treated with enzymes nowadays. Enzyme engineering recently tackled an endoglucanase obtained from Volvariella volvacea to understand the role of structural and functional domains of this enzyme. The recombinant production of this enzyme and a truncated variant (that retains the catalytic core) were carried in P. pastoris. After two-step chromatography purifications, the wild type and the truncated variant were compared in their specific activities toward soluble substrates and in actual deinking and biostoning experiments. The enzymatic deinking and biostoning comparisons showed that the full-length enzyme has higher performance efficiency compared to the truncated version suggesting a fundamental functional role of the deleted sequences (Wu et al., 2007). One of the oldest applications of enzymes within the textile industry is the use of α-amylases to remove starch sizing (coating) (Hao et al., 2013). Threads of fabric are often coated with starch in order to prevent them from breaking down during weaving; however, the sizing needs to be removed before further processing. The degradation of starch is achieved mainly by α-amylases from bacterial origin, obtained especially from B. subtilis (Raul et al., 2014). The power of structure-based rational design was recently used to introduce arginines to the surface of an alkaline α-amylase isolated from Alkalimonas amylolytica hence significantly improve its thermos-stability (Deng et al., 2014). Multiple arginines were introduced to the protein surface to replace seven residues in total (Gln166, Gln169, Ser270, Lys315, Gln327, Asn346, and Asn423). Five of the seven single-mutated enzymes (S270R, K315R, Q327R, N346R, and N423R) showed an enhanced thermos-stability. In a second round of site-directed mutagenesis engineering, multiple arginines were subsequently introduced to the protein surface, and the quintuple-mutated enzyme (S270R/K315R/Q327R/N346R/ N423R) showed a 6.4-fold improvement in its half-life at 60 C compared with those of wild-type enzyme. Furthermore, the mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. Similarly, an in silico rational design and systems engineering of disulfide bridges within the enzyme’s catalytic domain were used to improve the thermo-stability of an alkaline α-amylase obtained from A. amylolytica. Seven residue pairs (P35-G426, Q107G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation and the respective residues were replaced with cysteines. The final triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a sixfold increase in half-life at 60 C compared with the wild-type enzyme (Liu et al., 2014). In a different study, the possibility of improving the oxidative stability of an alkaline amylase obtained from A. amylolytica for the textile industry was probed by targeting

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methionine residues at the catalytic domains. Site-directed mutagenesis protocols replaced five methionines at positions 145, 214, 229, 247, and 317 in the amino acid sequence of this alkaline amylase with serines. The preliminary results indicated the potential of this approach to improve the oxidative stability of the targeted enzyme (Yang et al., 2012). Collectively, the earlier listed examples provide an effective strategy to improve the stability of amylases (thermal and oxidative) with potential applications within detergent and textile industries. Finally, dirty and gray cotton fabrics must be cleaned in order to make them hydrophilic and white, which makes dyeing and finishing processes successful (Kalantzi et al., 2008; Sawada and Ueda, 2001). Impurities such as waxes, pectins, and hemicelluloses can be eliminated with the help of another commercialized enzyme treatment, namely alkaline pectinase (EC 4.2.2.2) (Agrawal et al., 2007). It is worthy to mention here that enzymes can also be used for bleaching in “an indirect” fashion by producing hydrogen peroxide (H2O2), which facilitates cotton bleaching (Pricelius et al., 2011). Carbon capture is a process of capturing carbon dioxide (CO2) emissions to reduce the amounts of CO2 entering the atmosphere and to minimize the associated global warming and ocean acidification effects (Savile and Lalonde, 2011). Theoretically, CO2 can be collected at large-emission sources, and transported and stored in underground geological formations (Yadav et al., 2014). Carbon dioxide can also be absorbed to undergo different chemical transformations (carbon dioxide scrubbing for example). However, most of these methods are energy-consuming ones and not very feasible on the long term. Enzymatic carbon capture has recently become a very promising way to eliminate CO2 from atmosphere (Savile and Lalonde, 2011). In this technique, carbonic anhydrase catalyzes the transformation of CO2 molecules to bicarbonate HCO2 3 . The bicarbonate anion then can be combined with low-grade minerals to make inert carbonates. The final solid precipitates/products can be later used as building materials (such as bricks and pavers) or just deposited as solid matter (Pierre, 2012). The carbonic anhydrase enzymes (EC 4.2.1.1) accelerate the hydration of carbon dioxide to carbonate anion and proton (or vice versa). The catalytic rate of this reaction is one of the fastest of all enzymes (Savile and Lalonde, 2011). The catalytic constant of the reaction is 1 3 106 s21, which means that each molecule of the enzyme can hydrate 1 million molecules of carbon dioxide per second. The rate of this reaction is only limited by the rate of its substrate diffusion (Vinoba et al., 2012). There are five families of proteins, which are able to catalyze such a reaction, but they are structurally and genetically diverse. The active site of these enzymes has a single divalent metal ion such as Zn21, Fe21, Cd21, or Co21 in a tetrahedral coordination (Domsic and McKenna, 2010; Supuran, 2008). One of the first reported carbon-capturing technologies utilizing this enzyme was developed by CO2 Solution, Inc. (Ville de Que´bec, QC, Canada). It made use of an immobilized carbonic anhydrase on a surface of beads, packed in a reactor (Mauksch et al., 2001). In this process, water flows down from the top of the reactor. The CO2-containing gas mixture enters the reactor from the bottom of it. CO2 capture occurs when the flow of water and gas mix together and contact the surface of the catalyst. This process was tested in 2004 in an aluminum foundry for 1-month period of continuous operation. It captured 80% of CO2 emitted from the fumes. The process was found to be more economical than

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the conventional counterpart usually used to capture CO2 by an amine solution, which requires heating of the amine solution in order to recover CO2. The biggest drawback in utilizing carbonic anhydrase in CO2 capture is the enzyme’s short lifetime due to its low stability under alkaline pH, high temperatures, or when exposed to any shearing forces (Di Fiore et al., 2015). Bioengineering techniques are being utilized to address these issues and generate thermophilic enzymes starting from mesophilic scaffolds through disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions, and aromatic clusters. One study explored the effect of incorporating proline residues at positions 170 and 234 within human carbonic anhydrase on enzyme’s rigidity. Furthermore, the effect of surface loop (residues 230 240) deletion on the enzyme’s compactness was examined. The data collected (thermal stability, kinetic studies, catalytic efficiency) can provide future blueprints for rational thermal stability engineering of this enzyme (Boone et al., 2015). Most recently, Novozymes has engineered a thermostable version of carbonic anhydrase enzyme that remains fully functional under high pH values of 9 10.5. The engineered enzyme can also withstand harsh temperatures (40 45 C for months or 80 135 C for hours but with rapid inactivation rates). Furthermore, Akermin, Inc. (St. Louis, MO, United States) has introduced a proprietary polymer coating that protects the enzyme. In essence, the multipoint interactions between the enzyme and the silica-based polymer support occur through hydrogen, ionic, and van der Waals forces. This reduces the enzyme’s susceptibility to denaturation, resulting in improved activity and extended operational lifetime. The carbon capture enzyme technology developed by Akermin, Inc., was successfully tested at the National Carbon Capture Center (Wilsonville, AL, United States) for a period of 6 months. It captured more than 80% of CO2 emission from flue gas exhausts without any detectable decline in performance within the first 3 months of operations (Zaks, 2012). An artificial, bifunctional enzyme containing an immobilized carbonic anhydrase from Neisseria gonorrhoeae and a cellulose-binding domain from Clostridium thermocellum was bioengineered recently. The chimeric enzyme is of particular interest due to its binding affinity for cellulose and retained carbonic anhydrase activity (Liu et al., 2009). Another example for using enzymatic carbon dioxide capture is encountered within space stations. The process was developed by the National Aeronautics and Space Administration (NASA, Washington, DC, United States) to purify the ambient atmosphere of confined inhabited cabins. Here, CO2 is captured through thin aqueous films in which some carbonic anhydrase is dissolved (Ge et al., 2002; Cowan et al., 2003). The efficiency is sufficient for small-scale captures that suit outer space applications.

16.5 CONCLUSION AND FUTURE PERSPECTIVES The use of enzymes in practical applications is expanding on a daily basis. Our understanding of these fascinating biocatalysts coupled with some recent advances in the molecular biology field (such as the affordable whole-genome sequencing of microorganisms) promises to take this field to another unmatched level. Although enzyme discovery and optimization remains a challenging task, the growing list of leading technologies and

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helpful resources available on the market will shortly bring another technological revolution to usable agricultural and industrial applications. This is supported at large by consumers’ approval and the search for greener ways of production. This field provides also a golden opportunity for well-trained biochemists, molecular biologist, and bioinformaticians to carry out collaborative projects aiming at isolating new enzymes, optimizing the existing ones, and exploring novel applications.

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17 Biomedical Engineering: The Recent Trends Manju Sharma and S.M. Paul Khurana Amity University Haryana, Gurgaon, India

17.1 INTRODUCTION Biomedical Engineering (BME) is the combination of application of engineering principles and design concepts to biology and medicine for advanced healthcare in diagnostic, monitoring, and therapy. The word bioengineering was coined by British scientist and broadcaster Heinz Wolff in 1954. The basis of BME lies in the basic sciences—Mathematics, Physics, Chemistry, and Biology. Bioengineering encompasses two important areas of interest: (1) it applies the principles of engineering science to understand functioning of living organisms and (2) it applies engineering technologies to develop and design new devices like therapeutic or diagnostic instruments or formulation of novel biomaterials for medical applications, artificial tissue, or organs designing and framing of new delivery systems. With the initiation of bioengineering concepts, there has been a technological evolution in two imperative aspects of healthcare, i.e., in diagnostic imaging and implanted therapeutic medical devices which range from clinical equipment to microimplants, common imaging equipment such as EEGs and MRIs, regenerative tissue growth, therapeutic biologicals, and pharmaceutical drugs respectively. Overall bioengineering focuses on the uses of biomaterials or biocompatible prostheses or principles to improve the healthcare services. It includes: 1. Acquiring novel knowledge and understanding of living systems through the innovative and substantive application of experimental and analytical techniques in the light of engineering sciences. 2. The development of new devices, algorithms, processes, and systems that will advance biology and medicine and improve medical practice and healthcare delivery.

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3. The term BME research should be seen in a wide sense: it includes the relevant applications of engineering not only to medicine but also to the basic life sciences. Now medical science is becoming more technology based, a progressive shift is happening in industry to meet the demand. The collective efforts made in engineering and science has brought a shift from traditional technologies to new technologies. From the past few years, there has been a paradigm shift in Europe, United States, and United Kingdom. It is presently about 9% in Germany, ranges from 6% to 7% in the United Kingdom, and touching approximately 14% in the United States. In India bioengineering has limited scope due to the lack of integration between research institutions, hospitals, and universities.

17.2 AREAS OF BME BME has successfully made its signature in the different key areas (Fig. 17.1) with contribution and trying to handle various problems in light of recent developments.

17.2.1 Bioinstrumentation It is the application of electronics and measurement principles and techniques to develop devices used in diagnosis and treatment of disease. The recent publications in the field of bioinstrumentation talk about the following areas 1. In vivo bioelectrical measurements with coated electrodes—Initially electrodes were coated by polyethylene dioxythiophene doped with paratoluene sulfonate (Green et al., 2012). The electrodes were immersed in high serum content cell culture medium and were subjected to continuous electrical stimulation. The good and stable performance of these electrodes was seen characteristics during 1.3 billion pulses of stimulation. The constant efforts were made to fabricate entire electrodes from conducting polymers Biomechanics Tele-health Tissue engineering Instrumentation and medical devices Bioengineering areas

Biosignal processing

Biomaterials Neutral engineering Medical imaging

FIGURE 17.1

Computational modeling

Emerging areas of BME.

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using chemical deposition methods and/or printing procedures. These electrodes are highly flexible, thin, light weight, and have long-term stability due to the properties of material used, appear feasible for use in bioelectrical measurement but further safety measure should be required to be investigated (Cui et al., 2001; Sekine et al., 2010; Waltman and Bargon, 1986). These electrodes were successfully used in micro-biosensor and implanted into a rat brain to record the extracellular level of the superoxide anion radical induced by acute and repeated injections of cocaine (Rahman et al., 2012). 2. mHealth technology—mHealth stands for mobile health which is an important part of eHealth (electronic health) where mobile phones and personal digital assistant (PDA) communication devices are used to deliver various diagnostic information to doctor, patient, and researchers. The concept was first proposed by Martinez et al. (2008) and reviewed by Boulos et al. (2011). These devices are considered as handy bioinstruments for real-time monitoring of vital signs in patient, and direct rider of care via mobile telemedicine (Cipresso et al., 2012). Latest published reviews suggest that there are two types of instrumentation systems: (1) Type A where PDA combined with external healthcare device and (2) Type B in which PDA used to measure medical images and physiological signals with inbuilt charge-coupled device camera. 3. Noninvasive instruments for healthcare—Keeping the “super aging society” in mind, it is necessary to look for noninvasive instruments for early diagnosis and preventive medicine to deal with timely treatment for life style diseases. Two systems have been developed: (1) ambulatory or wearable physiological monitoring or (2) nonconscious physiological monitor. The Holter type electrocardiogram (ECG) recorder (Yamakoshi, 2011) and portable sphygmomanometer known as “ambulatory blood pressure monitor”, having base on the auscultation and/or cuff oscillometric method in clinical medicine. The mobile phone based diagnostic systems incorporate an external device to monitor physiological variables such as respiration rate, cardiac R-R intervals, blood oxygen saturation (Scully et al., 2012), and ECG (Lee et al., 2012). Recently, an alcohol-based vehicle ignition-interlock device was developed, for the noninvasive quantification of blood alcohol concentration using near-infrared light; this method is called as “pulse alcometry” (Yamakoshi et al., 2012). Wearable textile electrodes were used to monitor ECG (Rantanen et al., 2002), which could be worn for a long time in T-shirt. This device can communicate emergency messages, positioning, and navigation aids for user. Furthermore, a prototype of garment monitoring system was developed to record ECG and respiratory measures together (Paradiso et al., 2005). Recently, a wireless body area network was established putting a number of miniature wireless sensors having ECG, a pulse oximeter, trunk angle, and motion sensors.

17.2.2 Biomechanics This field applies means of mechanics to understand motion in human, animals, organs, cells, and even in devices. This area also tries to study transport of chemical constituents across biological and synthetic media and membranes. “Mechanics” is the branch of physics, which analyzes the actions of forces. Applied mechanics, remarkably mechanical engineering disciplines, includes continuum mechanics, mechanism analysis, and structural

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FIGURE 17.2

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Different fields of biomechanics.

analysis; kinematics and dynamics play prominent roles in the study of biomechanics. Different active fields of biomechanics (Fig. 17.2) are significantly dealing with orthopedic problems worldwide to make life moving. Biomechanics is widely used in orthopedic industry to design orthopedic implants for human joints, dental parts, external fixations, and other medical purposes. 17.2.2.1 Sports Biomechanics The laws of mechanics are applied to gain understanding of human movement of athletic performance, i.e., responses of action of human body while playing, and sports implements like hockey stick, cricket bat, and javelin (Bartlett, 1997). It takes care of muscular, joint, and skeletal actions of the body during the execution of a given task, skill, and/or technique. Systematic understanding of biomechanics relating to sport skills helps in good performance, rehabilitation, and injury prevention, along with acquiring mastery in sports (Michael, 2008). 17.2.2.2 Continuum Mechanics This is a branch of mechanics, which deals with the analysis of the kinematics and the mechanical behavior of materials modeled as a continuous mass rather than as discrete particles. Augustin-Louis Cauchy, the French mathematician in the 19th century, first mentioned about such models. The two major areas of continuum mechanics are as below:

Solid mechanics

Fluid mechanics

Continuum mechanics consider body as stress-free and it only has the presence of interatomic forces i.e., ionic, van der Waals, and metallic forces to hold the body together and maintain its shape in the absence of all external influences, including gravitational attraction. Solid mechanics has specific applications in many other areas, such as understanding the anatomy of living beings and the design of surgical implants and dental prostheses.

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Classification of Biotribology Research and Associated Research Focuses

Classification type

Significant investigations

Joint tribology

Articular cartilage, implant interfaces, hip joint, joint fluid, knee joint, restorative materials of joints, etc.

Skin tribology

Skin friction induced perception, skin irritation and discomfort, skin care, synthetic skin, skin in contact with articles (such as shaving devices, tactile texture, shoes, and socks) for daily use, various medical as well as sport devices, medical and cosmetic treatment, skin friction, grip of objects, etc.

Oral tribology

Natural teeth, implanted teeth, dental restorative materials, tongue, mandibular joints, saliva, toothpaste, swallowing, etc.

Tribology of the other human bodies or tissues

Bone, cells, contact lenses, ocular surfaces, capillary blood flow, etc.

After Zhou Z.R., Jinb Z.M., 2015. Biotribology: recent progresses and future perspectives. Biosurf. Biotribol. 1, 3 24.

17.2.3 Biotribology The main focus of biotribology research is on having the understanding of the working of natural biological systems and to know how development of diseases takes place, as well as how medical treatments and devices are optimized, often from an engineering point of view. Presently, this branch touches many areas like joint, skin, oral tribology, along with tribology of many other human tissues and organs as mentioned in Table 17.1 The poly (ether-ether-ketone) (PEEK) material offers choice over conventional polymer and metal materials in orthopedics, as PEEK is well known for its biocompatibility and low elastic modulus. The observations were made over the tribological behaviors of glass fiber reinforced PEEK (GFRPEEK) against ultra-high-molecular weight polyethylene (UHMWPE) and polytetrafluoroethylene in a ball-on-disc contact configuration in two different conditions: one in dry friction and the second with 25% (v/v) newborn calf serum lubricated conditions. The advantages of GFRPEEK on UHMWPE for their friction bearing tribological properties were noticed, so that they could be used as bearing materials for artificial cervical disc (Song et al., 2015). The real geometry (including radial clearance) of rubbing surfaces has been observed in a novel approach of in situ film formation within hip joint replacements (Vrbka et al., 2015).

17.2.4 Computational Biomechanics Computational biomechanics is a promising research field, which provides thorough treatment from foot, ankle, knee, hip, lower limb, spine, to head and teeth, as well as bone and muscle at the tissue level. It informs about complex biomechanical behaviors of normal and pathological human joints to surface new methods of orthopedic treatment and rehabilitation. It provides novel link between surgeons and machines, which enables them to map and carry out surgical interventions with more accuracy and lesser amount of trauma. Computer-integrated surgery systems could help to improve clinical outcomes and the efficient healthcare delivery (Zhang and Fan, 2014). It also helps in getting footing

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for computer-integrated medicine by extracting clinically important information regarding the physical status of the underlying biology at cell, tissue, organ, and organism level and integrating information with molecules of body.

17.2.5 Biofluid Mechanics Biofluid mechanics focuses on macrocirculation, microcirculation, and specialty circulation that flows through kidney, lungs, eyes, joints, diarthroses, and splanchnic circulation that are important in human body. It is necessary to understand fluid dynamic factors such as velocity distribution, pressure, flow rate ratio, flow behavior, velocity gradients, and stress on the wall and on blood cells to design medical device for recording and diagnosis purpose. Various mechanical factors mostly seem responsible for the deposition of blood cells and lipids, which lead to atherosclerosis (Liepsch, 2002). Studies in hemodynamics establish understanding for flows in artificial organs and implants like artificial heart valve prostheses, microfluidic filter systems, blood pumps, elastic silicon rubber models of the cardiovascular system with flow wire and stents, or patches for vessel surgery, and on flows in biological/human systems such as the circulatory blood system (Rubenstein et al., 2015).

17.2.6 Biomaterials Biomaterials describe both living tissue and materials used for implantation. They could be (1) synthetic (metals, polymers, ceramics, and composites), (2) derived from animal and plants, (3) hybrid or semisynthetic materials. Understanding the properties of the any biomaterial is vital in the design of implant materials. Biomaterials are engineered in a way that they could provide a perfect microenvironment to cells for their growth and differentiation. Utilization of diverse biomaterials for their applications in the area of tissue reconstruction, bioactive molecule, delivery cell guidance, and biomimetic coating is required with precision. The biomaterials are categorized into three generations (Table 17.2), since its development (from 1960s to 1970s). TABLE 17.2 Generations of Biomaterials in Use to Support Life S. No

Generations of biomaterials Materials used

1.

First

Synthetic—metals, polymers, and ceramics

Able to replace tissue with minimum toxicity

2.

Second

Bioactive—two approaches to make bioactive compounds: (1) coat metal with ceramics and (2) modify surface of metal chemically to interact with tissue/other materials

Able to interact with internal environment to enhance the biological response and the tissue/surface bonding

3.

Third

Bioactive and bioresorbable—metals with more focus on titanium and titanium alloys

As temporary 3D porous structures able to activate genes that stimulate regeneration of living tissue

Working

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First generation of biomaterials has given wattage in medical field (1) that should not elicit toxicity or carcinogenicity in living tissue, (2) having resistance to corrosion in aqueous environment, and (3) having mechanical properties (Hench, 1980). The secondgeneration biomaterials were tagged to be bioactive and resorbable. The bioactive biomaterials have application in orthopedic and dental care. Examples include bioactive glasses, ceramics, glass ceramics, and composites. Presently, resorbable biomaterials are in use as fracture fixation plates and screws used in orthopedic surgeries (Hench and Thompson, 2010). Biomaterials that can stimulate specific cellular response are considered as thirdgeneration biomaterials (Hench and Polak, 2002). For example, bioactive glass (third generation) and porous foams are designed in a way that they activate genes to stimulate regeneration of living tissues. Idea of scaffolding material development with nanoscale features is being implemented to mimic the native extracellular matrix of the host. Artificial tissues that have the same features as the natural counterpart can be developed as bio-materials and are on current focus for futuristic use (Bhat and Kumar, 2013). Bioceramics and biopolymer are good examples of biomaterials. Ceramics have also evolved its role as bioceramics for the repair and reconstruction of diseased or damaged parts of the body. It is commonly used in orthopedic surgery and dentistry, but they have a wide application within the industry of biomedical device (Liepsch, 2002). Bioceramics is categorized on the basis of use and its interaction with the host tissue, i.e., bioactive, resorbable, and bioinert (Yun, 2015). Biopolymers are polymers produced by living organisms. Proteins and peptides, cellulose and starch, and DNA and RNA are considered as biopolymers, having monomeric units such as sugars, amino acids, and nucleotides, respectively (Buehler and Yung, 2009). Interestingly, 33% of plant matter is cellulose, and it is the most common biopolymer as well as organic compound on Earth (Stupp and Braun, 1997; Klemm et al., 2005), Besides biopolymers and bioceramics, various other inert and nontoxic metals were used as biomaterials for medical treatment. The hollow gold nanospheres (HAuNS) with unique photothermal therapy capabilities are in medicinal use. This property of HAuNS attributed to their inert and nontoxic properties. Here, the electrostatic approach was successfully executed to absorb Chlorin e6 (Ce6) at low pH by forming HAuNS-pHLIPCe6 in antitumor treatment (Yu et al., 2016). Micro and nanoscale technologies were used to develop functional biomaterials for tissue engineering and drug delivery applications as delivery is affected by shape and size of different delivery agents (Fig. 17.3). It is necessary to control size and shape of the delivery agent as they may finally modulate parameters

FIGURE 17.3 Parameters of drug delivery.

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such as pharmacodynamics; bioavailability is necessary and so is true for cell-specific targeting (Tee et al., 2014). Champion et al. (2007) discussed the methods of particle preparation and the role of particle shape that plays an important role in drug delivery. Recently, a progress has been made on the design and fabrication of nanoparticles of various shapes and their unique properties associated to drug delivery (Liu et al., 2012). Zhang et al. (2013) has reviewed innovations made in the field of materials and processing of drug delivery systems. These advances have helped in the past and have the ability to influence the future of drug delivery.

17.2.7 Tissue Engineering This field deals with cells, science of engineering and materials to improve or replace various biological functions. This area has close association with biomaterials but has its own wide range of applications. One of its most important applications is in the field of repair and replacement of whole tissues or a portion of tissues such as cartilage, bone, skin, blood vessels, and muscles (Langer and Vacanti, 1993). The tissues used for engineering require having capability of maintenance and/or enhancing tissue functioning at site of treatment (MacArthur and Oreffo, 2005). Tissue engineering involves different steps from cell isolation to successful implantation (Fig. 17.4). The types of cells used for this purpose are isolated and recognized with their source (Table 17.3) Recently, the mesenchymal stem cells from bone marrow and fat have been found to be having the capacity to differentiate into a variety of tissue types, including bone, nerve, fat, and cartilage (Amini et al., 2012; Brown et al., 2013). Nanostructured calcium phosphate (CaP) can be grouped in three forms: nano-CaP coatings, calcium phosphate cement, and nano-CaP composites as biomaterials/scaffolds have specific similarity to inorganic components of bone. Nano-CaP biomaterials show different interactions with stem cells: 1. 2. 3. 4.

support stem cells to attach/proliferate and induce osteogenic differentiation, influence surface patterns on cell alignment, achieve better bone regeneration than conventional material, and microencapsulate the cell in nano-CaP scaffolds for quick bone tissue engineering (Cassidy, 2014; Wang et al., 2014). Bioactive factors + Stimulus

Repair & regeneration of Tissue

Scaffold

FIGURE 17.4

Tissue-engineered Implantation construct

Stepwise tissue engineering.

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Types of Cells Used for Tissue Engineering

Type of Cells

Source and Function

Autologous

Cells come from the same individual who requires the implantation; being the same source, they adjust easily and have no problem of pathogen transmission; however, severely ill person and person with genetic disease does not have sufficient amount of these cells

Allogeneic

The source of these cells is from the body of a donor of the similar species; besides ethical issues fibroblast cells from human foreskin are immunologically safe as well as a viable choice for tissue engineering of skin.

Xenogenic

Comes from individuals of another species, extensively for the experiments of construction of cardiovascular implants

Syngeneic or isogenic

From genetically identical organisms, such as highly inbred research animal models, clones, or twins

Stem cells

Undifferentiated cells can divide in culture and come up with different forms of specialized cells

17.2.8 Biorobotic Biologically enthused robots often hold sensory abilities, and their mobility and flexibility is also higher than traditional robots. Biorobotics cover a diverse array of disciplines with a large applicability. Considering surgery still as an option having involved danger of anesthetics, infections, organ rejection, and missed cancer cells which leads to failure if remains in the body. Biorobotic technologies have threefold applications. Till now the robotics developed is especially suitable for noninvasive or minimally invasive surgery and for better outcomes of surgery. Nowadays, medical practitioners have been using robots on a regular basis for heart, brain, spinal cord, throat, and knee surgeries at many hospitals in the United States (Shrotriya and Pandey, 2013). Diagnosis is the second major field using robotics in medicine. Robotic diagnosis minimizes invasiveness to the human body and improves the accuracy and scope of the diagnosis. For example, the development of the robotic capsular endoscope (Fig. 17.5A and 17.5B) is a boon for executing noninvasive diagnosis of gastrointestinal tract (Polo Sant’Anna Valdera of the Sant’Anna School of Advanced Studies, 2005, Italy). The robotic capsular endoscope hardly has any side effect with easy movement through digestive system due to its miniature size. It takes accurate and precise high-quality pictures, instantaneously sends them to recorder but sometime difficult to control the camera. It is made of a biocompatible material and does not make any harm to body. The third use of robotics is to provide assistance to accommodate a deficiency—either as fully functioning robots or highly advanced prosthetics to recover physical functions of human beings such as robotic prosthetic legs, arms, and hands. The investigation on powered leg or using electromyographic signals for movement control is on progress at the Technical University of Berlin. Science of highly advanced prosthetics represents area of neural engineering and biorobotics intersect as both disciplines are required to first induce signal and then translate it into movement. Tee et al. (2015) has recently developed artificial sensing skin which is power driven and having efficient mechanoreceptor with a flexible organic transistor circuit where transduction II. INDUSTRIAL AND ENVIRONMENTAL TECHNOLOGIES

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FIGURE 17.5 (A) Robotic capsular endoscope for examination of gastrointestinal tract. (B) Camera fitted inside the capsule.

of pressure directly converts into digital frequency signals. It is one step forward towards design and use of large-area organic electronic skins with neural integrated touch feedback for replacement limbs. Kaushik Jayaram and Robert Full (2016) have built a compressible robot based on their finding on cockroach’s movement through gaps just millimeters high.

17.2.9 Biosensors Biomedical signal processing involves the analysis of measurements made by instruments to provide useful information upon which clinicians can make decisions (B˘anic˘a, 2012). Communication between body and physiological instruments can be made so fruitful to record even less than 1% of values in blood pressure, heart rate, blood glucose, oxygen saturation levels, brain activity, nerve conduction, and so on. Patient . Receiving signals .Recording of signals . Processing of signals . Decision . Action

Biotransducers are considered as a recognition transduction component of a biosensor system and made of two components a biorecognition layer and a physicochemical transducer; action of both together converts a biochemical signal to an electronic or optical signal. The biosensors can be classified on the basis of biotransducer type used in biosensor to record signals (Fig. 17.6) Continuous monitoring of different metabolisms together needs to develop integrative minimal invasive technology. These innovative devices require a high-degree of integration, high reproducibility, security, and privacy in data transmission, long-term biocompatibility, low detection limit, high sensitivity, high reliability, and high specificity (Carrara et al., 2012; Sharma and Khurana, 2016). Glucose monitor is available commercially and can detect oxidized glucose by the electrode. This instrument relies on

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Electrochemical Optical Electronic Types of biosensor based on biotransducers

Piezoelectric Gravimetric Pyroelectric

FIGURE 17.6 Different types of biosensors based on biotransducers. TABLE 17.4

Placement of Biosensors

Biosensors

Placement

In vivo

It has functions inside the body (Kotanen et al., 2012)

In vitro

In a test tube, culture dish, or elsewhere outside a living organism

At line

Used in a production line where a sample can be taken, tested, and a decision can be made (Stoica Leonard, 2006)

In line

Can be placed within a production line to monitor a variable with continuous production and can be automated

Point of concern

Being at the location where the test is required

amperometric sensing of glucose by means of glucose oxidase, which produces detectable hydrogen peroxide. To overcome the limitation of amperometric sensors, a flurry of research is present into novel sensing methods, such as fluorescent glucose biosensors (Ghoshdastider et al., 2015). The placement of biosensors depends upon where the monitoring is required (Table 17.4).

17.2.10 Neuroengineering Also known as neural engineering, it uses engineering techniques to connect between the interface of living neural tissue and nonliving devices. It helps in the understanding of neural system, so action could be taken to replace, repair, and enhance the system. The combination of neuroscience and engineering together has scope in neuromechanics, neuromodulation, neural growth, and repair. Neuromechanics make connection in various areas, i.e., biomechanics, neurobiology, senses and sensitivity, where robotics is also an integral part of it (Edwards, 2010). Scientists all over the world are searching for advanced techniques and models to study neural tissues with different mechanical properties. They are keen to know how these mechanical properties affect the tissues’ ability to hold up and create force and movements as well as their vulnerability to traumatic loading (Laplaca and Prado, 2010). Microelectrode arrays as neuromodulator stimulators can stimulate and record brain function. Further efforts in this direction are focused on how to make these devices II. INDUSTRIAL AND ENVIRONMENTAL TECHNOLOGIES

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adjustable and responsive for drugs and other stimuli. Neuromodulator devices can improve nervous system dysfunction related to Parkinson’s disease, dystonia, obsessive compulsive disorder, tremor, Tourette’s syndrome, chronic pain, severe depression, and eventually epilepsy (Potter, 2012). The problems arise due to brain damage or malfunctions are getting solution with the collective investigations on peripheral and central nervous system function; progress in this area needs intervention of neuroscience and engineering to establish neural and rehabilitation stream of BME. Today, it seems possible to regenerate neural tissue with the intervention of engineering and develop materials and devices to facilitate the growth of neurons for specific applications such as the regeneration of the spinal cord tissue for spinal cord injury, peripheral nerve injury, and retinal tissue. The science of tissue engineering and genetic engineering are areas developing scaffolds for spinal cord to regrow across, thus addressing the neurological problems (Schmidt and Leach, 2003; Potter, 2012).

17.3 FUTURE DIRECTIONS The handshake between engineering and biomedical sciences is an emerging field giving bright hope for healthcare. The diverse field of engineering ranging from mechanical, computers, electronics, chemical, and nanotechnology is contributing in biomedical to improve health devices, diagnosis, and drug delivery system although the techniques developed under BME are facing some challenges. The bone tissue engineering field is aimed at functional bone, but the lack of sufficient vascularization at the defect site needs further investigations. The blending of starch/polymer is sometimes nonmiscible in many cases requiring chemical strategies, otherwise it leads to poor bone quality due to poor mechanical properties. Scientists have identified three major areas of biomaterials that need to be given maximum attention, like variation, poor immunogenic response, and the technological processing techniques. Designing of biosensors faces challenges that how to decrease the limit of detection and increase the sensitivity. In addition, it should also perform accurate analysis that could lead to increase in life span with comfort. Developing biosensors with noninvasive approach is in progress for future use. There is a paradigm shift in healthcare from “classical medical care” to that of “progressive medical care” with improved instrumentation, imaging, and diagnostics. Biomedical solutions are being seen as a beneficiary for the society at individual level as they have the ability to improve the life span of a person who is considered the most valuable in the society. Assistance or novice intervention to prevent disease before it starts has always been on priority in medical science. Bioengineering is being seen as a hope for sophisticated, sensitive, and highly evolved systems to improve human health.

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Rahman, M.A., Kothalam, A., Choe, E.S., Won, M.S., Shim, Y.B., 2012. Stability and sensitivity enhanced electrochemical in vivo superoxide microbiosensor based on covalently co-immobilized lipid and cytochrome C. Anal. Chem. 84, 6654 6660. Rantanen, J., Impio, J., Karinsalo, T., Malmivaara, M., Reho, M., Tasanan, M., et al., 2002. Smart clothing prototype for the arctic environment. Pers. Ubiquit. Comput. 6 (1), 3 16. Rubenstein, D., Yin, W., Frame, M.D. (Eds.), 2015. Biofluid Mechanics: An Introduction to Fluid Mechanics. Macrocirculation and Microcirculation Academic Press, Massachusetts, USA, 544 pages. eBook; ISBN: 9780128011690. Schmidt, C.E., Leach, J.B., 2003. Neural tissue engineering: strategies for repair and regeneration. Annu. Rev. Biomed. Eng. 5, 293 347. Scully, C.G., Lee, J., Meyer, J., Gorbach, A.M., Granquist-Fraser, D., Mendelson, Y., et al., 2012. Physiological parameter monitoring from optical recordings with a mobile phone. IEEE. Trans. Biomed. Eng. 59 (2), 303 306. Sekine, S., Ido, Y., Miyake, T., Nagamine, K., Nishizawa, M., 2010. Conducting polymer electrodes printed on hydrogel. J. Am. Chem. Soc. 132 (38), 13174 13175. Sharma, M., Paul Khurana, S.M., 2016. Cell free biosystems. In: Khan, M.S., Khan, I.A., Barh, D. (Eds.), Applied Molecular Biotechnology: The Next Generation of Genetic Engineering. Taylor and Francis/CRC press, Florida, USA, pp. 465 483. Shrotriya, S., Pandey, A. (Eds.), 2013. Imitating Humans: A Technical Approach. Lulu Press, Raleigh, NC, 9781300652892, 233 pages. Song, J., Liu, Y.H., Wang, S., Liao, Z.H., Liu, W.Q., 2015. Study on the wettability and tribological behaviors of glass fiber reinforced poly(ether-ether-ketone) against different polymers as bearing materials for artificial cervical disc. Biotribology 4, 18 29. Stupp, S.I., Braun, P.V., 1997. Molecular manipulation of microstructures: biomaterials, ceramics, and semiconductors. Science 277 (5330), 1242 1248. Tee, B.C.-K., Chortos, A., Dunn, R.R., Schwartz, G., Eason, E., Bao, Z., 2014. Tunable flexible pressure sensors using microstructured elastomer geometries for intuitive electronics. Adv. Funct. Mater. 24, 5427 5434. Tee, B.C.K., Chortos, A., Berndt, A., Nguyen, A.K., Tom, A., McGuire, A., et al., 2015. A skin-inspired organic digital mechanoreceptor. Science 350, 313 316. Vrbka, M., Neˇcas, D., Hartl, M., Kˇrupka, I., Urban, F., Gallo, J., 2015. Visualization of lubricating films between artificial head and cup with respect to real geometry. Biotribology 1-2, 61 65. Waltman, R.J., Bargon, J., 1986. Electrically conducting polymers: a review of the electro polymerization reaction, of the effects of chemical structure on polymer film properties, and of applications towards technology. Can. J. Chem. 64 (1), 76 95. Wang, P., Zhao, L., Liu, J., Weir, M.D., Zhou, X., Xu Hockin, H.K., 2014. Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells. Bone Res. 2, Article number: 14017. Yamakoshi, H., Dodo, K., Okada, M., Ando, J., Palonpon, A., Fujita, K., et al., 2011. Imaging of EdU, an alkynetagged cell proliferation probe, by Raman microscopy. J. Am. Chem. Soc. 133 (16), 6102 6105. Yamakoshi, T., Ogawa, M., Matsumura, K., Itasaka, Y., Miyazaki, S., Yamakoshi, Y., et al., 2012. A preliminary study on development of a novel optical instrument for non-invasive blood alcohol measurement: proposal of pulse alcometry. Trans. Jpn. Soc. Med. Biol. Eng. 50 (2), 237 247. Yu, M., Guo, F., Wang, J., Tan, F., Li, N., 2016. A pH-driven and photoresponsive nanocarrier: remotely-controlled by near-infrared light for stepwise antitumor treatment. Biomaterials 75, 25 35. Yun H.-S. (Ed.), 2015 Ceramics in bio-applications. Biomaterials: BioMed Central. Zhang, M., Fan, Y. (Eds.), 2014. Computational Biomechanics of the Musculoskeletal System. CRC Press, Bosa Roca, 404 pages; ISBN-10: 1466588039. Zhang, Y., Chan, H.F., Leong, K.W., 2013. Advanced materials and processing for drug delivery: the past and the future. Adv. Drug. Deliv. Rev. 65 (1), 104 120.

Further Reading Gerard, M., Chaubey, A., Malhotra, B.D., 2002. Application of conducting polymers to biosensors. Biosens. Bioelectr. 17 (5), 345 359. Liu, J., Wang, J., 2001. Improved design for the glucose biosensor. Food Technol. BioTechnol. 39, 55 58. Zhou, Z.R., Jinb, Z.M., 2015. Biotribology: recent progresses and future perspectives. Biosurf. Biotribol. 1, 3 24.

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18 Omics Approaches in Biofuel Technologies: Toward Cost Effective, Eco-Friendly, and Renewable Energy Vikas Y. Patade, Lekha C. Meher, Atul Grover, Sanjay M. Gupta and Mohammed Nasim Defence Institute of Bio-Energy Research, Haldwani, India

18.1 INTRODUCTION The global energy demand is steadily increasing with the economic growth combined with the population explosion. Among the energy consuming sectors, transportation consumes about 30% of the primary energy. The major sources of the energy are fossilderived fuels including petroleum, coal, and natural gases. According to the BP Statistical Review of World Energy conducted in 2010, the crude oil and natural gas, the major energy resources, may be run out respectively in another 45 and 60 years, with the current global energy consumption policy. Thus, the continued use of fossil-based fuels is not sustainable owing to its limited availability and emission of the greenhouse gases and other air contaminants including carbon dioxide (CO2), carbon monoxide (CO), sulfur dioxide (SO2), nitrogen oxides (NOx), particulate matter and volatile organic compounds, upon combustion. Furthermore, the volatile prices of the fossil-derived fuels are affecting the economic growth in the developing and underdeveloped countries. Therefore, for environmental and economic sustainability, renewable and carbon neutral efficient biofuels are needed to displace or supplement in long run, and complement the fossil-derived fuels in near future.

Omics Technologies and Bio-engineering: Towards Improving Quality of Life DOI: https://doi.org/10.1016/B978-0-12-815870-8.00018-8

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18.2 BRIEF OVERVIEW OF THE FIRST-GENERATION BIOFUEL TECHNOLOGIES First-generation biofuels are the fuels derived from sugar, starches, or vegetable oils. Biodiesel and bioethanol are widely known as first-generation or conventional biofuels. The use of vegetable oil as a fuel dates back to more than a century when Rudolph Diesel invented the compression-ignition (CI) engine. In order to lower the viscosity and improve volatility, the triglyceride oils require some chemical modifications such as transesterification or emulsification. Transesterification of the oil with a short-chain alcohol is preferred method to derive biofuel from the oil. The fatty acid methyl esters (FAME) obtained by transesterification of oil with methanol are popularly known as biodiesel. There is decrease in viscosity and improvement in the volatility as well as other fuel properties after transesterification. The physical properties of FAME resemble to petroleum-derived diesel fuel. Presently, biodiesel is derived from food-grade edible oils in many developed and developing countries, that is, soybean oil in the United States, canola in Canada, rapeseed in Europe, palm in Malaysia and Indonesia, and so on. India being one of the largest edible oil importers, it cannot divert these edible sources for fuel purpose. The nonedible oilseeds, that is, Karanja (Pongamia pinnata) and Jatropha (Jatropha curcas), are suitable feedstock for biodiesel in India. Recently, Center for Jatropha Promotion (CJP) has also identified other plants like Simarouba, Camelina, etc., to be equally potent sources of deriving biodiesel in India. The characteristic of biodiesel (B100) should be in accordance to the norms specified by EN 14214, ASTM D 6751, or IS 15607 as shown in Table 18.1. The biodiesel has similar fuel characteristics and can be blended with conventional diesel to fuel the CI engines. The bioalcohols are produced from sugar and starches through the process of fermentation by enzyme or microorganisms. Ethanol is the most common bioalcohol where biobutanol is known to a lesser extent. Bioethanol can be used in sparkignition engines in blends with gasoline. The biofuels are free from sulfur, aromatic compounds, and burns cleanly in the engine as a result, there is no SOx, unburnt hydrocarbon, or polyaromatics in the exhaust emissions. The catalyst used in the transesterification of oil plays the crucial role in process of synthesis of biodiesel. The hydroxides or alkoxides of sodium and potassium are used as catalyst for industrial scale biodiesel production. The use of above alkali catalysts requires the feedstocks to have specific quality having minimum level of free fatty acids and moistures. The acid catalyst process is suitable for high free fatty containing vegetable oils. The acid-catalyzed method has not gained much interest due to the fact that the reaction proceeds slowly, liquid acids corrode the reaction vessel and disposal of liquid acids have environmental concerns. The transesterification of oil catalyzed by heterogeneous catalyst has been reported in the literature (Kulkarni et al., 2006). The use of biocatalyst for vegetable oil transesterification is an alternate method to prepare biodiesel. Lipases from Mucor miehei, Candida antarctica, Thermomyces lanuginosus, Candida rugosa, Pseudomonas cepacia, etc., are known biocatalyst for biodiesel synthesis from oils (Table 18.2). The lipases are immobilized in solid support for their repeated use for biodiesel synthesis. The lipases-catalyzed transesterification proceeds with stepwise addition of methanol since the excess short-chain alcohol in the reaction medium inactivates the lipase. The lipase from C. antarctica is nonspecific and catalyzes the vegetable oil transesterification without acyl migration. The yield of esters

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TABLE 18.1

Specification of Biodiesel (B100) in European Countries, the United States, and India Specifications

S. no. Properties

Unit

EN 14214

ASTM D 6751

IS 15607

1

FAME content, min.

% (m/m)

96.5

n.s.

96.5

2

Density at 15 C

kg/m3

860 900

n.s.

860 900

3

Viscosity at 40 C

cSt

3.5 5.0

1.9 6.0

2.5 6.0

4

Flash point, min.



101

93

120

5

Sulfur content, max.

mg/kg

10

15

50

6

Carbon residue, max.#

% (m/m)

n.s.

0.05

0.05

7

Cetane number, min.

51

47

51

8

Sulfated ash content, max.

% (m/m)

0.02

0.02

0.02

9

Water content, max.

mg/kg

500

500

500

10

Total contaminants

mg/kg

24

n.s.

24

11

Copper strip corrosion (3 h at 50 C)

rating

Class 1

Class 3

Class 1

12

Oxidation stability, 110 C, min.

h

8

3

6

13

Acid value, max.

mg KOH/g 0.50

0.50

0.50

14

Iodine value, max.

gI2/100g

120

n.s.

To report

15

Linolenic acid methyl esters, max.

% (m/m)

12

n.s.

n.s.

16

Polyunsaturated ($4 double bonds) methyl esters, max.

% (m/m)

1

n.s.

n.s.

17

Methanol content, max.

% (m/m)

0.2

0.2

0.20

18

Monoglyceride content, max.

% (m/m)

0.7

0.4

n.s.

19

Diglyceride content, max.

% (m/m)

0.2

n.s.

n.s.

20

Triglyceride content, max.

% (m/m)

0.2

n.s.

n.s.

21

Free glycerol, max.

% (m/m)

0.02

0.020

0.02

22

Total glycerol, max.

% (m/m)

0.25

0.240

0.25

23

Group I metals (Na 1 K), max.

mg/kg

5.0

5

To report

24

Group II metals (Ca 1 Mg), max.

mg/kg

5.0

5

To report

25

Phosphorus content, max.

mg/kg

4.0

10

10

26

Cold soak filterability, max.

seconds

n.s.

200

n.s.

Cloud point



C

n.s.

To report

n.s.

Distillation temperature (AET, 90% recovery), max.



C

n.s.

360

n.s.

27 28

C

n.s., not specified. # On 10% distillation residue.

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TABLE 18.2 Biodiesel Production With Various Lipases Lipase source (immobilized)

Oil

Alcohol

Yield %

Reference

C. antarctica

Rapeseed

Methanol

91

Watanabe et al. (2007)

Rhizopus oryzae

Soybean

Methanol

80 90

Kaieda et al. (2001)

Chromobacterium viscosum

Jatropha

Ethanol

92

Shah et al. (2004)

M. miehei

Sunflower

Ethanol

83

Selmi and Thomas (1998)

P. cepacia

Palm kernel

Ethanol

72

Abigor et al. (2000)

T. lanuginosus

Sunflower

Methanol

90 97

Dizge et al. (2009)

Bacillus sp. S23

Microalgae

96

Surendhiran et al. (2015)

during enzymatic transesterification in solvent-free medium is listed in Table 18.2. In enzyme-catalyzed reaction, there is no negative influence of free fatty acids in the oil as free fatty acids are also converted into fatty acid alkyl esters. The immobilized lipases are easily recovered after completion of transesterification and reused several times. Bioethanol is the fuel, which has globally gained much interest and widely used in Brazil and the United States since last couple of decades utilizing the by-products of sugarcane. The ethanol industries in Brazil use sugarcane exclusively, the Brazilian sugarcane system of agro-energy represents the most efficient system. Currently 80% of vehicles are running with ethanol and also some jet engines. Ethanol has high octane rating than gasoline, which allows increase in engines’ compression ratio for increased thermal efficiency. The feedstocks commonly used for deriving ethanol are sugars and starches. The process of bioethanol production involves pretreatment, enzymatic digestion of starches to sugars followed by fermentation and further purification by distillation and drying. The pretreatment step includes washing, size reduction, extracting the juice, and separating the bagasse. The fermentation process involves the conversion of fermentable sugars, that is, hexoses to ethanol by the action metabolism of microorganisms. When sucrose is the substrate, fermentation is carried out by the yeast Saccharomyces cerevisiae while the bacteria Zymomonas mobilis is employed for fermentation of glucose to ethanol. The theoretical yield of ethanol is 0.511 g ethanol/g hexose and the real yield is around 0.485 g ethanol/g hexose. The fermentation is performed at temperature below 32 C, pH between 4 and 5, sugar concentration to be less than 16 Bx. The ethanol content in the fermentation medium is 7% 7.5% (w/w) and needs further distillation processes in several steps to obtain bioethanol (Soccol et al., 2011).

18.3 SECOND-GENERATION BIOFUEL TECHNOLOGIES The first-generation biofuels compete with inputs for food, so the alternative may be the advanced or second-generation biofuels, which uses cellulosic products such as wood, straw, long grass, or wood waste for biofuel production (Sims et al., 2010). Furthermore,

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these can meet the demand for fuel in a fair and eco-friendly manner. The advantage of second-generation biofuels is the ability to use the whole plant and not just its parts (e.g., grains), as is the raw material for the first generation. All plants contain lignin, hemicellulose, and cellulose. Lignocellulosic ethanol is made by freeing the sugar molecules from cellulose using enzymes, steam heating, or other pretreatments. These sugars can then be fermented to produce ethanol in the same way as first-generation bioethanol production (Limayema and Ricke, 2012). The by-product of this process is lignin, which can be burned as a carbon neutral fuel to produce heat and power for the processing plant and other purposes. Second-generation biofuel feedstocks that mainly include cereal and sugar crops, specifically grown energy crops, agricultural and municipal wastes and waste oils, etc. Green waste such as forest residues or garden or park waste may be used to produce biofuel via different routes. Examples include biogas captured from biodegradable green waste, and gasification or hydrolysis to syngas for further processing to biofuels via catalytic processes. Second-generation biofuels from lignocellulosic biomass can be broadly obtained through biochemical and thermochemical processes (Fig. 18.1) (Menon and Rao, 2012). Biochemical conversion uses biocatalysts, such as enzymes, in addition to heat and other chemicals, to convert the carbohydrate portion of the biomass into an intermediate sugar stream. Biochemical processes typically employ pretreatment to accelerate the hydrolysis process, which separates out the lignin, hemicellulose, and cellulose (Philbrook et al., 2013). Once these ingredients are separated, the cellulose fractions can be fermented into alcohols. Liquid biofuels from biomass can be obtained through thermochemical processing or by chemical treatment. Thermochemical treatment comprises thermal decomposition and chemical transformation of substrates by the action of the temperature in the presence of various concentrations of oxygen. The advantage of thermal treatment in relation to the

Technology

Biochemical/Physical Conversion -Chemical processing -Physical processing -Biochemical processing Lignocellulosic Biomass

End product

Second generation ethanol

Value added products Thermochemical Conversion -Pyrolysis -Gasification -Torrefaction

BtL Fuels

FIGURE 18.1 Second-generation biofuel processing ways and products from cellulosic biomass.

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18. OMICS APPROACHES IN BIOFUEL TECHNOLOGIES

biochemical is able to convert all organic ingredients, not just the polysaccharides, as is the case with chemical treatment (Thomas et al., 2009). Carbon-based materials can be heated at high temperatures in the absence (pyrolysis) or presence of oxygen, air, and/or steam (gasification). These thermochemical processes yield combustible gas and solid char. The gas can be fermented or chemically synthesized into a range of fuels, including ethanol, synthetic diesel, or jet fuel. Conversion of biomass into bio-oil, biochar, syngas, and others requires entirely thermochemical processes, such as torrefaction, carbonization, thermal liquefaction, pyrolysis, and gasification. The relative advantage of thermochemical conversion over biochemical is due to higher productivity and compatibility with existing infrastructure facilities (Zhang et al., 2010). However, the majority of these processes are still under development phase and trying to secure a market share due to various challenges, right from suitable infrastructure, raw material, technical limitations, government policies, and social acceptance (Elliott, 2008).

18.4 THIRD-GENERATION BIOFUEL TECHNOLOGIES The biofuels derived from microalgae, the unicellular algae, are referred as thirdgeneration biofuels. Microalgae are a promising feedstock for biofuels owing to their rapid growth rate and higher lipid productivity than the best oil producing terrestrial plants. Furthermore, the higher photosynthetic efficiency and wider adaptability to different environmental conditions are the other reasons for interest in microalgae for biofuels (Chisti, 2007). Microalgae do not need fertile land and can be grown in sewage water, thus eliminating or minimizing the competition with food crops for resources, consequently avoiding the food versus fuel conflict. In addition to source of different types of biofuels, microalgae are useful as nitrogen-fixing biofertilizers and in phyto-remediation (Munoz and Guieysse, 2006).

18.4.1 Microalgae Cultivation Microalgae can be cultivated in open/raceway ponds or closed photo-bioreactors (Fig. 18.2). In case of pond cultivation, operating cost is low but contamination risk and

FIGURE 18.2

Cultivation of microalgae under partially controlled condition for biofuel production.

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343

weather dependence are high, in addition to the low reproducibility, low biomass concentration and hence the higher harvesting cost. Contamination risk is low with closed photobioreactor cultivation and biomass concentration is higher along with lower harvesting cost. However, operating cost as well as start-up capital cost is very high. Therefore, cultivation system may be chosen depending on the requirements. Water is an important input for algae cultivation and depending on the microalgae species, freshwater or seawater can be used. Wastewater can be treated using microalgae for removal of nitrogen and phosphate from the effluents, which would otherwise result in eutrophication. Besides this, applications of microalgae for removal of several heavy metal contaminants have also been proven. Inadequate supply of carbon in the form of carbon dioxide is often the limiting factor in the microalgae productivity. To produce one unit of biomass with average chemical composition, algae use approximately 1.8 units of CO2. Naturally dissolved CO2 in water is not enough; therefore, bubbling of air into water to improve the dissolution is practiced as the pure CO2 is expensive. Alternatively, the waste source of CO2 like flue gases that typically contains 4% 15% CO2 can also be added to algae ponds without any harmful effects (Doucha et al., 2005). Besides CO2, the dissolved NOx can also be used by algae as nitrogen source. However, the requirement of flue gas needs to be optimized depending on the algae species, light intensity, and temperature. Furthermore, the altered pH due to dissolution of CO2 and SO2 needs to be controlled or buffered. Being photoautotrophic organism, microalgae rely on solar radiation for photosynthesis and thereby its growth. Therefore, depth of all the algae-culture systems is designed in a way to allow efficient harvesting of light by the algae. Closed photo-bioreactors are equipped with artificial light in the photosynthetic active radiations (PAR) (400 700 nm). Only 45% of solar radiation spectrum is PAR and maximum efficiency to use it during photosynthesis is 27% in algae. Therefore, the maximum theoretical conversion of light energy to chemical energy is approximately 11% (Gao et al., 2007). Furthermore, up to 25% of the photosynthates produced during the day time are utilized at night, as photosynthesis does not occur in dark/night, depending upon temperature and other conditions (Chisti, 2007). Besides, the carbon source and light-, micro-, and macronutrients are required for algae to grow. Among these, nitrogen and phosphorous are the most important nutrients. The nutrients can be supplemented in the form of pure chemicals, but can add significantly to the cost of cultivation. Synthetic media such as CHU, BG 11, and BBM suitable for microalgae cultivation are based on the pure chemicals for nutrient sources. Commercially available agricultural fertilizers can be a relatively low-cost alternative. Furthermore, wastewater effluents rich in these nutrients from different sources can also be used for economic cultivation of microalgae. Microalgae strains exhibit wider tolerance to temperature and other environmental conditions; however, average temperature of 25 C is favorable for optimal growth in general.

18.4.2 Microalgae Biomass Harvesting The smaller size of few micrometers makes harvesting and further concentration technically challenging and thus expensive contributing 20% 30% of the total cost of biomass production (Molina Grima et al., 2003). Gravity settling of the biomass is the simplest

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FIGURE 18.3 Harvesting of microalgae biomass by the process of flocculation.

technique for harvesting but the process is slow therefore requires substantial time in addition to the space. Filtration separates the algae cells from liquid medium by passing a suspension through permeable medium onto a screen of given aperture size that retains the cells. However, due to small mesh size (,100 µm), a pressure of compressed air or vacuum is required to apply across the medium in order to force fluid to flow through the filter. Filtration and microstraining may be cost-effective methods for harvesting microalgae but require large surface areas, high cost, formation of a filter-cake, which substantially increases head loss, prone to clogging, and require frequent maintenance. Centrifugation is commonly used to concentrate small-sized unicellular high-value algae, but generally is considered expensive for biofuel production as it contributes nearly 40% of the production cost. Alternatively, combination of gravity settling followed by centrifugation of the dense biomass as a secondary harvest method would reduce the cost considerably. Most of the algae are characterized as negative-charged surfaces. Because of the identical surface negative charges, microalgae cells repel each other, remain suspended, and do not get settled easily. The surface charge can be blocked by the treatments with flocculants, allowing the cells to adhere each other generating aggregates/flocs, thereby facilitating the sedimentation (Fig. 18.3). In case of electro-flocculation, electric charge is applied to aggregate microalgae cells. The technique has been proven to effectively remove up to 95% of algae in freshwater (Poleman et al., 1997). Main advantage of the technique is no need for flocculants; however, the technique suffers with a disadvantage that the cathodes are prone to fouling. Recently, bioflocculation of nonflocculating microalgae has been evaluated as a simple, effective, economic, and environmentally friendly promising alternative effective method for harvesting of microalgae (Ndikubwimana et al., 2016).

18.4.3 Lipid Extraction and Biodiesel Production The harvested biomass is first pretreated to alter degree of cell disruption, residual moisture content, and particulate size, which are known to affect the microalgal lipid extraction. The ideal technology for lipid extraction microalgae should be specific for

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lipids to minimize co-extraction of contaminants such as protein and carbohydrates. Furthermore, the technique should be more selective for acylglycerols than other lipid fractions to minimize downstream purification. Organic solvent and subcritical organic solvent are the commonly followed lipid extraction methods for microalgae biomass. Organic solvent extraction is based on the basic chemistry concept of “like dissolve like,” which means nonpolar (neutral) organic compounds are dissolved in the nonpolar solvents such as petroleum ether, and vice-versa. In recent years, efforts are being made by scientific community to enhance the kinetics of the lipid extraction by organic solvent method through various modifications including microwave assisted and supercritical organic solvent extraction. In case of supercritical solvent extraction method, the accelerated extraction kinetics and cellular disintegration are achieved at critical temperature and pressure values of the solvent. The emerging green technology appears suitable for lipid extraction from microalgae and has potential to replace the existing organic solvent extraction due to various benefits over the existing one. One of the major challenges for an industrial scale economic production of microalgaederived biodiesel is the nonavailability of the energy efficient cum economic microalgae harvesting, dewatering, and lipid extraction method. Alternate novel method, which does not require harvesting/dewatering the algal cells for biofuel production, is based on the concept of milking or in situ extraction (Yadugiri, 2009). In the process, biocompatible organic solvent (e.g., n-heptane) is re-circulated through the aqueous phase for mixing and lipid extraction purpose. The microalgae cells on repeated exposure to biocompatible solvent retained metabolic activity to continuously produce the compounds of interest without sacrificing of the cells. Feasibility of milking and re-milking the cells of microalgae species with biofuel potential for lipids or other readily usable forms of biofuels has been demonstrated (Zhang et al., 2011). OriginOil, Inc., developed a method of milking known as Live Extraction, in which algae cells are electrically stimulated for continuous oil extraction. The projected benefits of the method are: applicability to wide range of feedstock saves significant energy and time as dewatering of algae biomass is not required, chemical-free process therefore solvent recovery is not required, and the high-throughput method is highly scalable. Simultaneous extraction and transesterification of lipids is popularly known as in situ or direct transesterification. In this process, acid catalyst (sulfuric acid/acetyl chloride is commonly used) and methanol are added to the microalgal biomass. Lipids extracted with methanol are transesterified by the acid catalyst to produce FAME. The downstream processing steps followed are similar to that of the traditional transesterification. Microalgae cell debris is removed out by filtration, and methanol is recovered by distillation of the reaction mixture. After settling, the biodiesel or un-transesterified lipids form top phase, whereas glycerol settles down as a bottom phase. The top phase is decanted off and washed repeatedly with water to eliminate any acid catalyst. Use of solvents with higher polarity has resulted in higher FAME conversion yield (Im et al., 2014). H2SO4 has been proved very effective as a reaction catalyst for converting fatty acids and triacylglycerols (TAGs) from wet microalgae biomass. Performance of the process is regulated by key operating parameters such as ratio of methanol to dried biomass and reaction temperature.

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18.5 PRACTICAL CHALLENGES AHEAD IN BIOFUEL TECHNOLOGIES The first-generation biofuels are derived from food reserves such as vegetable oils, sugars, and starches. This creates food versus fuel scenario globally. In the context of developing countries such as India, which import edible oil to supplement the food demand, it is less likely to derive biofuels from food crops. Furthermore, the feedstocks for first-generation biofuel compete with food crops for land and water resources. The resources for first-generation biofuels are also limited for competing with fossil fuel. Therefore, the alternative may be the second-generation biofuels, which uses cellulosic products such as forest- or agro-based plant residues for biofuel production. Furthermore, the whole plant and not just its parts can be utilized for producing these biofuels, therefore feedstock availability is not a problem. However, the process involved requires huge capital investment and relatively simple conversion processes are not available consequently, and there is no proven commercial technology available today. Microalgae are a promising feedstock for the third-generation biofuels owing to their rapid growth rate and higher lipid productivity than the best oil producing terrestrial plants. Furthermore, the higher photosynthetic efficiency and wider adaptability to different environmental conditions are the other reasons for interest in microalgae for biofuels (Chisti, 2007). Microalgae, which can be grown even in wastewaters, successfully eliminate competition with food crops. However, the major challenges are cost-effective microalgae cultivation and harvesting technique for biofuel production. Furthermore, rapid and economical technique for biomass drying, extraction of lipid, and further conversion into biodiesel or other biofuels are not yet available. However, development of model-integrated production and biorefinery system will bring viable algae-based biofuels into the market.

18.6 OMICS ADVANCEMENT AND APPROACHES FOR COST-EFFECTIVE PRODUCTION OF RENEWABLE ENERGY Over the past few years, a massive amount of omics data has been generated to gain insight into biofuel production. Omics advancements contribute to the development of the fourth-generation biofuels from genetically engineered species (Grover et al., 2013, 2014a,b). The omics technological advancement has tremendous future scope to extract deeper biological knowledge and thereby cost-effective production of renewable energy. It mainly includes key area of research such as new strain development, improved cultivation, low-energy harvesting, and high-yield extraction-conversion technology. In order to advance the economic feasibility of the microalgae or other feedstocks, much attention is being given on genetic and metabolic engineering to increase the yield of biofuel relevant lipids without compromising the growth. The lipid accumulation in microalgae is usually triggered by exposure to some forms of stress including nutrient deficiency, salinity, temperature, etc. Therefore, research efforts to identify the lipid triggers and further engineering the algal strains with potential to produce more lipids

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throughout the whole growth cycle have received significant attention. Earlier efforts to increase lipid content and modify the fatty acid profile to optimize microalgae as a biodiesel feedstock by introducing heterologous plant fatty acid synthesis enzymes have resulted in relatively lower success. This emphasizes the necessity of comprehensive understanding of the algal fatty acid biosynthetic machinery. At present, scant available information on algal metabolic pathways and nonavailability of stable transformation protocol are the major challenges in engineering microalgae for high lipid content. Algal fatty acid biosynthesis pathway has been deduced based on homology with well-characterized plant system and the fatty acid synthase enzyme encoding genes have been annotated in many of the genome sequenced algae. Lei et al. (2012) cloned five genes viz. acylase carrier protein (ACP), ketoacyl-ACP synthetases (KAS), thioesterase (TE), fatty acid desaturase (FAD), and malonyl ACP transacylase (MAT) involved in fatty acid biosynthesis from Haematococcus pluvialis. Among the genes, transcript expression levels of ACP, KAS, and TE were in linear relationship with fatty acid synthesis in response to the different lipid biosynthesis-triggering stressors, thus were proposed as key rate limiting genes. As mentioned earlier, in general, microalgae accumulate lipids as an energy storage molecule on exposure to the environmental stresses; however, net lipid yield is reduced due to severe decline in biomass growth on exposure to the stresses. Fan et al. (2014) reported differential lipid accumulation and transcript expression of genes involved in, in response to nitrogen, phosphorous, or iron depletion in an oleaginous microalgae Chlorella pyrenoidosa. The transcript abundance of genes encoding ME (malic enzyme), Accase (acetyl CoA carboxylase), and DGAT (di-acyl glycerol acyl transferase) showed significant correlation with lipid accumulation. Thus, these genes are likely to exert great influence on lipid biosynthesis. ME being considered as a major supplier of a critical factor for intracellular fatty acid content, that is, NAPDH, overexpression of the encoding gene could have enhanced lipid content. Accase catalyzes the first rate-limiting step in the fatty acid biosynthesis pathway through formation of malonyl CoA from acetyl CoA, whereas DGAT enzyme plays an important role in acylation process of diacylglycerol into TAG in lipid biosynthesis. Therefore, the genes encoding these enzymes could be important targets for metabolic engineering of microalgae strains for enhanced biofuel production. Trentacoste et al. (2013) demonstrated the application of targeted metabolic engineering toward the enhancement in lipid accumulation in eukaryotic microalgae Thalassiosira pseudonana without compromising growth. In this study, expression of multifunctional lipase/phospholipase/acetyl transferase was silenced through targeted antisense knockdown approach. The knockdown microalgae strains thus developed exhibited greater than threefold higher lipid content as compared with the wild type without compromising growth during exponential growth phase. The lipid content was fourfold higher than the wild type after 40 hours of silicon starvation. In order to extract the lipids from microalgae for biofuel production, the biomass is handled in energy intensive steps of harvesting, drying, and then organic solvents extraction, severely affecting overall economics of the derived biofuels (Molina Grima et al., 2003). Therefore, to skip these steps, Liu et al. (2011) genetically engineered the cyanobacteria to produce and continuously secrete the free fatty acids, which can be further collected from the culture medium. Acyl-ACP thioesterase I encoded by tes A gene in

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Escherichia coli is normally a periplasmic protein due to presence of a signal sequence peptide. In absence of the signal peptide, the synthesized fatty acids are secreted in the culture medium (Cho and Cronan, 1995). The same concept is being industrialized for biofuel production using engineered E. coli by the biofuel company LS9 (Steen et al., 2010). Recently, Liu et al. (2011) applied the concept to cyanobacteria because being photosynthetic organism cyanobacteria has a big advantage over the E. coli. Alkanes (C4 C23) possess higher energy density and compatibility with existing liquid fuel engines and are the major constituents of gasoline, diesel, and jet fuels. Wang et al. (2013) genetically engineered cyanobacteria, which exhibit higher photosynthetic efficiency and growth rate than the eukaryotic microalgae, to achieve efficient photosynthetic production of alka(e)nes. Alkane biosynthetic genes [acyl-acyl carrier protein reductase (AAR), aldehyde deformylating oxygenase (ADO)] were over-expressed under the regulation of strong promoter Rubisco in cyanobacteria Synechocystis sp. PCC 6803. The overexpression resulted in more than eightfold enhanced alka(e)ne production in the engineered strains on dry weight basis than the wild-type strains. Furthermore, feasibility of the enhanced alka(e)ne production in the engineered strains was demonstrated by redirecting the carbon flux to acyl-ACP. Thus, the results demonstrate the power of metabolic engineering strategies to overproduce alka(e) nes in cyanobacteria. However, enhanced understanding on physiological roles and regulatory mechanism of native alka(e)nes in cyanobacterial cells need to be developed. As medium-chain alkanes are less toxic to the cells than the other nonnative products, huge scope lies in engineering cyanobacteria for enhanced alkane production. Genetic engineering approach has been widely used for improvement in biofuel traits in terrestrial plants as well. Members of NAC family genes contribute to enhanced stress tolerance and in secondary growth of the plants, thereby building biomass. Thus, overexpression of NAC transcription factor gene provides a possibility to tailor biofuel plants. Several studies on overexpression of NAC genes have shown improved biotic and abiotic stress tolerance as well as enhanced biomass production in the transformed plants (Singh et al., 2016). Phenomenon of photorespiration causes considerable losses in photosynthetic productivity of most C3 plants. Recently, Dalal et al. (2015) reported significant impact of photorespiratory bypass approach on increasing seed productivity for biofuel crop Camelina. On overexpression of photorespiratory bypass genes, photorespiration was reduced coupled with increased photosynthesis in the bypass expressing Camelina lines. In these lines, seed yield was increased by up to 70% without any loss in seed quality. Furthermore, the transgenic plants also produced more biomass with earlier flowering, seed setting, and maturity than the wild types. Thus, the approach may be useful in other C3 biofuel plants for early and higher biomass yield for biofuel production. Commercial application of genetically engineered species is, however, subject to strict biosafety regulations. In case of an aquatic species like microalgae, biosafety requirements such as sterilization of entire cultivation system to prevent release in open environment will have a detrimental impact on overall economics and energy balance of the system. Thus, the large-scale application of engineered microalgae in the current biosafety regimes does not appear viable.

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18.7 CONCLUSION AND FUTURE PERSPECTIVES Natural petroleum resources synthesized over millions of years are likely to be exhausted shortly. Although, a number of alternative fuels have been discovered, none of them is as usable as biofuels are, primarily because we do not need to change the way we currently use our fuels or energy resources. Both the cultivation of the raw material for biofuels and harnessing the yield are challenging as these so-called biofuel crops are relatively naı¨ve to our agricultural systems and understandings. Thus, huge amount of investments on research and development of these resources are required. For the ease of classification of various biofuel technologies, these have been classified into the first, second, third, and fourth generations, which in anyway does not indicate the superiority of one over the other. The limited potential of first-generation biofuels to make a significant contribution to displace fossil fuels and reduce GHG emissions highlighted by several studies unleashed a sense of urgency for the transition toward second-generation biofuels (Limayema and Ricke, 2012). While dedicated energy crops would still be competing for land with food crops, it is envisioned that either by using lesser quality soils (Jatropha) or by providing more utilizable biomass per unit of land (e.g., Camelina, switchgrass, or short tree rotations), the pressure for prime quality soils will be reduced. As compared to the case of first-generation biofuels, where feedstock can account for over two-thirds of the total costs, the share of feedstock in the total costs is relatively lower (30% 50%) in the case of second-generation biofuels. To date, there is no large-scale commercial production of second-generation biofuels. If external costs of production of fossil fuels were considered, the cost difference will generally be lower for many second-generation biofuels. At present, the second-generation biofuel technologies look most promising, as they do not compete for resources with the food crops, no special requirements for cultivation of the raw material exist, and technology for conversion of biomass to biofuels is nearing commercialization. Furthermore, the major drawbacks of these technologies that are energy input for thermochemical conversion can be overcome by maturation of third- and fourth-generation biofuels. Given the current state of technology, and the uncertainty remaining about the future breakthroughs that would potentially make some advanced-generation biofuels cost competitive, policymakers need to carefully consider what goals are to be pursued in providing support to different biofuels. Biofuels that simultaneously advance multiple policy goals could warrant greater support when designing incentive mechanisms. An integrated approach combining economically sustainable rural development, climate change mitigation, and alternative energy provision provides a good policy framework for advanced-generation biofuels. It is also necessary to consider regional and international developments in policies and trade in order to maximize the potential benefits achievable through the policies implemented (Pacheco, 2007).

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References Abigor, R.D., Uaudia, P.O., Foglia, T.A., Haas, M.J., Jones, K.C., 2000. Lipase-catalyzed production of biodiesel fuel from some Nigerion lauric oils. Biochem. Soc. Trans. 28, 979 981. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294 306. Cho, H., Cronan Jr., J.E., 1995. Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J. Biol. Chem. 270, 4216 4219. Dalal, J., Lopez, H., Vasani, N.B., Hu, Z., Swift, J.E., Yalamanchili, R., et al., 2015. A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa. Biotechnol. Biofuels 8, 175. Dizge, N., Keskinler, B., Tanriseven, A., 2009. Biodiesel production from canola oil by using lipase immobilized onto hydrophobic microporous styrene-divinylbenzene copolymer. Biochem. Eng. J. 44, 220 225. Doucha, J., Straka, F., Livansky, K., 2005. Utilization of flue gas for cultivation of microalgae (Chlorella sp.) in an outdoor open thin layer photobioreactor. J. Appl. Phycol. 17, 403 412. Elliott, D., 2008. Biofuels research opportunities in thermochemical conversion of biomass. In: Conference on Cellulosic Biofuels. 1. http://scholarworks.umass.edu/timbr/1. Fan, J., Cui, Y., Wan, M., Wang, W.Q., Li, Y., 2014. Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors. Biotechnol. Biofuels 7, 17. Gao, K.S., Wu, Y.P., Li, G., Wu, H.Y., Villafane, V.E., Helbling, E.W., 2007. Solar UV radiation drives CO2 fixation in marine phytoplankton: a double edged sword. Plant Physiol. 144, 54 59. Grover, A., Patade, V.Y., Kumari, M., Gupta, S.M., Arif, M., Ahmed, Z., 2013. Omics approaches in biofuel production for a green environment. In: Barh, D., Zambare, V., Azevedo, V. (Eds.), OMICS: Applications in Biomedical, Agricultural and Environment Sciences. CRC Press, Taylor & Francis Group, LLC, Boca Raton, FL, pp. 623 636. ISBN: 9781466562813, Catalog No.:K15973. Grover, A., Patade, V.Y., Kumari, M., Gupta, S.M., Arif, M., Ahmed, Z., 2014a. Bio-energy crops enter the omics eraIn: Barh, D. (Ed.), OMICS Applications in Crop Science. CRC Press, Taylor & Francis Group, LLC, Boca Raton, FL, pp. 549 562. ISBN: 9781466585256. Available from: doi:10.1201/b16352-18. Grover, A., Singh, S., Pandey, P., Patade, V.Y., Gupta, S.M., Nasim, M., 2014b. Overexpression of NAC gene from Lepidium latifolium L. enhances biomass, shortens life cycle and induces cold stress tolerance in tobacco: potential for engineering fourth generation biofuel crops. Mol. Biol. Rep. 41, 7479 7489. Im, H., Lee, H., Park, M.S., Yang, J.W., Lee, J.W., 2014. Concurrent extraction and reaction for the production of biodiesel from wet microalgae. Bioresour. Technol. 152, 534 537. Kaieda, M.T., Kondo, S.A., Fukuda, H., 2001. Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent free system. J. Biosci. Bioeng. 91, 12 15. Kulkarni, M.G., Gopinath, R., Meher, L.C., Dalai, A.K., 2006. Solid acid catalyzed biodiesel production by simultaneous esterification and transesterification. Green Chem. 8, 1056 1062. Lei, A., Chen, H., Shen, G., Hu, Z., Chen, L., Wang, J., 2012. Expression of fatty acid synthesis genes and fatty acid accumulation in Haematococcus pluvialis under different stresses. Biotechnol. Biofuels 5, 18. Limayema, A., Ricke, S.C., 2012. Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Prog. Energ. Combust. 38, 449 467. Liu, X., Sheng, J., Ill, R.C., 2011. Fatty acid production in genetically modified cyanobacteria. Proc. Natl Acad. Sci. 108, 6899 6904. Menon, V., Rao, M., 2012. Trends in bioconversion of lignocellulose: biofuels, platform chemicals and bio-refinery concept. Prog. Energ. Combust. 38, 522 550. Molina Grima, E., Belarbi, E.H., Acie´n, Ferna´ndez, F.G., Robles, Medina, A., et al., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Resour. Conserv. Recycling 19, 1 10. Munoz, R., Guieysse, B., 2006. Algal-bacterial processes for the treatment of hazardous contaminants: a review. Water Res. 40, 2799 2815. Ndikubwimana, T., Zeng, X., Murwanashyaka, T., Manirafasha, E., He, N., Shao, W., et al., 2016. Harvesting of freshwater microalgae with microbial bioflocculant: a pilot-scale study. Biotechnol. Biofuels 9, 47. Pacheco, M.A., 2007. Overview of biofuel technologies. In: TAPPI International Renewable Energy Conference, 10 11 May 2007, Atlanta, Georgia.

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Philbrook, A., Alissandratos, A., Easton, C.J., 2013. Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass. In: Petre, M. (Ed.), Environmental Biotechnology New Approaches and Prospective Applications. InTech. Available from: http://dx.doi.org/10.5772/55309. Available from: https://www.intechopen.com/books/environmental-biotechnology-new-approaches-and-prospective-applications/ biochemical-processes-for-generating-fuels-and-commodity-chemicals-from-lignocellulosic-biomass. Poleman, E., De Pauw, N., Jeurissen, B., 1997. Potential of electrolytic flocculation for recovery of micro-algae. Resour. Conserv. Recycling 19, 1 10. Selmi, B., Thomas, D., 1998. Immobilized lipasecatalyzed ethanolysis of sunflower oil in a solvent free medium. J. Am. Oil Chem. Soc. 75, 691 695. Shah, S., Sharma, S., Gupta, M.N., 2004. Biodiesel preparation by lipase-catalyzed transesterification of jatropha oil. Energy Fuels 18, 154 159. Sims, R.E.H., Mabee, W., Saddler, J.N., Taylor, M., 2010. An overview of second generation bio-fuel technologies. Bioresour. Technol. 101, 1570 1580. Singh, S., Grover, A., Nasim, M., 2016. Biofuel potential of plants transformed genetically with NAC family genes. Front. Plant Sci. 7, 22. Soccol, C.R., Faraco, V., Karp, S., Vandenberghe, L.P.S., Soccol, V.T., Woiciechowshi, A., et al., 2011. Lignocellulosic Bioethanolbioethanol: Current status and future perspectives. In: Pandey, A. (Ed.), Biofuels: Alternative Feed Stocks and Conversion Processes. Academic Press, Salt Lake City, UT, ISBN: 978-0-12-385099-7. Steen, E.J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., et al., 2010. Microbial production of fattyacid-derived fuels and chemicals from plant biomass. Nature 463, 559 562. Surendhiran, D., Sirajunnisa, A.R., Vijay, M., 2015. An alternative method for production of microalgal biodiesel using novel Bacillus lipase. 3 Biotech 5, 715 725. Thomas, D.F., Andy, A., Dutta, A., Phillips, S., 2009. An economic and environmental comparison of a biochemical and a thermochemical lignocellulosic ethanol conversion processes. Cellulose 16, 547 565. Trentacoste, E.M., Shrestha, R.P., Smith, S.R., Gle, C., Hartmann, A.C., Hildebrandt, M., et al., 2013. Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc. Natl. Acad. Sci. 110, 19748 19753. Wang, W., Liu, X., Lu, X., 2013. Engineering cyanobacteria to improve photosynthetic production of alka(e)nes. Biotechnol. Biofuels 6, 69. Watanabe, Y., Pinsirodom, P., Nagao, T., Yamauchi, A., Kobayashi, T., Nishida, Y., et al., 2007. Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase. J. Mol. Catal. B Enzym. 44, 99 105. Yadugiri, V.T., 2009. Milking diatoms—a new route to sustainable energy. Curr. Sci. 97, 748 750. Zhang, F., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2011. Screening of biocompatible organic solvents for enhancement of lipid milking from Nannochloropsis sp. Process Biochem. 46, 1934 1941. Zhang, L., Xu, C., Champagne, P., 2010. Overview of recent advances in thermo-chemical conversion of biomass. Energ. Convers. Manage 51, 969 982.

Further Reading Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., et al., 2016. Peptide mediated microalgae harvesting method for efficient biofuel production. Biotechnol. Biofuels 9, 10. Oey, M., Ross, I.L., Stephens, E., Steinbeck, J., Wolf, J., Radzun, K.A., et al., 2013. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii. PLoS One 8, e61375. Park, W., Feng, Y., Ahn, S.J., 2014. Alteration of leaf shape, improved metal tolerance, and productivity of seed by overexpression of CsHMA3 in Camelina sativa. Biotechnol. Biofuels 7, 96. Radakovits, R., Eduafo, P.M., Posewitz, M.C., 2011. Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum. Metabolic Eng. 13, 89 95. Yao, L., Qi, F., Tan, X., Lu, X., 2014. Improved production of fatty alcohols in cyanobacteria by metabolic engineering. Biotechnol. Biofuels 7, 94.

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19 Omics-Based Bioengineering in Environmental Biotechnology Tehreem Tanveer1, Kanwal Shaheen1, Sajida Parveen2, Zujaja T. Misbah2,3, Mustafeez M. Babar3 and Alvina Gul2 1

Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan 2National University of Sciences and Technology (NUST), Islamabad, Pakistan 3Shifa Tameer-e-Millat University, Islamabad, Pakistan

19.1 INTRODUCTION Environmental omics, a very active research field, has recently gained extensive attention due to its application in the study of genes, protein metabolites, RNAs, and related functions as a result of their exposure to environmental chemicals. This emerging research avenue is now of pivotal importance owing to its contribution to understanding the effects of environmental chemicals on health and environment. For the identification of environmental threats, their classification and prioritization, screening for toxicity, and mode of their toxic action, environmental omics utilizes transcriptomics, proteomics, metabolomics, and genomics. In addition, it helps in identifying or monitoring the harmful effects of exposure to these chemicals on living organisms and environment. Moreover, they help in understanding and identifying the toxicity pathways, biomarkers, and genetic signatures. Owing to its vital role, there is a constant need to improve its current understanding by highlighting the milestone developments. This would encourage the exploration of new means to broaden its applications for future studies (Ge et al., 2013). Our understanding about how the ecosystem and living systems are affected by environmental toxins has been enhanced by omics-based technologies for identification of metabolic, proteomic, and genetic profiles. Determination of chemical toxicity, assessment of associated health risks, and the field of environmental toxicology are endowed by advancement in omics. In order to understand the effects of these chemicals on health and environment, means to gap the outcomes of omics and toxicity data should be developed.

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The integration of the two fields would help in ensuring the real challenges and issues of environment including identification of health and environmental biomarkers, establishment of innovative approaches of environmental omics, and chemical mixture toxicology characterization. These methods would help in enhancing our knowledge of chemical toxicity, monitoring and evaluation of ecological and health hazards, and exploitation of natural resources in ways that sustain living organisms and the environment. Moreover, the screening and early discovery of various environmental toxins that can potentially contribute to the development of diseased conditions can also be investigated (Ge et al., 2013). Application of environment omics includes the study of environmental toxins, their toxicity mechanism, and their significant effects on the health and the ecosystem. It, additionally, investigates both the short- and long-term effects of these toxins. The broad range of applications in this field has also helped in the clinical surveillance of environmental stress factors and their effect on biological systems. For instance, for the study of mechanisms involved in arsenic carcinogenesis, the techniques of metabolomics, proteomics, and genomic technologies have been widely employed (Lau and Chiu, 2006; Tsuchiya et al., 2005). In environmental toxicology and health hazard research, these fields are of utmost importance. By decoding mechanism of toxicity and their mode of action, omics helps to broaden the scope of investigation of health and ecological risks (Mei et al., 2007; Ortiz et al., 2010; Wang et al., 2012). Moreover, it is also used for the identification of exposure and toxicity biomarkers by studying environmental diseases and toxic effects (Matheis et al., 2011; Vineis et al., 2009; Yasokawa and Iwahashi, 2010). Cross-species toxicity patterns can, hence, also be investigated (Eaton et al., 2006; Ralston-Hooper et al., 2011). An application of mixture of bioinformatics, genomic, and proteomic methods has also been used to study the lethal mode of action of fungicides and endocrine imbalance (Ge et al., 2011; Kishi et al., 2006). A detailed insight of the toxic mechanisms involved in the pathogenesis has also been provided by an integrated omics approach. Experimental data obtained as a result of these studies have been used in the establishment of toxicity prediction methods for chemical extrapolation, endpoint determination, and also for the assessment of health hazards of these chemicals. Another vital application of omics on environmental toxicology and related effects on living organisms is recognition of changes at metabolite, protein, and gene level as a function of expression profiling (Bruno et al., 2009; Mezhoud et al., 2008; Yi and Pan, 2011). This chapter provides an insight into the omics-based bioengineering and its application in the field of environmental biotechnology. Ecology of soil microbiology, means to control environmental pollution, identification of chemical-induced toxicity, and the geno-proteomic modifications associated with the environmental stress factors have been discussed in the light of the recent developments in the field.

19.2 APPLICATION OF OMICS IN SOIL MICROBIAL ECOLOGY Development of flawless experimental methodologies, systems, and technologies for estimating the various unknown microbes in nature is very urgent in order to meet the environmental challenges and to maintain a healthy natural environment. Application

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of omics-based approach to systematic analysis of environmental microbial communities has been used to recognize unique catalysts, which can reduce environmental toxins. Moreover, it has also contributed to the sanitation of polluted natural resources to form unique bio products that can be employed for the monitoring of environmental risk factors. Thus, the significance of omics has led to the emergence of new research fields including metametabolomics, metaproteomics, and metagenomics. The fields are still in their infancy and considerable efforts need to be done to improve their understanding. A number of research groups are currently working to decipher the mechanisms involved in the interaction of microbes, the environment, and the health of living organisms. Tringe et al. (2005) explored the combined environmental signature gained from various microbial ecosystems by using a gene-based-bioinformatics method (Tringe et al., 2005). This work suggested that the predicted protein complement of a community is affected by its environment. A number of other studies have been carried out to provide novel insights related to the field of metaproteomics. A few of these include the investigation of protein expression profiles from activated sludge (Wilmes and Bond, 2006; Wilmes et al., 2008), exposure of freshwater samples to heavy metals (Lacerda et al., 2007), filthy soil and groundwater (Benndorf et al., 2007), endosymbiont (Markert et al., 2007), lake water (Pierre-Alain et al., 2007), and extracellular proteins in activated sludge (Park et al., 2008). Among the recent advancements, based on a similar approach, a novel protein was isolated as an essential component of energy conservation in acid mine biofilms (Ram et al., 2005). Hence, integrating the data obtained from the molecular applications on the microbes has helped in gaining a deeper understanding of the mechanisms involved in their adaptation under various environmental conditions. Although, there are only a few studies on metaproteomics but this field has already gained importance for its use in the functioning of microbial ecosystem. An approach of metaproteomics to study the microbial community collected from the Chesapeake Bay was introduced by Kan et al. (2005). Dissolved proteins in seawater have also been examined for the same purpose. In the light of these studies, metaproteomics analyses were conducted on numerous marine environmental samples. Consequently, several metabolic and physiological activities including nutrient consumption and environmental adaptation were discovered (Morris et al., 2010). Metaproteomics, hence, opens a new window for marine microbial oceanography and microbial biogeochemistry through investigation of protein expression in complex marine environmental samples (Wang et al., 2011). Based on the oil spill degradation rate, scientists have also predicted that a new species of microbes that feed on the petroleum hydrocarbons in deep-water horizon may exist (Beazley et al., 2012).

19.2.1 Metagenomics and Soil Function On the basis of the relative abundance of genes, metagenomics approaches characterize specific microbial communities. The main purpose of these approaches is, hence, to deliver a broad and complete view of these communities, even though the emphasis is mainly on a portion of the physiological functions represented. There are a number of molecular techniques used so far such including next generation sequencing, genetic

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polymorphism studies, and differential genetic sequence patterning. Although these techniques have provided a detailed insight into the learning of metagenomics yet, they are susceptible to certain limitations (Lombard et al., 2011). Soil metagenomics is vulnerable to limitations in the quantification of sequenced genes and their annotation. This leads to issues related to the complete coverage of soil metagenome. For example, in a study even by using 300 Gbp of sequence data, complete coverage of soil community was not attained (Howe et al., 2014). By using quantitative methods, metagenomics data can be explained in terms of the relative abundance of functional genes. This can help in the prediction of the activity of these genes and their relative functional contribution, for example the comparison of the activity of DNA gyrase/citrate lyase with ammonia monooxygenase. Moreover, the main purpose for using metagenomics has been to exploit the association of difference in functional gene abundance with the difference in the rate and of their associated processes. A number of recent studies have investigated the relative gene expression as a function of the biochemical process of the microbes, for instance, the changes in the characteristics of soil following fire (Ta¸s et al., 2014), presence of Sphagnum spp. (Jacquiod et al., 2013), and chitin amendment (Bragina et al., 2014) in various environmental samples.

19.2.2 Metatranscriptomics and Soil Function The main function of metagenomics study is to identify the microorganisms present in the soil sample. It also helps in the gaining information related to the seed bank and the microbes in the environmental samples. To the contrary, metatranscriptomics can provide us the information related to the actual physiological activity of the microbes and the rate at which they are performing the function. At the technical level, metatranscriptomics is based on the RNA extraction from a community of microbes, followed by mRNA sequencing or cDNA synthesis and ultimately its amplification. Thereafter, an in-depth information related to the relative abundance of gene transcripts is obtained by employing a combination of sequencing and computational tools (Carvalhais et al., 2012; Tveit et al., 2013).

19.2.3 Extracting Value From Metatranscriptomics The availability and abundance of gene transcript gives us the indication that whether the protein encoded by the gene has a current quantitative or qualitative activity or there may be an activity by the protein in the future. It can, hence, be said that it is responsible for giving us information regarding the impact of external environment on the microbial community. An indication of up and down regulation of a gene can be seen by the measurement of change in the abundance of gene transcripts helping in the prediction of the metabolic rates. However, there is a limitation in the reliability of this information as there is a lack of detailed information regarding the dynamics of production, decomposition of transcripts, and the products encoded by these transcripts. For example, the abundance of transcripts of 16S rRNA delivers information regarding the future activity but there are a number of limitations related to the calculation of the protein kinetics (Blazewicz et al., 2013).

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19.2.4 Niche Specialization and Differentiation Specialization and differentiation of niche develops connections between heterogeneity of soil, activity, and structure of the microbial community. It creates a connection between the ecological and evolutionary theories along with providing a detailed information and explanation about the composition of microbial community in the soil. Most of the studies on niche specialization assume that there is a deep connection between the function and phylogeny as niche specialization is a widely accepted concept in soil microbial ecology. Niche specialization forms the foundation for the investigation of correlation between characteristics of soil and metagenomes. It also explains the relationship of communities to ecosystem function and the response of environment to the communities. In order to answer these questions, an in-depth investigation to gain knowledge is necessary. These would include the study of environmental selection patterns, speciation, invasion and dispersal, and the investigation of connection between phylogeny, physiology, mutation, and recombination. A number of research groups have investigated the impact of the environment on the microbial communities. The conceptual pretext employed in them is that the members of different phylogenetic groups are evolved in such a way that they will respond in the same manner to a particular environmental fluctuation. All these analyses reflect that the composition of a community has an enormous impact on the ecosystem and its function. Specialization and differentiation of niche results in the genetic diversity that is followed by a particular selection pattern. Both the ecosystem and the genetic diversity are based on the activity of the microbes and their response to environmental threats. Hence, alternate means should be employed to study the assembly of the microbial community (Ofi¸teru et al., 2010).

19.3 APPLICATION OF OMICS IN CONTROLLING POLLUTION The accumulation of substances, such as pesticides and other xenobiotics, in exposed tissues of organisms can be evaluated by a number of biomonitoring techniques. Organisms like filter feeders have great ability to gather pollutants. These organisms are, hence, used as sentinel organisms. For monitoring at environmental level, specialized and diverse biomarkers are used as there may be less interrelated pollutants in environment causing numerous outcomes. In order to recognize ecological biomarkers and to develop some significant interconnections among disease, exposure, and their outcomes, omics technologies are now being employed. They generate a large amount of data in an efficient and cost-effective manner. Environmental biomarkers used for evaluating exposure and toxicity have been found by comparison between omics profiles of exposed organisms, tissues, and cells. The data are, then, compared on the database with profiles that were subjected to known hazardous substances (Williams et al., 2011). Recently, omics-based techniques for the evaluation of ecological toxicity markers are frequently used ecotoxicology research. Various biomarkers for ecotoxicology monitoring are now available (Martı´nDı´az et al., 2008). The techniques currently employed are, however, still in the evaluation phase. A few limitations in this field include the availability of less number of model organisms for environmental data sequences and absence of criteria for checking accuracy

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FIGURE 19.1

Tracking insights of the bioremediation using omics-based approaches.

of ecological biomarkers (Ge et al., 2013). Efforts should, hence, be made to develop newer, more efficient methods for the monitoring of eco-toxicity. Fig. 19.1 summarizes the process of bioremediation for addressing the issue of environmental pollution. Contaminated environmental sample is collected and DNA from organisms is extracted. DNA microarrays are done using transcriptomics, it is followed by proteomics and interactomics. Extraction of proteins using this approach can be used to screen new molecules during the process of mineralization.

19.4 APPLICATION OF OMICS-BASED BIOENGINEERING FOR CHEMICAL TOXICITY SCREENING Several procedures are employed for the health risk assessment after exposure to a particular chemical. Unfortunately, these procedures are not well-suited to be used simultaneously for multiple chemical entities or their mixtures. Newer and more

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sophisticated omics techniques can be used for the risk assessment, thereby, providing an opportunity to develop advanced assessment and calculation of joint outcomes. Omics technology can potentially be employed for interpreting the changes that occur as a consequence of unfavorable environment conditions on an organism at the DNA, RNA, protein, and metabolite level. Moreover, the chemicals and their mixtures, which are considered hazardous for the health and the environment, need to be recognized. This is done by understanding and recognition of molecular targets, pathways, and ecological outcomes to organisms after an exposure. Above all, in different mixtures of chemicals, using the omics tools, the recognition, classification, and threat assessment are done owing to their efficiency in recognizing the endpoints of toxicity of novel molecules (Iwahashi et al., 2007; Hook et al., 2008). Therefore, for the characterization of interactions between chemicals and health risk associated with these chemicals, metabolomics and proteomics are now widely employed (Merhi et al., 2010; Pelletier et al., 2009; Wu and Wang, 2010). Studies are carried out to identify a particular chemical in a mixture and analyzing its capability to induce an adverse effect. Based upon the monitoring data, these studies are helpful in developing models that can be used to analyze the level of toxicity of chemical mixtures and to develop safer alternates. Therefore, it is imperative that genomics, proteomics, and metabolomics play a significant role in analyzing and improving the chemical mixtures that can ultimately pose a threat to living organisms and their environment. Moreover, they can directly be employed for environmental health risk monitoring by improving the experimental design, analyzing the data, and developing alternate models for the prediction of toxicity associated with the environmental toxins.

19.5 OMICS APPLICATIONS IN ENVIRONMENTAL STRESS-RELATED GENE AND PROTEIN MODIFICATIONS Omics-based technologies can be employed for the recognition of genes and modification of proteins as a function of environmental toxins. For instance, they can be employed for the study of oxidative stress and oxidation of proteins caused by the action of the environmental chemicals at the cellular level. Reactive oxygen species are produced by the cell and, then, absorbed by proteins in very high concentration as a result of ecological toxicity (Sheehan and McDonagh, 2008). Thus, proteomic analysis can also be employed for the study of adverse effects of environmental toxicity via oxidative stress that is manifested in the form of protein modifications. Fig. 19.2 represents the integrative omics approaches that help in deciphering the changes caused by environmental contaminants in the plant body. In addition, redox proteomics study can also be employed for the identification and quantization of changes occurring in proteins due to environmental toxicity. Sheehan (2006) designed a procedure for the identification and measurement of redox-based toxicity by using simple electrophoresis separations (Sheehan, 2006). The application of this methodology can help in improving the prediction of stress-related outcomes, for instance, active thiol sepharose is used to detect proteins or redox variants in shotgun proteomics (Hu et al., 2010). The protein phosphorylation and oxidation of processes are similar to

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FIGURE 19.2

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A schematic overview of study of abiotic stress in plants using common system biology.

each other as both are reversible. However, the oxidation of cysteine, containing the thiol groups, does not disrupt function of proteins (Held and Gibson, 2012). The thiol protein modification process is a crucial signaling pathway that plays a role in regulation of redox reactions and, hence, the study of toxicity (Winterbourn and Hampton, 2008). Similarly, carbonylation process is also being investigated and studied as a marker of environmental toxicity (Chora et al., 2010; Tedesco and Sheehan, 2012). It has, recently, been established that the modification by means of carbonyls leads to an increased oxidative stress (Sheehan and McDonagh, 2008). Thus, carbonylation is considered to be responsible for loss of entire function of protein and, thus, can be used for identification of damage caused by oxidation (Cannizzo et al., 2011). As discussed, oxidized proteins are easier to identify by using the omics techniques that can further be developed to detect toxicity biomarkers. However, newer and alternate methods need to be developed for the direct detection of oxidative stress and related changes as a result of environmental chemicals. Proteomics can be employed in a manner to detect the particular compounds causing a toxicity as well as for the exact protein modifications that take place in response to an environmental toxin. In order to identify the toxic agents and mechanisms, it is crucial to isolate the oxidized proteins. The proteins can then be used for understanding the structural and functional alterations, and an environmental toxin might have caused. All of these mechanisms can be studied via the analysis of modifications observed in the RNAs and metabolites. Therefore, in addition to the alterations in the proteins, changes in transcriptome and metabolome of an organism can also function to decipher the mechanistic linkages between environmental toxins and the related outcomes. They can, hence, serve as important biomarkers of environmental exposure, toxicity, and effects. However, further efforts to identify newer and more specific indicators in this regard are needed (Ge et al., 2013).

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Among the various branches of omics, genomics provide the most basic information about the toxicity profiles of various environmental toxins. It is aimed at the identification of effects of various chemicals and their metabolites at the genetic level, for instance, the changes in the gene expression in various models (Iwahashi et al., 2006). Moreover, the mutations in sequences and the resulting patterns are generated and compared. Apart from the advancements in the field of genomics and the availability of genomic data, the outcomes of these studies are not precise enough to predict the level of toxicity under in vivo circumstances. It is, therefore, imperative that the genomics should be studied in conjunction with the transcriptomics, proteomics, and metabolomics data (Kishi et al., 2006; Lau and Chiu, 2006).

19.6 CONCLUSION AND FUTURE PERSPECTIVE In recent years, the field of omics has considerably advanced in terms of techniques, robustness, and cost-effectiveness. Its application to ecotoxicology and risk assessment of toxicity in organisms is used as reference models. For improved application of omics techniques, a deeper understanding of means to employ genomic and proteomic data to environmental toxicology is required. Moreover, the consideration of association between the outcomes of environmental conditions and biomarkers for ecological assessment is necessary. It is, therefore, expected that in future the advancements in the application of omics-based technologies would contribute to the development of better quality crops under abiotic stress conditions.

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Ge, Y., Bruno, M., Foth, H., 2011. Applications of proteomic technologies to toxicology. Gen. Appl. Syst. Toxicol. 2011, 197 215. Ge, Y., Wang, D.-Z., Chiu, J.-F., Cristobal, S., Sheehan, D., Silvestre, F., et al., 2013. Environmental omics: current status and future directions. J. Integrat. Omics 3 (2), 75 87. Held, J.M., Gibson, B.W., 2012. Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes. Mol. Cell. Proteomics 11 (4), R111. 013037. Hook, S.E., Skillman, A.D., Gopalan, B., Small, J.A., Schultz, I.R., 2008. Gene expression profiles in rainbow trout, Oncorhynchus mykiss, exposed to a simple chemical mixture. Toxicol. Sci. 102 (1), 42 60. Howe, A.C., Jansson, J.K., Malfatti, S.A., Tringe, S.G., Tiedje, J.M., Brown, C.T., 2014. Tackling soil diversity with the assembly of large, complex metagenomes. Proc. Natl Acad. Sci. 111 (13), 4904 4909. Hu, W., Tedesco, S., McDonagh, B., Ba´rcena, J.A., Keane, C., Sheehan, D., 2010. Selection of thiol-and disulfide-containing proteins of Escherichia coli on activated thiol-sepharose. Anal. Biochem. 398 (2), 245 253. Iwahashi, H., Ishidou, E., Kitagawa, E., Momose, Y., 2007. Combined cadmium and thiuram show synergistic toxicity and induce mitochondrial petite mutants. Environ. Sci. Technol. 41 (22), 7941 7946. Iwahashi, Y., Hosoda, H., Park, J.-H., Lee, J.-H., Suzuki, Y., Kitagawa, E., et al., 2006. Mechanisms of patulin toxicity under conditions that inhibit yeast growth. J. Agric. Food Chem. 54 (5), 1936 1942. Jacquiod, S., Franqueville, L., Ce´cillon, S., Vogel, T.M., Simonet, P., 2013. Soil bacterial community shifts after chitin enrichment: an integrative metagenomic approach. PLoS One 8 (11), e79699. Kan, J., Hanson, T.E., Ginter, J.M., Wang, K., Chen, F., 2005. Metaproteomic analysis of Chesapeake Bay microbial communities. Saline Syst. 1, 7 15. Kishi, K., Kitagawa, E., Onikura, N., Nakamura, A., Iwahashi, H., 2006. Expression analysis of sex-specific and 17β-estradiol-responsive genes in the Japanese medaka, Oryzias latipes, using oligonucleotide microarrays. Genomics 88 (2), 241 251. Lacerda, C.M.R., Choe, L.H., Reardon, K.F., 2007. Metaproteomic analysis of a bacterial community response to cadmium exposure. J. Proteome Res. 6 (3), 1145 1152. Lau, A.T.Y., Chiu, J.F., 2006. Proteomic and biochemical analyses of in vitro carcinogen-induced lung cell transformation: synergism between arsenic and benzo [a] pyrene. Proteomics 6 (5), 1619 1630. Lombard, N., Prestat, E., van Elsas, J.D., Simonet, P., 2011. Soil-specific limitations for access and analysis of soil microbial communities by metagenomics. FEMS Microbiol. Ecol. 78 (1), 31 49. Markert, S., Arndt, C., Felbeck, H., Becher, D., Sievert, S.M., Hu¨gler, M., et al., 2007. Physiological proteomics of the uncultured endosymbiont of Riftia pachyptila. Science 315 (5809), 247 250. Martı´n-Dı´az, M.L., DelValls, T., Riba, I., Blasco, J., 2008. Integrative sediment quality assessment using a biomarker approach: review of 3 years of field research. Cell Biol. Toxicol. 24 (6), 513 526. Matheis, K., Laurie, D., Andriamandroso, C., Arber, N., Badimon, L., Benain, X., et al., 2011. A generic operational strategy to qualify translational safety biomarkers. Drug Discov. Today 16 (13), 600 608. Mei, N., Guo, L., Liu, R., Fuscoe, J.C., Chen, T., 2007. Gene expression changes induced by the tumorigenic pyrrolizidine alkaloid riddelliine in liver of Big Blue rats. BMC Bioinform. 8 (Suppl. 7), S4. Merhi, M., Demur, C., Racaud-Sultan, C., Bertrand, J., Canlet, C., Estrada, F.B.Y., et al., 2010. Gender-linked haematopoietic and metabolic disturbances induced by a pesticide mixture administered at low dose to mice. Toxicology 267 (1), 80 90. Mezhoud, K., Praseuth, D., Francois, J.-C., Bernard, C., Edery, M., 2008. Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin. Hormonal Carcinogenesis V. Springer, New York, pp. 419 426. Morris, R.M., Nunn, B.L., Frazar, C., Goodlett, D.R., Ting, Y.S., Rocap, G., 2010. Comparative metaproteomics reveals ocean-scale shifts in microbial nutrient utilization and energy transduction. ISME J. 4 (5), 673 685. Ofi¸teru, I.D., Lunn, M., Curtis, T.P., Wells, G.F., Criddle, C.S., Francis, C.A., et al., 2010. Combined niche and neutral effects in a microbial wastewater treatment community. Proc. Natl. Acad. Sci. 107 (35), 15345 15350. Ortiz, P.A., Bruno, M.E., Moore, T., Nesnow, S., Winnik, W., Ge, Y., 2010. Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles. J. Proteome Res. 9 (3), 1268 1278.

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Park, C., Novak, J.T., Helm, R.F., Ahn, Y.-O., Esen, A., 2008. Evaluation of the extracellular proteins in full-scale activated sludges. Water Res. 42 (14), 3879 3889. Pelletier, G., Masson, S., Wade, M.J., Nakai, J., Alwis, R., Mohottalage, S., et al., 2009. Contribution of methylmercury, polychlorinated biphenyls and organochlorine pesticides to the toxicity of a contaminant mixture based on Canadian Arctic population blood profiles. Toxicol. Lett. 184 (3), 176 185. Pierre-Alain, M., Christophe, M., Se´verine, S., Houria, A., Philippe, L., Lionel, R., 2007. Protein extraction and fingerprinting optimization of bacterial communities in natural environment. Microb. Ecol. 53 (3), 426 434. Ralston-Hooper, K.J., Sanchez, B.C., Adamec, J., Sepu´lveda, M.S., 2011. Proteomics in aquatic amphipods: can it be used to determine mechanisms of toxicity and interspecies responses after exposure to atrazine? Environ. Toxicol. Chem. 30 (5), 1197 1203. Ram, R.J., VerBerkmoes, N.C., Thelen, M.P., Tyson, G.W., Baker, B.J., Blake, R.C., et al., 2005. Community proteomics of a natural microbial biofilm. Science 308 (5730), 1915 1920. Sheehan, D., 2006. Detection of redox-based modification in two-dimensional electrophoresis proteomic separations. Biochem. Biophys. Res. Commun. 349 (2), 455 462. Sheehan, D., McDonagh, B., 2008. Oxidative stress and bivalves: a proteomic approach. Invertebrate Surviv. J. 5, 110 123. Ta¸s, N., Prestat, E., McFarland, J.W., Wickland, K.P., Knight, R., Berhe, A.A., et al., 2014. Impact of fire on active layer and permafrost microbial communities and metagenomes in an upland Alaskan boreal forest. ISME J. 8, 1904 1919. Tedesco, S., Sheehan, D., 2012. Protein thiols as novel biomarkers in ecotoxicology: a case study of oxidative stress in Mytilus edulis sampled near a former industrial site in Cork Harbour, Ireland. J. Integrat. Omics 2 (2), 39 47. Tringe, S.G., Von Mering, C., Kobayashi, A., Salamov, A.A., Chen, K., Chang, H.W., et al., 2005. Comparative metagenomics of microbial communities. Science 308 (5721), 554 557. Tsuchiya, T., Tanaka-Kagawa, T., Jinno, H., Tokunaga, H., Sakimoto, K., Ando, M., et al., 2005. Inorganic arsenic compounds and methylated metabolites induce morphological transformation in two-stage BALB/c 3T3 cell assay and inhibit metabolic cooperation in V79 cell assay. Toxicol. Sci. 84 (2), 344 351. Tveit, A., Schwacke, R., Svenning, M.M., Urich, T., 2013. Organic carbon transformations in high-Arctic peat soils: key functions and microorganisms. ISME J. 7 (2), 299 311. Vineis, P., Khan, A.E., Vlaanderen, J., Vermeulen, R., 2009. The impact of new research technologies on our understanding of environmental causes of disease: the concept of clinical vulnerability. Environ. Health 8 (1), 54 64. Wang, J., Wang, Y.-Y., Lin, L., Gao, Y., Hong, H.-S., Wang, D.-Z., 2012. Quantitative proteomic analysis of okadaic acid treated mouse small intestines reveals differentially expressed proteins involved in diarrhetic shellfish poisoning. J. Proteomics 75 (7), 2038 2052. Wang, M., Wang, Y., Wang, J., Lin, L., Hong, H., Wang, D., 2011. Proteome profiles in medaka (Oryzias melastigma) liver and brain experimentally exposed to acute inorganic mercury. Aquat. Toxicol. 103 (3), 129 139. Williams, T.D., Turan, N., Diab, A.M., Wu, H., Mackenzie, C., Bartie, K.L., et al., 2011. Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach. PLoS Comput. Biol. 7 (8), e1002126. Wilmes, P., Bond, P., 2006. Towards exposure of elusive metabolic mixed-culture processes: the application of metaproteomic analyses to activated sludge. Water Sci. Technol. 54 (1), 217 226. Wilmes, P., Andersson, A.F., Lefsrud, M.G., Wexler, M., Shah, M., Zhang, B., et al., 2008. Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal. ISME J. 2 (8), 853 864. Winterbourn, C.C., Hampton, M.B., 2008. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 45 (5), 549 561. Wu, H., Wang, W.-X., 2010. NMR-based metabolomic studies on the toxicological effects of cadmium and copper on green mussels Perna viridis. Aquat. Toxicol. 100 (4), 339 345. Yasokawa, D., Iwahashi, H., 2010. Toxicogenomics using yeast DNA microarrays. J. Biosci. Bioeng. 110 (5), 511 522. Yi, C., Pan, T., 2011. Cellular dynamics of RNA modification. Acc. Chem. Res. 44 (12), 1380 1388.

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Further Reading Duque, A.S., Farinha, A.P., da Silva, A.B., de Almeida, A.M., Santos, D., da Silva, J.M., et al., 2013. Abiotic stress responses in plants: unraveling the complexity of genes and networks to survive. In: Vahdati, K., Leslie, C. (Eds.), Abiotic Stress—Plant Responses and Application in Agriculture. InTech Open Access Publisher, Rijeka, pp. 49 101. Kumavath, R.N., Deverapalli, P., 2013. Scientific swift in bioremediation: an overview. In: Patil, Y.B., Rao, P. (Eds.), Applied Bioremedition—Active and Passive Approaches. Intech OpenScience, InTech Open Access Publisher, Rijeka, pp. 377 396. Powell, M.J., Sutton, J.N., Del Castillo, C.E., Timperman, A.T., 2005. Marine proteomics: generation of sequence tags for dissolved proteins in seawater using tandem mass spectrometry. Mar. Chem. 95 (3), 183 198.

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C H A P T E R

20 Biochar for Carbon Sequestration: Bioengineering for Sustainable Environment S.P. Sharma Punjab Agricultural University, Ludhiana, India

20.1 INTRODUCTION 20.1.1 What Is Environmental Sustainability? Environment is a complex of myriad of resources and factors (physical, chemical, and biotic) that act upon and affect an organism or an ecological community thereby determining its form and survival. It is adorned with a unique ability to replenish and reorder itself in its natural form (Atkins et al., 1998). However, need arises for the maintenance of the intricate balance among the resources, factors, and the organisms when this self-sustenance property is perturbed by several anthropological activities, particularly agricultural and industrial interventions, which disrupt the equilibrium leading to dire ecological consequences. Maintenance of the natural resources both as “source” of inputs as well as “sink” for waste disposal is the prima-facie of environmental sustainability (ES) promotion strategies (Goodland, 1995). Hence, ES protocols can be summarized as amalgamation of conservation strategies and other management techniques, which aim for the maintenance if not enhancement of ecosystem functions, its valuable components, or qualities of earth’s physical environment required for sustaining human life on earth.

20.1.2 Why There Are Increasing Concerns? The start of the 20th century witnessed logarithmic rise in agriculture, urbanization, and industrial activities. These activities are resource demanding and have caused rapid

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depletion of natural resources beyond the self-repair and replenishment capability of the natural system. This has also climaxed down to aberrant climate changes as a consequence of global warming. There are recurrent episodes of sudden droughts (hot and cold both), temperature extremes, acid rains, flooding, and many more in the past few decades. Moreover, ES concept has always been debated due to the clash with the rapid exploitation of the natural resources to harness higher economic benefits in short-time period. The world population is rising with the numbers doubling in less than a decade. The projected figures for the world’s population in the year 2020 are expected to be 7.584 billion, which is an alarming scenario as natural resources will be utilized at much faster rates for food production. Hence, there is a vicious circle involving natural resource exploitation and issues regarding overriding of the system sink disposal capacities, which are resulting in environment disruption and therefore jeopardizing the very existence of the human kind.

20.1.3 How to Address ES-Related Issues? A paradigm shift is required in environmental management approaches to address the ES-related problems and to help place at the forefront. However, keeping the account of the population big bang globally and the economic benefits of agricultural practices, which also have to be taken care of, ES remains at the backdrop. Therefore, the current scenario demands application of environment friendly, resource-conserving cultivation techniques apart from employing green power generation methods, and fabrication of less power hungry machineries. The use of multiplexed strategies such as interconnected waste management (agriculture, industry, and urban), resource-conservation, nutrient management, and power generation technologies is need of the hour. Biochar technology embraces all the facets of environmental sustainability criteria (Fig. 20.1). It is substantiated by active merger of green and white biotechnology to develop a carbon-negative bio-economy (Vanholme et al., 2013). It involves the use of plant biomass and agriculture-to-urban waste material for green power generation. The charred residue of the power generation or biochar production unit has a variety of application benefits such as vehicles for balanced and sustained nutrient release and management in arable/forest soils, for retention of water, remediation of inorganics, organic compounds, and toxic materials, and finally for enhancing growth/yield of vegetable and other crops.

20.2 WHAT IS BIOCHAR? The maintenance of the soil organic carbon (SOC) pool is critical for sustainability in crop production (Johnston et al., 2009). However, the SOC is affected by a variety of soil abiotic and edaphic factors. Subsequently, even minute alterations in the SOC contents may lead to disproportionate larger variations in the soil physical properties (Spokas et al., 2012). “Biochar” is also a carbon-based product. Precisely, it is a type of recalcitrant amorphous graphitic carbon domains produced by thermochemical oxygen-limited combustion of lignocellulosic crop residue/wood/other solid biomass at relatively lower temperature, that is, below or around 700 C (Lehmann and Joseph, 2015). The pyrolysis of plant biomass in oxygendepleted conditions results in formation of a carbon skeleton having quite low hydrogen and

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FIGURE 20.1 The biochar, its application benefits, and ES.

oxygen functional groups or low oxygen-to-carbon ratio. The major carbon components in plant biomass include cellulose, hemicellulose, and cyclic phenolic compounds primarily the “lignin,” which are subjected to decomposition and rearrangement of bonds between carbon atoms with high order of aromaticity due to polycondensation (Enders et al., 2012). This imparts resistance to microbial degradation and therefore, it functions as a stable carbon sink on soil application (Spokas et al., 2012). There are a variety of carbon black derivatives or char that are being used for a variety of purposes. However, biochar is specifically a bio-carbon soil amendment/amelioration to enhance soil fertility for increasing crop growth and productivity (Sohi et al., 2010). The idea for soil biochar application sprouted from the higher soil fertility and crop productivity quotient of the famous tropical Amazonian lowland humid “Terra Preta” terrain soils (Filiberto and Gaunt, 2013; Mann, 2005).

20.2.1 What Are Its Types? The biochar is a process-specific and rather a process-driven product, thereby it can be categorized on basis of the type of feedstock, production/operation

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characteristics, and/or the extent of the pyrolysis. Evidently, biochar can be broadly classified on basis of biomass used (can be plant biomass, i.e., agricultural residue, wood, or energy crops, and animal biomass, i.e., compost, sewage sludge, municipal, or urban waste) (Spokas et al., 2012) and the feedstock utilized for generating biochar. The properties of the produced biochar vary according to the variations in the amount of the alkaline and alkali earth metal content, readily decomposing cellulose/ hemicelluloses and lignin content of the feedstock. The types of biochar on basis of production conditions are given in Section 20.2.2. However, these can be subcategorized as low- and high-temperature pyrolysis-derived biochar with the former being easily assimilable by microbial attack and has high volatile content due to thermal decomposition of cellulose and hemicellulose. The latter becomes highly hydrophobic owing to high amount of aromatic C compound, thereby exhibits recalcitrance and resistant to microbial decomposition or carbon-sink/carbon-sequestration potential and shows enhanced adsorption capabilities due to high surface area and alkaline pH (Jindo et al., 2014). Therefore, the physical and chemical properties, which determine application efficacy and associated benefits of particular biochar, vary with the type of biochar.

20.2.2 How Biochar Can be Produced? Biochar is a product of pyrolysis involving thermochemical decomposition of organic matter usually agricultural waste (Spokas et al., 2012). This thermochemical decomposition involves release or absorption of heat upon reaction. Since organic biomass contains high amount of carbon, the biochar generation occurs at low temperature without use of catalyst. The biochar production conditions are critical because these influence the physical and chemical properties of the biochar and therefore vary according to the thermochemical conditions and the feedstock used (Enders et al., 2012). Among the production condition, the temperature and heating/residence time are very critical (Jindo et al., 2014). For the synthesis of biochar, about a dozen advanced techniques (torrefaction, slow, fast, and flash pyrolysis, gasification, hydrothermal carbonization, and microwave-assisted pyrolysis) are available, which can be customized with the type of feedstock used (Spokas et al., 2012). These techniques can be broadly classified as slow and fast pyrolysis with the former being a continuous process involving slow heat transfer followed by removal of gaseous volatiles. The latter generates char particles by fast heating (Sohi et al., 2010). A more comprehensive review on synthesis of biochar by Spokas et al. (2012) outlines and compares various techniques for biochar production. The morphological characteristics of the biochar generated from plant biomass also vary according to the production conditions (Wang et al. 2011) as it involves stepwise loss of weight of the treated biomass (Fig. 20.2). The initial weight loss occurs due to loss of water below 100 C followed by degradation of cellulose, hemicelluloses, and lignin above 220 C, and final weight loss due to burning of the carbonaceous residues formed due to degradation products formed $ 220 C (Markovska and Lyubchev 2007).

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FIGURE 20.2 Aggregate morphology of the rice waste-based biochar as depicted by Scanning Electron Microscopy.

20.2.3 Why Biochar Can be a Possible Solution for ES? Biochar provides multitude of benefits for ensuring ES. Owing to its varied dynamics, a new term, “biocharculture,” has been coined to encompass the applicational and environmental benefits of biochar (Reddy, 2014). It is a very useful soil conditioner. It alters a variety of soil physical properties (improvement of the moisture retention and enhanced air permeability), changes chemical properties (better cation exchange capacity, increased buffering capacity of SOC), and influences the soil microbial activity (Jindo et al., 2014; Spokas et al., 2012). Biochar application also helps in emission mitigation or reversal by active management of agricultural land. Therefore, the “carbon foot-printing” of a particular land use has to be discerned before aiming for decrease in emission mitigation by increasing “carbon sequestration.” As “carbon sequestration” aims for mitigation of global warming by locking the greenhouse gases (GHGs) (CO2, NO2) and other forms of carbon (largely organic) for long term in soil, it can be accomplished because biochar predominantly contains cyclic carbon with high aromaticity and depleted H and O, which imparts

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resistance to microbial attack on application in soil. This enhanced recalcitrance feature helps in curbing the emission of CO2 by microbial decomposition and carbon mineralization of organic biomass. Therefore, biochar production and application fundamentally harness the natural process of photosynthesis for plant biomass generation apart from removing atmospheric CO2 by autotrophic organisms, that is, plants, reduction of CO2 to carbohydrate, and other products. This plant biomass on thermochemical pyrolysis in O2-depleted environment generates recalcitrant biochar as carbon sink or longwave geoengineering option (Downie et al., 2012). There are a myriad of application benefits of this locked carbon repository to soil, which will be discussed in the following sections. As far as the cleaning or remediation dimensions of biochar are concerned, it is useful for filtration of soil percolating water (Lehmann and Joseph, 2009), in sequestration and therefore biosorption of the contaminating heavy metals for land reclamation (Zhang et al., 2013), removal as well as sorption and leaching of nutrients like nitrogen and phosphate from wastewater, municipal waste, and soil (Foereid, 2015; Yao et al., 2012), and adsorption of organic xenobiotics such as pesticides and polyaromatic compounds (Lehmann and Joseph, 2015; Ogbonnaya and Semple, 2013) from contaminated soils. The fundamental phenomena governing the remediation role of biochar are “adsorption at liquid solid interface” due to van der Waals (physiosorption) and electrostatic (chemisorption) forces (Foereid, 2015). The carbon black and its forms like charcoal are already known to possess high-adsorption kinetics for organic compounds. Therefore, enhancing the carbon aromaticity characteristics further increases the adsorption capabilities of biochar. Chemical tempering of the biochar either by functionalization or augmentation with certain inorganics alters the adsorption preferences or kinetics for cations/anions (Fang et al., 2014). The adsorption further reduces the mobility and bioaccessibility of the adsorbed moieties leading to sequestration of the contaminant and its environmental toxicity (Ogbonnaya and Semple, 2013).

20.3 BIOCHAR-BASED BIOENGINEERING TECHNOLOGIES 20.3.1 Biochar and Various Use Efficiency Strategies Many nutrients are required in different amounts for the proper growth and development of a crop plant. These nutrients are either to be replenished by natural system or to be furnished through the application of inorganic/organic fertilizers in actively agarable soils. The native soil nutrients as well as applied fertilizers form complexes with organic carbonaceous matter (SOC including humic and fulvic acids) and with the inorganic aluminosilicate clay minerals. While the unbound nutrients are lost through leaching, photovolatilization and surface run off, the locking and subsequent release of both types of nutrients will be an ecological benefit for maintenance of ES. Vegetables being high yielding and short season are high-nutrient grazers and therefore forge large amounts of essential nutrients from the soil. The most practical approach for maintaining the soil nutrients in plant-available form for a consistent and longer period demands the use of nutrient delivery vehicle like biochar.

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20.3.1.1 Biochar as Nutrient Delivery Vehicle The porous characteristic of biochar imparts it with unique property such as good adsorption/sequestration and subsequent slow release of adsorbed nutrients. Biochar can also sorb nutrients such as N, P, and K from soil solution thus enabling their prolonged retention in the soil by avoiding leaching (Kim et al., 2014). This way biochar contributes to enhance the nutrient use efficiency of the nutrients applied as inorganic fertilizers. Invariably, biochar augmented or co-composted with the bio-waste like green manure, compost, or farm-yard manure shows higher N retention, which is later released in a controlled manner to obtain yield benefits over the individual application of the compost or biochar (Kammann et al., 2015). Although a thoughtful experimentation is required before the final use of a particular type of biochar yet, biochar is an excellent vehicle or carrier for controlled and long-term nutrient delivery to the growing crops. The porosity, lignin content, and the pyrolysis temperature are the three major factors that can be tempered to design specific biochar engineered for delivering nutrients in a particular soil type and pH conditions (Joseph et al., 2013). For example, by controlling the lignin content and pyrolysis temperature in switch grass biochar pellets, the controlled release of adsorbed P and K nutrients can be ensured (Kim et al., 2014). 20.3.1.2 Biochar Amendments Affecting Soil Nutrient Status and Enhancing Nutrient Use Efficiency As discussed in Section 20.2 and its subsections, the physical and chemical characteristics of biochar depend upon the feedstock and pyrolysis conditions. The generated biochar can be utilized to adsorb nutrients for their subsequent release in a pH- and temperature-dependent manner. This implies that adsorption of various nutrient ions on the biochar surface (nutri-sorption) and their release occurs due to alteration in cation exchange capacity and pH of the biochar-amended soils (Filiberto and Gaunt, 2013). Yao et al. (2012) reported that biochar amendment substantially decreased the adsorption of N and P as nitrate/ammonium and phosphate ions decreasing their occurrence in soil leachates by substantial percentage. The soil characteristics such as texture, clay-to-sand contents, SOC, and pH can alter the biochar nutri-sorption potentials (Yao et al., 2012). Moreover, the nutrient mobility also changes the biochar adsorption and subsequent release characteristics. It can be observed that biochar application enhances the nutrient use efficiency of the applied fertilizers also. The nitrogen dynamics, primarily involving a decrease in nitrate transformation for subsequent decreased N loss, occurs in response to biochar addition, which can be considered important for better nitrogen use efficiency (Jindo et al., 2014). Thus, biochar-nutrient complexes may lock nutrients from not only leaching, run-off, microbial mineralization, and physical volatilization phenomena but also help in temperature and pH-dependent slow release of adsorbed nutrients. Therefore, crop plants can efficiently use nutrients as these will exist in plant-available forms near the root zone for ready uptake. A long-term improvement of the physical properties of soil on biochar application including better aggregate formation, changes in soil microbial diversity, and activities impart an indirect effect on retention of highly and moderately mobile nutrients like N and P.

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20.3.1.3 Biochar for Enhancing Water Use Efficiency Water scarcity is one of the daunting issues related to climate change and global warming (Batool et al., 2015). The agricultural sector utilizes about 75% of the freshwater resources for irrigation, whereas only about 1/3rd to 1/7th fraction of this water is used by the growing crop (Yu et al., 2013). Biochar is a promising soil conditioner that can be utilized for enhancing the water use efficiency by water retention (WR) in coarse and light soils to guarantee crop yield stability under drought or water shortage conditions (de Melo Carvalho et al., 2014). The soil WR characteristics include water potential and water content, which may or may not be altered by biochar amendment (Brantley et al., 2015). The biochar addition alters the soil physical properties particularly significant enhancement in formation of macroaggregates and increase in saturated hydraulic conductivities of the soils (Castellinia et al., 2015; Ouyang et al., 2013). Largely, the biochar Soil Water Retention (SWR) capacity depends on the type of soil, sandy soils exhibit better response over clay soils, and type of biochar and the biochar application rate. The water-holding capacity can even double by mass using biochar (Yu et al., 2013). A soil study involving application of Eucalyptus wood biochar in the aerobic rice system resulted in increase in plant-available water in the upper soil layer for 2 3 years of biochar application (de Melo Carvalho et al., 2014). The biochar application in vegetable crops under water shortage conditions is very useful as it will not only enhance productivity but it will also reduce crop water requirements. Moreover, it also curbs leaching of WUE-affecting mobile nutrients, particularly K from the top soil layer helping in retaining water for shallow root system vegetable crops (Liu et al., 2017). The biochar soil amendment not only helps plant to tolerate water deficient conditions, it also enhances the overall WUE and growth (Kammann et al., 2011). Use of biochar at 0% and 5% by weight in tomato grown in pots under water deficient conditions enhanced the soil moisture contents resulting in improved physiological, yield, and quality traits (Akhtar et al., 2014). In a similar 6-week pot study, Batool et al. (2015) observed improvement in WUE of Abelmoschus esculentus L. plants stressed due to low water application (at 60% field capacity) over untreated control plants by application of biochar.

20.3.2 Biochar and Climate Change Abatement: Curbing Greenhouse Gas Emissions An increase in 4.0 C in temperature is estimated by the end of 21st century. These climate changes are as a result of increase in the atmospheric concentration of GHGs (IPCC, 2007). These GHG emissions have to be curbed apart from the atmospheric removal of GHGs. Biochar is expected to possess both the desirable requirements for climate change abatement. Biochar is produced from the plant or animal biomass, which helps in atmospheric removal of CO2, and due to the recalcitrant nature it locks the carbon becoming a part of the huge carbon-negative economy (Vanholme et al., 2013). Soil application of biochar curbs the emission of not only CO2 but also other about hundred manifold potent GHGs particularly nitrous oxide (N2O) and methane (CH4) (Downie et al., 2012). Moreover, the enhanced agronomic benefit in terms of higher crop growth and yield fosters the locking of the atmospheric carbon as plant biomass and its slow pyrolysis to

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biochar and bioenergy generation. Many studies advocate significant reduction in emission of CO2, N2O, and CH4 GHGs due to biochar amendment in land use under vegetable cultivation (Sun et al., 2014; Jia et al., 2012).

20.3.3 Biochar-Based Bioengineering of Ecological Niches Several ecological niches are suffering from accumulation of heavy metals/xenobiotics or of synthetic chemicals in mammoth amounts, particularly the industrial and mine sites, and the landfills (Lu et al., 2014). Such surplus chemical compound additions to the environment overruns the carrying/balancing capacities of the environment. Similarly, the energy production plants with special mention to the ones using radionuclides and fossil fuels (as coal and natural gas) are potential sources of radionuclide and coal ash wastes and are required to be remediated. Moreover, the indiscreet or overapplication of inorganic fertilizers and pesticides in the agricultural lands is posing serious plant, animal, and human health hazards. All these dissimilar and varied categories of wastes can be handled prudently by using wood/agricultural/urban waste based biochar (Table 20.1). Biochar can be used for the remediation of a variety of organic and inorganic contaminants from soil and water because of the carbon chemistry derived from pyrolysis. It can be used for adsorption and subsequent immobilization of the contaminants and may contribute to some extent for the altered microbial degradation due to variation in the microflora profile on biochar amendment (Yang et al., 2006). 20.3.3.1 Heavy Metal Removal The rapid industrialization has resulted in ecological problems pertaining to substandard/careless industrial effluent disposal causing rampant contamination of surface and subsurface soil as well as the groundwater aquifers due to mobilization and leaching of heavy metals such as zinc, cadmium, copper, lead, chromium, nickel, arsenic, and many more. The concerns aggravate as heavy metals get bioaccumulated in food web with concentrations that increase across the trophic levels (biomagnification), and their bioavailability severely affects the growth and development of plants and plant grazers and poses serious health risks to humans (Paz-Ferreiro et al., 2014; Uchimiya et al., 2010). Higher heavy metal concentrations are detrimental to diversity in number and functionality as well as health and properties of soil microflora (Paz-Ferreiro et al., 2014). Moreover, the techniques for the effective concurrent removal of these contaminants are still in their infancy (Jiang et al., 2012). Hence, soil-amendment products, particularly biochar, could have a vital role for reducing the environmental risk regarding soil heavy metal contamination. It will serve as a soil amendment having specific chemical properties, which promote adsorption or precipitation of several soil contaminants including heavy metals (Bolan et al., 2014). Heavy metal contaminants are nonbiodegradable, marginally volatizable (such as arsenic and mercury) and highly toxic in even few hundred ppb or ppm concentrations; their remediation strategies need to be devised for in situ stabilization or immobilization via adsorption/biosorption on soil organic matter (humic and fulvic acids), and microbial extracellular compounds/plant root exudates (organic acids and other carbon sources to

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TABLE 20.1 Wastewaters Biochar source/ feedstock

Effect of Biochar Amendment for Remediation of Heavy Metals and Organic Pollutants and for Adsorption of Nutrients From Municipal/Industry

Pyrolysis temperature

Biochar particle size

Contaminating chemical/moiety/ compound

Type of study

Biochar application rate

Mechanism of remediation

Outcome/results

Reference

Lu et al. (2014)

AGRICULTURAL WASTE Rice straw

$ 500 C

,0.25 and ,1 mm

Sandy loam paddy soil naturally cocontaminated with Cd, Cu, Pb, and Zn

Pot experiment

Applied at three rates (0%, 1%, and 5%, w/w)

Adsorption of metal ions on biochar

Significant decrease in solubility and occurrence of Cd, Cu, Pb, and Zn in soil and in aboveground biomass of Sedum plumbizincicola, increase in aboveground biomass of Sedum plumbizincicola

Rice straw

At 400 C

,150, 150 250, and 250 425 μm

Cd and sulfamethoxazole (SMX)

Laboratory batch adsorption isotherm experiment

Applied at 0.1%

Precipitation and the formation of surface complexes between Cd or SMX and carboxyl or hydroxyl groups

Han et al. Maximum (2013) adsorption of Cd at pH 5.0 and of SMX at 3.0, Cd helped in adsorption of SMX in binary system

Wheat straw

Obtained by burning wheat straw in open conditions

Ash and biochar components fractionated on basis of relative density in organic solvents

Diuron, a substituted urea herbicide (concentration range of 0 6 mg/L)

Stuttgart silt loam soil in laboratory experiment to obtain adsorption isotherm

Applied at 0.01%, 0.02%, 0.05%, 0.1%, 0.5%, and 1% ash

Sorption of diuron by biochar

Sorption increased by a factor of 4 on a unit mass basis

Yang and Sheng (2003)

Wheat straw

Step-wise temperature increase starting from 200 C, followed by consecutive elevation to 250, 300, and 500 C maintained for 1.5 h at each point

Sieved through 0.15-mm mesh

Hexachlorobenzene (HCB), a persistent organic pollutant at 50 2000 g/L

Laboratory tube adsorption isotherm study using ferri-udic argosols collected from a vegetable field

5 and 50 mg per tube to sorb 60% 80% of applied HCB in terms of mass ratio

Immobilization and rapid reduction in HCB bioavailability

42 times higher HCB sorption by biochar than that by soil, reduced HCB dissipation, volatilization and earthworm HCB uptake from soil

Song et al. (2012)

Wheat straw

At 350 550 C

Ground to pass through 2 mm sieve

Cd contaminated soils

Field experiment in ferricaccumulic stagnic anthrosol

Applied at 10 40 t/ha to soil by plowing and raking at a depth of 0 15 cm

Due to increase in soil pH and soil organic content

Reduction in both soil extractable and wheat straw uptake Cd contents

Cui et al. (2012)

Corn straw

At 100 600 C

Milled to pass through 0.15 mm sieve, demineralization with HCl, centrifugation, neutralization, and dried at 70 80 C

Simazine, a triazine herbicide (concentrations varying from 0.006 to 3.3 mg/L)

Laboratory batch sorption experiment conducted in glass vials

Applied at 1%

Adsorption and portioning of simazine on soft and glassy carbon dominated and no-carbon components of the biochar

Simazine sorption regulated by BC fractions obtained at different pyrolytic temperatures

Zhang et al. (2011)

Plant biomass of four different phytoremediation plants

Pyrolysis at 500 C under N2 atmosphere for 2 h followed by activation at three different temperatures (500, 600, and 700 C) in CO2 atmosphere for 2 h

30 mg/L of N and P solutions

Batch adsorption experiment

Applied at 0.2 g per 50 mL of the ammonium or phosphate solutions

Chemical interactions particularly CEC and physical sorption of nutrient on biochar

Thalia dealbata biochar (600 C pyrolysis temperature) most promising sorbent for removing contaminants (N and P) from aqueous solution

Zeng et al. (2013)

Polycyclic aromatic hydrocarbons

Laboratory study of creosotepolluted soil (40 g dry mass)

Applied at 2.5% sequentially along with wheat straw immobilized Pleurotus ostreatus

Effective PAH biodegradation and immobilization due to mycological and biochar amendments, respectively

Better PAH biodegradation rate and the second lowest bioavailable fraction and soil eco-toxicity compared to mycoremediation strategy alone

Garcı´aDelgado et al. (2015)

Mesophilic biogas plant digesting piggery manure pretreated slurry samples containing following nutrients in mg/L, NH4-N (1390 1450), NO3-N (47 54), NO2-N (34 56), PO4-P (15 20), and TOC (226.1)

Batch adsorption experiment

Applied at 0.1 5 g

Adsorption/removal of 60% and 53% NH4-N by wood and rice husk biochar, respectively

Sorption capacity of 2.86 and 0.23 mg ammonium and phosphate, respectively, per gram of biochar and 10% 50% utilization of available excess biomass

Kizito et al. (2015)

WOOD-BASED BIOCHAR Pine woodchip biochar

At 450 C

Mixed wood cuttings and rice husks

Slow pyrolysis at 600 C for a retention time of 10 h

Dry grinding to reach particle size of 0.25 mm

(Continued)

TABLE 20.1 Biochar source/ feedstock

(Continued)

Pyrolysis temperature

Biochar particle size

Contaminating chemical/moiety/ compound

Type of study

Biochar application rate

Mechanism of remediation

Outcome/results

Reference

ANIMAL AND URBAN WASTE Sewage sludge (SS)

At 400 550 C in electrical furnace using a horizontal quartz reactor

BC sieved through 10 mm sieve before application

Soil (4.5, 24.2, 42.0, 32.3, 20.5, 97.9 mg/kg) and SS (18.0, 23.0, 42.0, 20.0, 165.0, 703 mg/kg) contained higher As, Ni, Pb, Cr, Cu, and Zn contents

Pot experiment using orchard silt (sandy) loam soil sampled from depth of 0 20 cm

Applied at different BCto-soil mass ratios (1:2, 1:4, 1:6)

Physical surface area and microporosity structure of the biochar

Heavy metal leaching increased with increased pyrolysis temperature, however, higher uptake and accumulation of Zn, Cu, and Pb only in garlic

Song et al. (2014a,b)

Sewage sludge

At 550 C for 8 h

,2 mm

Soil contaminated with Zn and Cd (at 250 and 0.3 mg/kg)

Greenhouse pot experiment using 0 20 cm top soil from a farmland near an iron refinery plant

Applied at 2%, 5%, and 10% on a dry weight basis

Potentially toxic elements (PTE) decrease due to alteration in soil pH, cation exchange, and DOC values, presence of oxygen functional groups, which form complexes with PTEs, immobilization in soil, and then reduced bioaccumulation in plants

Significant decrease in available concentrations of As (52% 67%), Pb (21% 50%), Cu (7% 48%), and Zn (2% 8%), while Cd concentration increased slightly (2% 14%) as compared to the control

Waqas et al. (2014)

Sewage sludge

At 550 C for 8 h

,2 mm

Soil contained Σ16PAH (9.9 mg/kg21) and Σ7PAH (5.5 mg/kg)

Greenhouse pot experiment using 0 20 cm top soil from a farmland near an iron refinery plant

Applied at of 2%, 5%, and 10% on a dry weight basis

Dilution, compound repartitioning, and adsorption of contaminating PAHs

Significant decrease in PAH availability and 44% 57% reduced accumulation in Cucumis sativa L. Reduction increased with increase in application rate

Waqas et al. (2014)

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form insoluble metal organic acid salts like metal oxalates) (Tangahu et al., 2011), chemical complexation with metal chelators (phyto- and microbial siderophores, melanin-like polymers) (Monachese et al., 2012) or ionic exchange with soil clay minerals like alumino-, tecto-, and phyllosilicates compounds (Li et al., 2015). This physical and chemical containment of the inorganic heavy metal pollutant restricts its lateral and vertical mobilization and leaching, thereby curbing extended contamination of the subsurface soil and groundwater aquifers. Biochar-based remediation of heavy metals hails for depleting pernicious ecological concentration of contaminant as a consequence of altered pH, ionic strength, functional group of the adsorbing organic compound, cation exchange capacity, and relative abundance of type of clay mineral in resident soil (Paz-Ferreiro et al., 2014; Zhang et al., 2013). However the physical, chemical, and heavy metal remediation properties of biochar are determined and hence vary according to the type of feedstock, pyrolysis temperature, degree of crystallization of the inorganic metal components of the biochar or the biochar ash content (Zhang et al., 2013). Mechanistically, biochar amendment works by exchange of divalent cations (Ca21, Mg21, and others) present on the surface, functional group complexation, and surface adsorption and precipitation to insoluble heavy metal phosphates and carbonates (Lu et al., 2012). The absorption potential of biochar can be modified to create a composite. Song et al. (2014a,b) have developed a modified corn straw biochar/MnOx by KMnO4 oxidation at high temperature (600 C) to enhance removal of copper ions from wastewater and therefore to develop low-cost adsorbent for Cu21 ions. Similarly, the heavy metal adsorption capability of the pristine biochar can be enhanced to twice and more by supplementation with nanomaterials or by development of novel synthesis protocols for generation of ionic-biochar nanocomposites. In a batch sorption experiment, Gan et al. (2015) observed twofold enhanced chromium (VI) removal efficiency from wastewater by fabrication of Zn-biochar nanocomposite. Vegetables have voracious nutrient and water requirements and therefore, these are most prone to exhibit high heavy metal absorption, uptake, and bioaccumulation capabilities on growing in heavy metal contaminated soils. The choice of the biochar amendment for vegetables needs to be relatively scrutinized such that the biochar itself has lowest risk of heavy metal carrying properties. Moreover, the biochar application rates need to be managed as the enhanced aromaticity obtained due to high-temperature pyrolysis and the type of the feedstock affects the macronutrient availability on soil application of biochar particularly the nitrogen dynamics (both nitrates and ammonium ions) in the soil (Clough et al., 2013). Application of biochar at varying soil-amendment rates in different vegetable crops has been known to cause heavy metal immobilization, decreasing their bioavailability, leaching, and uptake by the test plants. Recent reports accentuate biochar application to help in stabilization/ immobilization of heavy metals in contaminated soils leading to significantly lower heavy metal contents in turnip, lettuce, spinach, fenugreek, and brassica over the control plants (Khan et al., 2015; Kim et al., 2015; Younis et al., 2015; Fiaz et al., 2014). Similarly, Hossain et al. (2010) reported better yield and heavy metal content in permissible limits in cherry tomatoes (Lycopersicon esculentum) on application of sewage sludge derived biochar.

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20.3.3.2 Organic Pollutant Removal Several organic compounds, including persistent (polychlorinated biphenyls, polyaromatic hydrocarbons) and emerging (phthalate acid esters, naturally released estrogenic steroid hormone and its metabolites, pharmaceutical and personal care products), are known to be serious soil and water pollutants (Zhang et al., 2013). As these could be carcinogenic and recalcitrant, there is an ardent need for either adsorption or remediation/catabolization using a variety of bioremediation strategies (Zhang et al., 2013). The soil and water pollution caused by such pollutants can be detrimental for both agricultural production/productivity and ecological health so that it culminates to posing serious threat to human survival. Therefore, the strategies involving the application of soil amendments appear to be instrumental in practical in situ remediation of a variety of contaminants. Biochar, one of the soil ameliorant and amendment, being a pyrolyzed carbon based material may help in remediation of different types of organic pollutants (Zhang et al., 2013; Sarmah et al., 2010) (Table 20.1). The adverse impacts of organic soil contaminants can be managed/decreased by application of geosorbents like biochar through a myriad of mechanisms such as adsorption of pollutant on surface of the biochar, immobilization followed by decreased bioaccessibility for plant uptake and soil accumulation. The organic contaminant sorption may be several 10-fold higher in biochar-amended soils over control soils. Moreover, biochar amendment also decreases dissipation, volatilization as well as final uptake of the contaminant by plant roots or soil meso- and macrofauna particularly earthworms (Song et al., 2012). A recent pot study also concluded higher retention of di-ethylhexyl phthalate in biochar-amended soil (He et al., 2016). This feature is of prime importance for actively arable soils facing inappropriate use of pesticide so that it will curb biomagnification and pesticide leaching phenomena. Interestingly, there exists variability in the effectivity of different feedstock-derived biochars for decreasing the bioavailability and accumulation of Polyaromatic hydrocarbons (PAHs) and heavy metals in contaminated soil (Khan et al., 2015). For example, woody biomass-derived biochar produced by slow pyrolysis exhibit low sorption of organic contaminants particularly of PAHs (Fabbri et al., 2013). Moreover, the fate of the organic contaminant also depends on the SOC content as higher SOC helps in higher retention of organic contaminant. Biochar amendment in higher SOC soils will not help in uptake of organic contaminant (He et al., 2016). The uptake and accumulation of organic contaminants also vary according to the type of vegetable crop like organochlorine uptake, which occur through passive and diffusive processes, varied in the root vegetable test crops (Florence et al., 2015). Similar to enhancement of heavy metal adsorption, organic pollutants remediation can be made more effective by amending or coating the pristine biochar with nano-based synthetic allotrope of carbon (SWCNT/MWCNT/graphene) (Inyang et al., 2015; Tong et al., 2013; Zhang et al., 2012) or by treatment with natural clay minerals as alumina and montmorillonite (Li et al., 2015). These amendments may result in enhancement of the thermal stability and absorption properties (Zhang et al., 2012) or pore expansion by doubling of the surface area and hence provision of increased adsorption sites for the pollutant capturing or immobilization (Li et al., 2015). Dip coating of cotton wood by graphene before pyrolysis into biochar caused 64-fold enhancement in the thermal decomposition

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temperature of graphene-coated biochar than pure biochar as well as increased aqueous methylene blue absorption ability (Zhang et al., 2012). 20.3.3.3 Sorption of Excess N or P From Wastewater Biochar can be used to adsorb nutrients from aqueous systems (Tan et al., 2015). This characteristic is most useful in adsorption of macronutrients such as nitrogen and phosphorus from wastewater from municipal or diary industry plants and anaerobic digestate slurry (Foereid, 2015; Kizito et al., 2015). The surface adsorption of the nitrogenous species, particularly ammonium ions and nitrate ions to a lesser extent on biochar, is largely governed by the particle size, internal structure, chemical composition, and surface characteristics of the adsorbing biochar (Zhou et al., 2015) apart from the contact time, temperature, pH, and NH1 4 -N concentration (Kizito et al., 2015). However, a comparative study using pine bark (feedstock), its biochar and zeolite suggested that the particle size has no effect on ammonium ion sorption properties with biochar having potential as cost effective commercial sorbent (Hina et al., 2015; Panayotova, 2015). Interestingly, the NH1 4 -N sorption occurs as physical entrapment in biochar pores, whereas its desorption and release is three to fourfold less than the NH2 3 -N, which indicates the formation of complex bonds with the biochar (Jassal et al., 2015). Similar to nitrogen nutrient, phosphorus is also required by the vegetable crops for growth and development. As phosphorus fertilizer prices are increasing and there are concerns for the unavailable, leached, or organic P, there is much research attention for recycling of lost P to ensure sustainable crop production. Biochar can be a promising alternative for P sorption and recovery from wastewaters (Fang et al., 2014). The adsorption potentials of biochar can be enhanced by magnesium (Mg) modification (Fang et al., 2014). The nutrients adsorbed on biochar can be subsequently used as fertilizers as biochar 1 tends to slowly release nutrient PO2 4 -P and NH4 -N to soil to improve soil properties and crop productivity (Hale et al., 2013).

20.3.4 Biochar Soil Microbial Community Interactions: Possible Implications Biochar amendment of soil alters many of the soil physical and chemical properties, which consequently alters the soil micro-, meso-, and macrofauna and -flora (Ameloot et al., 2013). The soil microorganisms play a vital role in decomposition of organic waste and mineralization processes, which are essential for maintenance of the biogeochemical nutrient cycles (Lehmann et al., 2011). As discussed, biochar comprises of carbon-negative recalcitrant organic biomass product. However, it comprises of a microbial decomposition labile amorphous carbon content also apart from more decomposition resistant cyclized aromatic carbon compounds (Ameloot et al., 2013). Therefore, biochar addition to soil may stimulate microbial number and diversity as even dormant microflora is activated (Warnock et al., 2007). Apart from providing carbon nutrition, it provides habitat for the growing soil microflora owing to occurrence of pores, protects microbes from toxic contaminants like heavy metals, xenobiotics, and likewise, alteration in the soil pH conditions and by making available several nutrients like N and P as well as moisture retention (Ameloot et al., 2013). The porous properties of biochar can be harnessed for development of inoculum carrier for the plant probiotic and beneficial microorganisms II. INDUSTRIAL AND ENVIRONMENTAL TECHNOLOGIES

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(Douds Jr et al., 2014). These alterations have a profound positive impact on the fertility and plant growth promotion of the amended soils particularly the vegetable crops as reported in tomato (Pane et al., 2015; Nzanza et al., 2012), red clover (Mia et al., 2014), and pepper (Kolton et al., 2011). The biochar soil microbial interface interactions also result in enhancing the resistance and resilience of diverse microbial types to environmental disturbance leading to higher functional stability (Lehmann et al., 2011).

20.4 AGRONOMIC EFFECTS OF BIOCHAR AMENDMENTS IN VEGETABLES 20.4.1 Biochar-Plant Growth Effects and Yield Impacts Biochar application in soil results in positive impacts on the plant growth and final yield (Table 20.2), whereas it may also result in no positive yield (Vaccari et al., 2015; TABLE 20.2 Effect of Biochar Amendment on Growth and Yield Characteristics of Vegetable Crops Biochar application rate

Biochar source/ feedstock

Pyrolysis temperature

Crop in which biochar applied

Green waste (cotton trash/ stalks, grass cutting, plant prunings)

At around 450 C

Radish (Raphanus sativus var. Long Scarlet)

Pot experiment in Alfisol (chromosol) carried out in temperaturecontrolled glasshouse

Applied at 280% increase the rate of over biochar 10 100 t/ha unamended soil

Chan et al. (2007)

Poultry litter

At 450 C Radish (Raphanus (unactivated sativus var. Long biochar) and Scarlet) 550 C (activated biochar by using hightemperature steam)

Pot experiment in hardsetting Alfisol (chromosol) carried out in temperaturecontrolled glasshouse

42% at 10 t/ha to Applied at 96% at 50 t/ha the rate of 10 100 t/ha biochar application rates

Chan et al. (2008)

Cherry tomato (Lycopersicon esculentum)

Pot experiment (pot size 19 3 15 3 20 cm) in temperaturecontrolled glasshouse

Applied at the rate of 10 t/ha

64% improvement in production of cherry tomatoes over unamended soil

Hossain et al. (2010)

Garlic (Allium sativum L.)

Pot experiment using orchard silt (sandy) loam soil sampled from depth of 0 20 cm

Applied at different BC-to-soil mass ratios (1:2, 1:4, 1:6)

Faster growth in biochar-amended soil and higher final dry matter yields than those planted in the reference soil

Song et al. (2014a,b)

Wastewater sludge At 550 C

Sewage sludge

At 400 550 C

Type of study

Outcome/results

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Williams and Qureshi, 2015) or even negative impact on the yield (Baronti et al., 2010). Moreover, these growth effects vary on basis of type of biochar, its application rate, soil type, and other edaphic properties. Largely, biochar amendment results in positive agronomic benefits as given in the detailed review by Spokas et al. (2012).

20.5 CONCLUSION Biochar is an essential facet for attaining ES and better vegetable crop production. Its profound benefits for climate change mitigation, enhanced agronomic, and remediation of a variety of environmental contaminants ranging from gases to solids have been well documented in the literature summarized in the earlier sections, which attenuate its wider acceptance and usage. However, few bottlenecks have to be addressed, particularly the generation of economically viable biochar in amounts required for field application. If used at higher application rates under field conditions, the total cost of generation and proper application becomes prohibitively expensive for the marginal and resource poor vegetable growers in developing nations. The creation of the carbon-negative economy also demands closer and flexible networking between biomass generation, conversion to biochar, bioenergy formation, and then transportation and supply to the actual users (Huggins et al., 2014; Vanholme et al., 2013).

Acknowledgments Author graciously thanks the Head, Department of Vegetable Science and Dean, College of Agriculture for providing necessary infrastructural and research facilities.

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FURTHER READING

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Vanholme, B., Desmet, T., Ronsse, F., Rabaey, K., van Breusegem, F., De Mey, M., et al., 2013. Towards a carbonnegative sustainable bio-based economy. Front. Plant Sci. 4, 174. Available from: https://doi.org/10.3389/ fpls.2013.00174. Wang, W., Martin, J.C., Zhang, N., Ma, C., Han, A., Sun, L., 2011. Harvesting silica nanoparticles from rice husks. J. Nanopart Res. 13, 6981 6990. Available from: https://doi.org/10.1007/s11051-011-0609-3. Waqas, M., Khan, S., Qing, H., Reid, B.J., Chao, C., 2014. The effects of sewage sludge and sewage sludge biochar on PAH and potentially toxic element bioaccumulation in Cucumis sativa L. Chemosphere 105, 53 61. Warnock, D.D., Lehmann, J., Kuyper, T.W., Rillig, M.C., 2007. Mycorrhizal responses to biochar in soil—concepts and mechanisms. Plant Soil 300, 9 20. Available from: https://doi.org/10.1007/s11104-007-9391-5. William, K., Qureshi, R.A., 2015. Evaluation of biochar as fertilizer for the growth of some seasonal vegetables. J. Bioresour. Manag. 2 (1), 41 46. Yang, Y.N., Sheng, G.Y., 2003. Enhanced pesticide sorption by soils containing particulate matter from crop residue burns. Environ. Sci. Technol. 37, 3635 3639. Yang, Y.N., Sheng, G.Y., Huang, M., 2006. Bioavailability of diuron in soil containing wheat-straw-derived char. Sci. Total Environ. 354, 170 178. Yao, Y.N., Gao, B., Chen, H., Jiang, L., Inyang, M., Zimmerman, A.R., et al., 2012. Adsorption of sulfamethoxazole on biochar and its impact on reclaimed water irrigation. J. Hazard Mater. 209 210, 408 413. Younis, U., Qayyum, M.F., Shah, M.H.R., Danish, S., Shahzad, A.N., Malik, S.A., et al., 2015. Growth, survival, and heavy metal (Cd and Ni) uptake of spinach (Spinacia oleracea) and fenugreek (Trigonella corniculata) in a biochar-amended sewage-irrigated contaminated soil. J. Plant Nutr. Soil Sci. 178, 209 217. Available from: https://doi.org/10.1002/jpln.201400325. Yu, O., Raichle, B., Sink, S., 2013. Impact of biochar on the water holding capacity of loamy sand soil. Int. J. Energ. Environ. Eng. 4, 44. Available from: http://www.journal-ijeee.com/content/4/1/44. Zeng, Z., Zhang, S., Li, T., Zhao, F., He, Z., Zhao, H., et al., 2013. Sorption of ammonium and phosphate from aqueous solution by biochar derived from phytoremediation plants. J. Zhejiang Univ. Sci. B (Biomed. Biotechnol.) 14 (12), 1152 1161. Zhang, G., Zhang, Q., Sun, K., Liu, X., Zheng, W., Zhao, Y., 2011. Sorption of simazine to corn straw biochars prepared at different pyrolytic temperatures. Environ. Pollut. 159, 2594 2601. Zhang, M., Gao, B., Yao, Y., Xue, Y., Inyang, M., 2012. Synthesis, characterization, and environmental implications of graphene-coated biochar. Sci. Total Environ. 435 436, 567 572. Zhang, X., Wang, H., He, L., Lu, K., Sarmah, A., Li, J., et al., 2013. Using biochar for remediation of soils contaminated with heavy metals and organic pollutants. Environ. Sci. Pollut. Res. 20, 8472 8483. Available from: https://doi.org/10.1007/s11356-013-1659-0. Zhou, Z., Yuan, J., Hu, M., 2015. Adsorption of ammonium from aqueous solutions on environmentally friendly barbecue bamboo charcoal: characteristics and kinetic and thermodynamic studies. Environ. Prog. Sustain. Energy 34 (3), 655 662. Available from: https://doi.org/10.1002/ep.

Further Reading Ghezzehei, T.A., Sarkhot, D.V., Berhe, A.A., 2014. Biochar can be used to capture essential nutrients from dairy wastewater and improve soil physico-chemical properties. Solid Earth 5, 953 962. Hardie, M., Oliver, G., Bound, S., Clothier, B., Close, D., 2014. Effect of biochar application on soil water availability and hydraulic conductivity. Soil Sci. Aust. Natl Soil Sci. Confer 2014, 1 4. Yachigo, M., Sato, S., 2013. Leachability and vegetable absorption of heavy metals from sewage sludge biochar. Soil Processes and Current Trends in Quality Assessment. InTech, Croatia, pp. 399 416. https://doi.org/ 10.5772/55123. Yilangai, M.R., Manu, A.S., Pineau, W., Mailumo, S.S., Okeke-Agulu, K.I., 2014. The effect of biochar and crop veil on growth and yield of Tomato (Lycopersicum esculentus Mill) in Jos, North central Nigeria. Curr. Agric. Res. 2 (1). Zhou, Y., Gao, B., Zimmerman, A.R., Fang, J., Sun, Y., Cao, X., 2013. Sorption of heavy metals on chitosanmodified biochars and its biological effects. Chem. Eng. J. 231, 512 518.

II. INDUSTRIAL AND ENVIRONMENTAL TECHNOLOGIES

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A A56, 23 AB Enzymes, 306 Abiotic stress, in plants, 362f Acacia spp., 148t Academia industrial gap, 268 Accase (acetyl CoA carboxylase), 349 Accessory genes, 6f, 7 Acetyl CoA carboxylase, 164 Acid catalyst process, 340 342 Acid-catalyzed method, 340 342 Acidithiobacillus ferrooxidans, 6 Acquired resistance, 196 197 Acremonium alternatum, 183 Acrodontium crateriforme, 183 Acrylamide, 316 Acyclovir, 203 Acyl-ACP thioesterase I, 349 350 Acyl-acyl carrier protein reductase (AAR), 350 Acylase carrier protein (ACP), 348 349 Adeno-associated vectors, 258 259 Affinity fusion based protein purification, 77 78 Agar, 38 39 Agaricus bisporus, 64t Agaropectin, 38 39 AgrenVec, 83t Agricultural biotechnology, 89 92 crop improvement, genetic engineering strategies for, 92 93 plant genetic modification, 92 plant transformation techniques, 92 93 crop quality, genetic manipulation for, 96 101 concerns about transgenic plants, 101 current status of GM plants, 99 100 eco-social impact of genetically modified crops, 98 99 goals of genetic engineering in crop improvement, 100 101 golden rice, 97 98 transgenic for improved fruit storage, 96 97 future prospects, 105 genetically modified crops, applications of, 93 96 resistance to abiotic stresses, 95 96

resistance to biotic stress, 94 95 genetic assisted plant breeding, 101 104 MABC breeding, 103 104 molecular markers, 101 104 variety identification and seed purity analysis, 103 origin and definition of, 89, 90f application of modern agriculture, 91 92 plant breeding program, 90 91 Agricultural waste, 376t Agrigenomics, 5 Agrobacterium tumefaciens, 93, 135 137, 188, 220 Agrobacterium-mediated plant transformation, 93 Agrocin 84, 183t Alcalase, 313 Aldehyde deformylating oxygenase (ADO), 350 Algae, 77 Algal biotechnology, 31 microalgae, 31 36 seaweeds (macroalgae), 36 47 Algal fatty acid biosynthesis pathway, 348 349 Algal metabolic pathways, 348 349 Alginate, 39 Alkalimonas amylolytica, 317 318 Alkaline cellulase K enzyme, 305f Alkaloid biosynthesis, 147 Alkane biosynthetic genes, 350 Alkanes (C4 C23), 350 Allium cepa, 148t Allylamines, 202t α-amylases, 300 301, 306 307, 311, 314, 317 α-galactooligosaccharides (α-GOS), 113 114 α-naphthyl acetate, 240 α-pinene, 148t Amano Pharmaceuticals, 306 Ambulatory blood pressure monitor, 327 Aminoclay NPs, 218 219 1-Aminocyclopropane-1-carboxylic acid (ACC), 96 97 Aminoglycosides, 200 201 Ampelomyces quisqualis, 183 Amphiroa fragilissima, 37f Amplified length polymorphism, 102t Amylases, 309, 313 Anabaena, 34

387

388 Anabaena flosaquae, 35 Anabaena oryzae, 35 Angiotensin-converting enzyme (ACE) inhibitory peptides, 111 Animal and plant pathogens seaweeds as biological control in, 43 44 Animal and urban waste, 376t Animal biotechnology, 277 Antagonistic secondary metabolites, 188 Anthocyanin, 148t Anthrax vaccine, 138 Antibiosis, 182 Antibiotics, 182, 183t, 193, 202t Antifolates, 203t AntiJen, 8t Antimicrobial resistance (AMR), 193 194, 205 206, 208 209 Antioxidant enzymes, 113 Antisense RNA technology, steps of, 98f Applied mechanics, 327 328 Arabidopsis, 154 155, 158 160 Arabinoxylan, 309 310 Arachidonic acid (AA), 33 34 Arachis hypogea, 164 Aragonite (CaCO3), 46 47 ArrayTrack, 291 Arsenic carcinogenesis, 356 Artemisia annua, 146 147, 164 Artemisinin derivatives, 203t Arthrobotrys oligospora, 181 Artificial neural networks (ANNs), 7 10 Artificial selection methods, 301 Aryl amino alcohol, 203t Ascomycetes, 57 Ascophyllum nodosum, 39 Aspergillus, 304 Aspergillus niger, 59 Aspergillus oryzae, 276, 304, 313 Assisted reproductive technologies (ART), 278 Astaxanthin, 33 Aulosira, 34 Auricularia polytricha, 64t Autoimmune diseases, 139 Azole class of drug, 201, 202t

B Bacillomycin D, 182, 183t Bacillus, 304 Bacillus anthracis, 14, 138 Bacillus cereus strain UW85, 182 Bacillus licheniformis, 300 301 Bacillus sp. S23, 342t Bacillus subtilis AU195, 182 Bacillus subtilis QST713, 182

INDEX

Bacillus thuringiensis, 95, 224 Bacteroides thetaiotaomicron, 16 Bacteroides Prevotella Porphyromonas, 118 Batch cultivation, 241 BCIpep, 8t Beam-mediated transformation, 138 Beauveria bassiana, 181 Benzenoid biosynthesis, 147 BepiPred, 7 10, 8t β-carotene, biosynthesis of, 98f β-glucanases, 309 310 β-glucuronidase, 120 Bifidobacteria, 276 277 Bifidobacterium bifidum, 276 277 Bifidobacterium bifidum MF 20/5, 112 Bifidobacterium longum, 16 Bifidobacterium longum KACC91563, 112 Bifidus, 277 Bio/BA yogurt, 277 Bioactive peptides, 111 112, 282 Biocatalysts/enzymes, 239 241 advances in enzyme industry, 242 243 application of enzyme, 243 enzyme production methods, 241 242 purification of enzyme, 242 Bioceramics, 331 Biochar, 368 372 affecting soil nutrient status and enhancing nutrient use efficiency, 373 biochar soil microbial community interactions, 381 382 bioengineering of ecological niches, 375 381 and climate change abatement, 374 375 for enhancing water use efficiency, 374 as nutrient delivery vehicle, 373 plant growth effects and yield impacts, 382 383 as possible solution for ES, 371 372 production, 370 types, 369 370 and various use efficiency strategies, 372 374 Biocharculture, 371 372 Biochemical conversion, 343 Biocontrol agent (BCA), 183t, 184t, 189 Biocontrol technology, 179 biopesticides versus chemical pesticide, 179 future prospects, 189 omics in, 186 189 genomics, 186 187 metabolomics, 187 188 proteomics, 187 secretomics, 188 189 for plant disease management, 181 185 antibiosis, 182 competition, 184 induced resistance in host plants, 184 185

INDEX

mycoparasitism, 183 strain improvement, 185 186 mutagenesis, 185 protoplast fusion, 186 transformation, 186 therapy in organic farming, 180 181 Biodiesel, 245 production, 346 347 specification of, 340, 341t Bioenergy, 46 Bioethanol, 186, 340, 342 Biofertilizers, 34 Biofluid mechanics, 330 Biofuel technologies, 339 first-generation, 339 342 future perspectives, 351 omics advancement and approaches for costeffective production, 348 350 practical challenges in, 348 second-generation, 342 344 third-generation, 342 lipid extraction and biodiesel production, 346 347 microalgae biomass harvesting, 345 346 microalgae cultivation, 344 345 Biofuels, 46, 245 247 Biohurt, 276 277 Bioinformatics, 4, 10 11, 59 60, 63, 263 264 Bioinspired/nano-enabled plants, 224 227 Bioinstrumentation, 326 327 in vivo bioelectrical measurements with coated electrodes, 326 327 mHealth technology, 327 noninvasive instruments for healthcare, 327 Biolistic method, 137 Biolistics technique, 219 Biolsilico, 161 Biomass-to-liquid process, 246 Biomaterials, 325 326, 330 332 generations of, 330t Biomechanics, 327 328 continuum mechanics, 328 sports biomechanics, 328 Biomedical engineering (BME), 325 Biomedical signal processing, 334 Biomineralization, 46 47 Bionanocrystallization, 47 Biopesticides, 179 versus chemical pesticide, 179 Biopolymers, 331 Bioreactors, 248 249 -based plant systems, 76 77 plants as, 74 78, 75f Biorefinery, 246, 248 Bioremediation, 44 Biorobotic, 333 334

Biosensors, 334 335 placement of, 335t types of, 335f Biotechnology, 3, 12 agricultural. See Agricultural biotechnology history of, 235 236 microbial omics in. See Microbial omics Biotic and abiotic stresses, 94f Biotransducers, 334 Biotribology, 329 Bone tissue engineering field, 336 Botryococcus, 35 Botryococcus braunii, 32f Brassica napus, 155 156 Breast cancer, 121 Brown seaweeds, polysaccharides of, 39 40 alginate, 39 fucoidan, 40 laminarin, 39

C C60 fullerenes/Fullerols, 225t Cadmium, 12, 218 219 Cadmium sulfide, 218 219 CaDXMT, 165 Caenorhabditis elegans, 23 Calcareous algae (CA), 46 47 Calcite (CaMgCO3), 46 47 Calcium phosphate NPs, 218 219 Calf rennet, 306 307 Camelina, 340 342 CaMV (Cauliflower mosaic virus), 140 CaMXMT, 165 Candida albicans, 61 62 drug resistance in, 201 202, 201f, 202t Candida antarctica, 340 342, 342t Candida rugosa, 340 342 Candida sp. infections, 56 Capreomycin-resistant (CPM) cell, 199 Carbon capture, 318 319 Carbon dioxide (CO2), 339, 344 345 Carbon dioxide capture, 318 319 Carbon foot-printing, 371 372 Carbon monoxide (CO), 339 Carbon nanotube (CNT), 214 Carbon sequestration, 371 372 biochar for. See Biochar Carbon-based materials, 225t, 343 344 Carbonic anhydrase, 319 Carbonylation process, 361 362 Carboxylases, 314 CaroRX, 82t Carotenoid, 151 152, 163 164 Carrageenan, 39

389

390

INDEX

Caseinate, 308 CASTER program, 240 CASTing (combinatorial active site saturation test), 240 Cauchy, Augustin-Louis, 328 CaXMT, 165 CDNA synthesis, 358 Cell line development, 260 261 Cell wall degrading (CWD) enzymes, 312 Cell-based screenings, 27 Cellic CTec3, 312 Cellular processes, 26 27 Cellulase, 243 244, 314, 316 317 Cellulose, 243 244, 316 317 Cellulosic biomass, 246, 343, 343f Centella asiatica, 152 Center for Jatropha Promotion (CJP), 340 Centrifugation, 345 346 Cephalosporium acremonium, 186 Ceramics, 331 Cereals, 286 287 Cerezyme, 81 82 CGA Platform, by Complete Genomics, 4 Challenges in omics-for-industry, 267 268 Cheese, 275 276 Chemical toxicity screening, 360 361 Chinese Hamster Ovary (CHO) cells, 260 Chitosan, 56 Chlamydomonas reinhardtii, 77 Chlamydomonas sp., 32f Chlorella minutissima, 33 34 Chlorella protothecoides, 35 Chlorella pyrenoidosa, 349 Chlorella sp., 32f Chlorella vulgaris, 32f, 34 35 Chlorella zofingiensis, 33 Chloroplast genome engineering for pharmaceuticals, 82 84, 85t Chlorosulfolipid, 34 Cholera, 141 Chromatography tandem mass spectrometry (LC-MS/MS), 278 Chromobacterium viscosum, 342t Chymosin, 275 276 Cidofovir, 204t CIGB, 83t Circular permutation, 239 Classical medical care, 336 Climate change abatement biochar and, 374 375 Cloning, 92 Clostridium thermocellum, 319 CO2 sequestration, 36 Coelastrella striolata, 33

Coffee rust (1970), 179 Cofilin-1, 14 Co-immunoprecipitation-based mass spectrometry, 23 Co-inertia Analysis (CIA), 266 267 Colitis model, acute, 16 17 Colloidal coomassie blue, 279 280 CollPlant, 83t Colorectal cancer (CRC), 119 120 Colorimetric assays, 301 Combined omics approaches, 264 267 Commercial status, 81 82 Comparative genome profiling, 28 Comparative genomics, 5 7 Competition, 184 Computational biology, 59 Computational biomechanics, 329 330 Computer-integrated surgery systems, 329 330 Contaminated environmental sample, 360 Continuous chromatography, 242 Continuous cultivation, 241 Continuum mechanics, 328 Conventional vaccines, 133 versus edible vaccines, 138 Corallina elongate, 37f Coralline alga,, 37f Core genome, 6f, 7 Corn and cellulose processing, 310 312 Corynebacterium glutamicum, 308 309 Cosmetics and cosmeceuticals, 34, 42 43 4-Coumarate CoA ligase, 164 CRISPR/Cas system, 262 CRISPR/Cas9 system, 249 Crohn’s disease (CD), 115 116 Crohn’s illness, 62 Crop adaptation, 92 Crop bioengineering, 213 214 applications of engineered NMs in, 215f Crop improvement, genetic engineering strategies for, 92 93 plant genetic modification, 92 plant transformation techniques, 92 93 Crop improvement, metabolic engineering and, 157f Crop quality, genetic manipulation for, 96 101 concerns about transgenic plants, 101 current status of GM plants, 99 100 eco-social impact of genetically modified crops, 98 99 goals of genetic engineering in crop improvement, 100 101 golden rice, 97 98 transgenic for improved fruit storage, 96 97 Crypthecodinium cohnii, 33 34 Cultured dairy products, 121 122 Cyanobacteria (BGA), 34

INDEX

D

E

Dactylaria spp., 181 Dactylella spp., 181 Dairy and food processing technology Biohurt, 276 277 biotechnological developments in, 275 276 cheese, 275 276 future prospects, 291 292 integrating omics, 292 transcriptomics, proteomics, and metabolomics, 291 292 proteomics of milk and milk products, 282 284 Danisco, 306 Data depository and bioinformatics tools, 263 264 DbMHC, 8t Dead algae drift, 37 Decoding mechanism, 356 Deep sequencing, 289, 303 Denim, 316 317 Desiccation-based transformation, 138 Destreak, 279 280 Detergents, 313 315 DGAT (di-acyl glycerol acyl transferase), 349 2,4-Diacetylpholoroglucinol, 183t 2,4-Diactylphloroglucinol (DAPG), 182 Diesel, Rudolph, 340 Difference gel electrophoresis (DIGE), 57, 279 280 Differential genetic sequence patterning, 357 358 3,4-Dihydroxylphenyl alanine (DOPA), 315 Direct RNA sequencing, 289 DiscoTope, 8t DNA fingerprinting methods, 103 DNA functionalized silica-coated gold NPs, 223 DNA gyrase, 199 DNA ligases, 92 DNA MAB, 102 103 DNA marker techniques, 101 DNA microarrays, 360 for gene-expression profiling, 290f DNA sequencing, 5 Docosahexaenoic acid (DHA), 33 34 DOF4, 154 155 Dolomite (CaMg(CO3)2), 46 47 Drug delivery, 46 Drug inactivation, by enzymatic attack, 197 Drug penetration into target cells, failure of, 197 Drug-resistant pathogens, 197 200 Dry seed cereals and grain legumes, 76 DSM, 306 Dunaliella primolecta, 35 Dunaliella salina, 32 33 Dunaliella sp., 32f Dunaliella tertiolecta, 31 32 Dynamic simulations, 160

Ebola vaccines, 84 Ebola virus, 26, 81 82 Echinocandins, 202t Ecological niches, bioengineering of, 375 381 heavy metal removal, 375 379 organic pollutant removal, 380 381 sorption of excess N or P from wastewater, 381 Edible vaccines, 80 81, 81f, 135 138 applications of, 139 140 autoimmune diseases, 139 gastrointestinal disorders, 139 hepatitis B, 140 malaria, 140 measles, 140 clinical trials and research studies, 140 141 conventional vaccines versus, 138 current developments, 141 142 banana, tomato, and potato, 142 disadvantages of, 139 electroporation/electrotransfection, 137 future prospects, 142 143 gene gun or biolistic method, 137 lipofection, 137 138 mode of action of, 138 patents on, 142 plasmid/vector mediated, 135 137 production of using genomics, 134 using metabolomics, 135 using proteomics, 134 135 using transcriptomics, 134 second-generation, 141 eHealth (electronic health), 327 Eicosapentaenoic acid (EPA), 33 34 Elastin-like polypeptides (ELPs), 78 Electrical power plants, 36 Electroporation, 137 Electrospray ionization (ESI), 281 Electrotransfection, 137 Elelyso, 82t Endogluconases, 243 244 Endonuclease, 244 245 Energy demand, 339 Engineered NMs, 224 Enterobacter cloacae, 184t Enterobacter species, 200 Enterococcus faecalis strains, 111 Enterococcus faecium CRL 183, 119 Enterotoxigenic E. coli (ETEC), 141 Entire genome sequencing, 303 Environmental biotechnology, 247 application, 247 omics-based bioengineering in, 355

391

392

INDEX

Environmental biotechnology (Continued) chemical toxicity screening, 360 361 environmental stress-related gene and protein modifications, 361 363 future perspective, 363 pollution control, 359 360 soil microbial ecology, 356 359 Environmental stress-related gene and protein modifications, 361 363 Environmental sustainability (ES), 367 368 addressing ES-related issues, 368 Enzyme discovery and engineering, 299 future perspectives, 319 320 for industrial applications, 300 303, 306 319 animal feed industry, 309 310 corn and cellulose processing, 310 312 food industry, 306 309 organic synthesis, 315 316 surfactants and detergents, 313 315 textile and carbon capture industries, 316 319 molecular engineering, 304 305 novel enzyme(s) isolation and optimization, 300f phage-display approach, 302f Enzymes as additives in food and feed, 67t advances in enzyme industry, 242 243 application of, 243 in different sectors, 68t associated with food and feed bio-processing, 66t definition, 241 production methods, 241 242 purification of, 242 Epigenomics, 277 278 Epithelial damage, 120 Epitome, 8t Error-prone polymerase chain reaction (epPCR), 239 240 Erwinia phytoene desaturase gene, 163 164 Escherichia coli, 14, 139, 151 152, 300 302, 349 350 Essential fatty acids (EFAs), 33 34 Esterases, 240 241, 244 245 Esterfip-H, 245 Ethanol, 245 production of from lignocellulosic biomass, 246 from sugars and starches, 245 246 Ethionamide/Prothionamide, and Thiomides, 199 Ethyl-tertiary-butyl-ether (ETBE), 245 246 Euglena gracilis, 31 32 European Bioinformatics Institute, 263 264 Exopolysaccharides (EPS), 112 113 Expression profiling, 134 135 Expression proteomics, 274 275 Extracellular polysaccharides, 112 113

F FAE1, 155 156 Faecalibacterium prausnitzii, 16 17 Fast Blue/Fast Red, 240 Fatty acid desaturase (FAD), 348 349 Fatty acid methyl esters (FAME), 340 Fatty acids, 33 34 Fe2O3 NPs, 225t Fed-batch cultivation, 241 Fermentation, 236 238, 299, 342 culture conditions, 238 fermentors, 238 inoculum (microorganisms), 237 product, 238 substrate, 237 238 types, 237 Fetal bovine serum (FBS), 260 261 Fighting against superbugs, 193 antibacterial mechanism of nanoparticles, 208 drug resistance in Candida albicans, 201 202 future perspectives, 208 209 human actions contributing towards MDR development, 194 195 industrially important drug-resistant pathogens, 197 200 MDR biotechnological interventions to counter, 206 207 in Herpes Simplex Virus (HSV), 203 205 in malaria, 203 in Pseudomonas aeruginosa, 200 201 in tuberculosis, 197 200 microbial drug resistance, global perspective of, 194 molecular basis of resistance, 196 197 acquired resistance, 196 197 strategies to control AMR, 205 206 infection prevention and control at personal and community level, 205 innovations, fostering, 206 political commitment, 206 First-generation biofuel technologies, 339 342, 348 Fishing, 267 Flammulina velutipes, 64t Flavin adenine dinucleotide (FAD/FADH), 315 Flavones, 148t Flavonoid biosynthesis, 147 Flavonols, 148t Fleming, Alexander, 235 236 Fluorescence, 279 280 Fluorescent glucose biosensors, 334 335 Fluoroquinolone (FQ), 197, 199 FQ-resistant Escherichia coli and Neisseria gonorrhea, 194 Flux analysis, 264 265

INDEX

Flux balance analysis (FBA), 160 161 Flux model-based analysis, 160 161 Fluxomics, 262, 266t Food and feed bio-processing, enzymes associated with, 66t, 67t Food industry, enzymes in, 306 309 Food omics, challenges and opportunities in, 292 293 Food process technology, 247 248 Food safety, transcriptomics in, 288 291 Food technology, proteomics of, 284 287 Formate dehydrogenase (FDH) system, 315 Foscarnet, 204t Frankidiniella occidentalis, 165 Fruit and vegetable crops, 76 Fucoidan, 40 Fucus vesiculosis, 40 Functional food biotechnology, 107 definitions, 107 108 lactic acid bacteria (LAB), 108 109 nutraceutical production by LAB, 109 114 antioxidant enzymes, 113 beneficial enzymes, 113 114 bioactive peptides, 111 112 exopolysaccharides, 112 113 vitamins, 109 111 probiotic effects of LAB, 115 122 Functional genomics, 11 17 metabolomics, 15 17 proteomics (interactomics), 13 14 transcriptomics, 11 12 Functional proteomics, 274 275 Functionalized magnesium phyllosilicate, 218 219 Fungal omics, 53 54 bioinformatics, 59 60 genomics, 54 55 industrial perspective, 62 63 medical perspective, 60 62 fungi and the gut microflora, 61 62 role of fungi on immunocompromised patients, 61 metabolomics, 58 59 proteomics, 56 58 sample preparation challenges, 60 transcriptomics, 55 56 Fusarium graminearum, 188 189 Fusarium sp., 55

G Gadopentaacetic acid (Gd-DTPA), 222 Gain-of-function method, 27 28 γ-linolenic acid (GLA), 33 34 Ganciclovir, 204t Ganoderma lucidum, 64t Gastrointestinal disorders, 139

393

Gel-based proteomics, 279 280 Gel-free proteomic approach, 280 281 Gene gun/biolistic method, 137 Gene knockout strategies, 259 260 Gene Ontology (GO), 291 Gene silencing, 259 260 Gene transformation, defined, 216 217 Gene-based-bioinformatics method, 357 Genencor enzymes, 306 Genetic assisted plant breeding, 101 104 MABC breeding, 103 104 molecular markers, 101 103 for hybrid vigor, 104 variety identification and seed purity analysis, 103 Genetic distance analysis, 103 Genetic engineering, defined, 92, 350 Genetic polymorphism studies, 357 358 Genetically engineered enzymes, 166t, 167t, 168t Genetically modified (GM) crops, 93 96, 213 214 eco-social impact of, 98 99 naming and sorting, 223 224 resistance to abiotic stresses, 95 96 herbicide resistance, 96 tolerance to water-deficit stresses, 96 resistance to biotic stress, 94 95 disease resistance, 95 insect resistance, 94 95 virus resistance, 95 GenMAPP, 263 264 Genome Analyzer, by Solexa/Illumina, 4 Genome based analysis, 158 159 Genome sequencing, 292 Genome-scale model-based analysis, 161 Genome-wide polymorphism, 101 102 Genomic databases, 162t Genomics, 5, 54, 255 256, 266t in biocontrol technology, 186 187 functional, 11 17 fungal, 54 55 production of edible vaccines using, 134 structural, 4 11 Geobacillus stearothermophilus, 300 301 Geranylgeranyl transferase gene, 145 Gerbera jamesonii, 217t Glass fiber reinforced PEEK (GFRPEEK), 329 Gliocladium virens, 183 Gliotoxin, 182, 183t Glucoamylase, 312 Glucose isomerase, 312 Glucose monitor, 334 335 Glucuronic acid, 40 Glutamic acid decarboxylase (GAD67), 139 Glycine max, 148t

394 Gold NPs, 218 219 Golden rice, 97 98 GPX-Macrophage, 8t Gracilaria edulis, 37f Gravity settling of biomass, 345 346 Great Bengal famine (1943), 179 Green seaweeds, polysaccharides of, 40 41 food, 41 ulvan, 40 41 Green waste, 343 Greenhouse gases (GHGs) emission, 371 372 curbing, 374 375 GS FLX, by Roche/454 Life Technologies, 4 Gut microflora, fungi and, 61 62 Gut-associated lymphoid tissue (GALT), 116 117

H H5, 82t H5 intradermal seasonal influenza vaccine, 82t HA vaccine, 82t Haematococcus pluvialis, 33, 348 349 Hairy root cultures, 76 77 Ham, 308 HaptenDB, 8t Harvested biomass, 346 347 Health Canada, 309 Heavy metal contaminants, 375 379 Heavy metals, effect of biochar amendment for remediation of, 375 379, 376t Hemicellulases, 244 245, 307 Hemodynamics, 330 Hepatitis B, 140 Hepatitis B surface antigen (HBSAg), 140 Herbicolin, 183t Herpes Simplex Virus (HSV) antiviral agents used as a treatment against, 204f multidrug resistance in, 203 205, 204t Hidden Markov Models (HMM), 263 264 High-resolution real-time imaging, NMs for, 221 222 High-throughput methods, 11 High-throughput omics experiments, integration of, 158 160 HIV antibody, 82t HLA peptidome scanning chip based mass spectrometry approach, 23 HLArestrictor, 8t Hollow gold nanospheres (HAuNS), 331 332 Holter type electrocardiogram (ECG) recorder, 327 HPtaa, 8t Human Microbiome Project, 249 250 Hybrid vigor, molecular markers for, 104 Hybridization, 24 microarray techniques, 24 subtractive, 24

INDEX

Hydrolytic assays, 240 241 Hydroxyapatite NPs, 218 219 Hydroxynitrile lyases, 240 241 Hyper Spectral Imaging, 222

I Iduronic acid, 40 IEDB, 8t IEDB-3D, 8t IL2Rgbase, 8t Illumina sequencer, 257 Illumina sequencing technology, 290 Immobilization, 305 Immobilized pH gradient (IPG) strip, 279 Immune Epitope Database and Analysis Resource IEDB, 7 10 Immunity, 7 Immunogenomics, 7 10 Immunoglobulin A (IgA), 283 Immunologic databases, 8t Immunomodulation, 112 Immunoproteomics, 56 Immunosuppressive illnesses, 56 In planta transformation, 93 In silico modeling and simulation of plant metabolism, 160 161 flux model-based analysis, 160 161 genome-scale model-based analysis, 161 kinetic model-based analysis, 160 tools and databases for, 161 162 integrated metabolic database system, 161 integrated metabolic networks, 161 162 In situ/direct transesterification, 347 In vitro direct molecular evolution techniques, 239 Induced resistance in host plants, 184 185 Industrial biocatalysts, 239 Industrial biotechnology, implications for, 255 256 Industrial biotechnology and bioprocess engineering, omics approaches in, 253 cell line development, 260 261 challenges in, 267 268 combined omics approaches, 264 267 data depository and bioinformatics tools, 263 264 future perspectives, 268 implications for, 255 256 mutagenesis, 258 259 next-generation sequencing (NGS), 257 258 reverse genetics, 259 260 synthetic biology, 261 262 Infinite enzymes, 83t Inflammatory bowel disease (IBD), 61 62, 113, 115 Inflammatory cytokines, 115 116 Influenza virus infection, 26 Innovations, fostering, 206

INDEX

Insulin-dependent diabetes mellitus, 139 Integrated Microbial Genomes, 303f Interactomics, 13 14, 254 255 Interconnected waste management, 368 Interleukin-10 (IL-10), 115 116 International Immunogenetics Information System (IMGT), 8t, 10 IMGT/HLA, 8t IMGT/LIGM-DB, 8t International Scientific Association for Probiotics and Prebiotics (ISAPP), 108 Inverse PCR, 258 Ion Torrent PGM, by Life Technologies, 4 Ionization techniques, 281 IPD-ESTDAB, 8t IPD-HPA (Human Platelet Antigens), 8t IPD-KIR (Killercell IG-like Receptors, 8t IPD-MHC, 8t Irish famine (1845), 179 Iron oxide NPs, 218 219 Isoelectric focusing (IEF), 279 Isoflavonols, 148t Isoniazid (INH), 197 198 isoprenoid biosynthetic pathways, 151 152 Isozymes, 102t, 304 305 Iterative saturation mutagenesis (ISM), 240 Iturin A, 182, 183t

J Jatropha (Jatropha curcas), 340 Jerner, Edward, 133 Joint tribology, 329t

K Kappa casein, 275 Kappaphycus alvarezii, 39 Kappaphycus striatum, 39 Karanja (Pongamia pinnata), 340 KBP, 83t Kefiran, 112 113 KEGG Pathway, 291 Ketoacyl-ACP synthetases (KAS), 348 349 Kinetic model-based analysis, 160 Klebsiella pneumonia, 200

L Laccase, 68t, 243 245 Lactase, 306 307 Lactic acid bacteria (LAB), 108 109 nutraceutical production by, 109 114 antioxidant enzymes, 113 beneficial enzymes, 113 114 bioactive peptides, 111 112

395

exopolysaccharides, 112 113 vitamins, 109 111 Lactobacillus acidophilus, 120, 276 277 Lactobacillus casei BL23, 113 Lactobacillus casei CRL 431, 117, 122 Lactobacillus casei DN 114001, 117 Lactobacillus casei strain Shirota, 120 121 Lactobacillus coryniformis CECT5711, 122 Lactobacillus curvatus CRL 705, 112 Lactobacillus delbrueckii subsp. bulgaricus, 110 Lactobacillus delbrueckii subsp. lactis CRL 581, 112 Lactobacillus fermentum CRL 722 and CRL 251, 113 114 Lactobacillus gasseri CECT5714, 122 Lactobacillus gasseri SBT2055, 118 119 Lactobacillus helveticus 416, 119 Lactobacillus helveticus MIMLh5, 117 118 Lactobacillus helveticus R389, 111 Lactobacillus plantarum CRL 725, 109 110 Lactobacillus rhamnosus GG, 116, 118, 120 Lactobacillus sakei CRL 1862, 112 Lactobacillus sakei NR28, 118 Lactobacillus salivarius UCC118, 115 Lactococcus lactis, 14 Lactococcus lactis subsp. cremoris, 111 Lactoferrin, 283 Laminaria digitata, 39 Laminaria hyperborea, 39 Laminaria japonica, 122 Laminarin, 39 Laundry detergents, 313 Lavender (Lavandula latifolia red.), 163 Leafy crops, 76 Lentinula edodes, 64t Lettuce, 142 Leucine dehydrogenase, 315 Leuconostoc citreum KACC 91035, 112 113 Leuconostoc mesenteroides KC51, 114 Lignin, 243 244, 342 343, 368 369 Lignin biosynthesis, 147, 153 154 Lignin peroxidase, 68t Lignocellulose, 312 Lignocellulosic biomass, 246 Lignocellulosic ethanol, 342 343 Linalool, 148t, 165 Lipases, 240 241, 307, 313 315, 340 342 biodiesel production with, 342t Lipid extraction method, 346 347 Lipofection, 137 138 Lipofection polyethylene glycol combination method, 137 Lipolase, 304, 313 Liposome-mediated transformation technique, 137 Liquefaction, 300 301, 311 Liquid biofuels, 343 344 Liquid chromatography (LC), 13

396

INDEX

Listeria monocytogenes, 117, 289 290 Locteron, 82t Loss-of-function method, 28 LT-B gene, 139 Lyases, 316 Lycopene β-cyclase (LYCB), 97 98, 163 164 Lycopersicon esculentum cv. Micro-Tom, 217t

M Macroalgae. See Seaweeds Macrofungi, medicinal and nutritional properties of, 64t Magnaporthe grisea, 60 Magnetic NPs, 218 220 Magnetic resonance imaging (MRI), 222 Major histocompatibility complex (MHC) class I and class II, 7 10 Malaria, 140 multidrug resistance in, 203 Malonyl ACP transacylase (MAT), 348 349 Manganese, 243 244 Manganese NPs, 225t Manganese peroxidase, 68t Mannanases, 243 244 MAPP66, 82t Marker-assisted backcrossing (MABC), 103 104 Mass analyzers, 281 282 Mass spectrometry, 22 24, 250, 281, 301 co-immunoprecipitation based, 23 HLA peptidome scanning chip based, 23 MS based proteomics, 13, 57 tandem affinity purification (TAP) based, 23 Matrix-assisted laser desorption ionization (MALDI), 281 Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, 13, 287 ME (malic enzyme), 349 Measles, 140 Meat glue, 308 Mechanics, defined, 327 328 Medicago truncatula, 158 Medical nanomaterials, 206 MEGAWHOP, 239 Melissa officinalis L., 217t Mentha spicata, 163 Mentha spp., 148t 4-Mercaptobenzoic (4-MBA) acid, 222 Merozite surface protein (MSP) 4/5, 140 Mesenchymal stem cells, 332 Mesoporous silica nanoparticles (MSN), 217 218 Metabolic flux analysis (MFA), 157 158, 264 265 Metabolic pathway databases, 162t Metabolic pathway flux, 151 152

Metabolically engineered plants, 153f Metabolome analysis, 15, 15f, 159 160 steps of, 16t Metabolomics, 15 17, 58, 254 255, 266t in biocontrol technology, 187 188 fungal, 58 59 production of edible vaccines using, 135 MetafluxNet, 161 162 Metagenomics, 22, 55, 356 357 Metal/metal oxide NPs, 225t Metametabolomics, 356 357 Metaproteomics, 356 357 Metarhizium anisopliae, 181 Metatranscriptomes, 55 Metatranscriptomics analysis, 25 26 metagenomics joined with, 26 (S, R)-2,3-p-Methoxyphenylglycyclic acid (MPGA), 315 mexAB-oprM, 200 201 mexEF-opN, 200 201 mexXY-oprM, 200 201 MGT-GENE-DB, 8t MHCcluster, 8t MHCPEP, 8t MHC-Peptide Interaction Database, 8t mHealth technology, 327 Microalgae, 31 36, 344 345, 348 biological importance of, 31 36 antibacterial, 35 anticancer activity, 34 antifungal, 35 antiviral, 35 biofertilizers, 34 biofuel, 35 bioremediation/phycoremediation, 36 CO2 sequestration, 36 cosmetics, 34 fatty acids, 33 34 feed, 33 foods, 31 33 wastewater treatment, 36 lipid accumulation in, 348 349 morphological structure of, 32f Microalgae biomass harvesting, 345 346 Microalgae cells, 347 Microalgae cultivation, 344 345, 344f Microarray, 24, 54 55, 289 Microarray databases, 162t Microbial biotechnology, 3 4, 10 11, 243 Microbial drug resistance, global perspective of, 194 Microbial genome sequencing, 292 Microbial metabolism, 16 Microbial omics, 1 functional genomics, 11 17

INDEX

metabolomics, 15 17 proteomics (interactomics), 13 14 transcriptomics, 11 12 structural genomics, 4 11 comparative and pan-genomics, 5 7 immunogenomics, 7 10 post-genomics, 10 11 Microbial rennet, 275 276, 276t Microbiome, 22 and personalized medicine, 249 250 Microelectrode arrays, 336 Microinjection-based method, 138 Microprojectile bombardment method. See Gene gun/ biolistic method MicroRNA, microarray technology for, 277 278 Milk proteins, 111, 282 proteomics of, 282 284 Milk-clotting activity (MCA), 275 MinION, 221 Mobile phones, 327 Modern agriculture, application of, 91 92 crop adaptation, 92 enhancement of compositional traits, 91 yield increase, 91 Modern plant breeding, 91 Molecular farming of plants, 73 Molecular markers, 101 103 advantages and disadvantages, 102t for hybrid vigor, 104 Monoseptic fermentation, 237 Morchella elata, 64t Morchella esculenta, 64t Morchella semilibera, 64t Moss, 77 mRNA sequencing, 358 MTT-based bioassay, 301 Mucor miehei, 340 342, 342t MudPit (multidimensional protein identification technology), 280 281 MUGEN Mouse Database, 8t Multicomponent edible vaccines, 133 Multidisciplinary Antimicrobial Management Teams, 206 Multidrug resistance (MDR), 193 194 biotechnological interventions to counter, 206 207 nano-silver, 206 207 zinc oxide nanoparticles as synergic antimicrobials, 207 in Herpes Simplex Virus, 203 205 human actions contributing towards, 194 195, 195f in malaria, 203 policy, cost, and surveillance of MDR pathogens, 206 in Pseudomonas aeruginosa, 200 201 in tuberculosis, 197 200, 198f

397

Multidrug-resistant Staphylococcus aureus (MRSA), 35 Multiple drug-resistant organisms (MDRO), 193 195 Multiwall CNTs (MWCNTs), 218 219, 225t, 226 227 Mutagenesis, 185, 258 259 Mutagenesis assistant program (MAP), 240 MYB11, 154 155 MYB34, 154 155 Mycobacterium tuberculosis, 197, 199t Mycoparasitism, 183, 184t Mycosubtilin, 183t

N NAC, 154 155 NAIST, 83t Nannochloropsis oculata, 34 Nanobarcoding, 223 224 Nanobioimaging, 221 222 Nanobiolistics, 219 Nanocides, 214 216 Nano-enabled chemical techniques, 220 Nano-enabled diagnostics, 222 223 Nano-enabled plant gene transformation, 217 218 Nanogrowth enhancers, 224 Nanomaterials (NMs), 214 applications of, in PBT and crop bioengineering, 215f to enhance seed germination and plant growth, 224 as explant sterilants in plant tissue culture, 214 216 as gene/protein delivery vehicles, 216 220 for high-resolution real-time imaging, 221 222 Nanoparticles, antibacterial mechanism of, 208 Nanopore-based gene, 221 Nanoprojectile-based gene gun technique, 219 Nanoscience and technology (NS and NT), 214 Nanosequencing, 221 Nano-silver, 206 207 Nanostructured calcium phosphate (nano-CaP), 332 333 Nanotechnology in bioengineering, 213 Nanotheranostics, 222 223 Nanovehicles, 216 220 National Center for Biotechnology Information (NCBI), 59, 263 264, 291 National Enzyme Company, 306 Natural petroleum resources, 351 NBM, 83t Nearly isogenic line (NIL) isogenic strategies, 104 Neisseria gonorrhoeae, 319 Neisseria meningitides, 14 NetChop, 8t NetCTL, 8t NetCTLpan, 8t NetMHC, 8t NetMHCcons, 8t

398

INDEX

NetMHCII, 8t NetMHCII 2.2 server, 7 10 NetMHCIIpan, 8t NetMHCpan, 8t NetMHCstab, 8t Neuroengineering, 335 336 Neuromechanics, 335 336 Neuromodulator devices, 336 New Castle disease, 133 New generation technologies (NGS), 12 Next-generation sequencing (NGS) techniques, 5, 53 54, 157 158, 221, 250, 257 258, 289, 303, 357 358 NHL vaccine, 82t Nicotinamide adenine dinucleotide (NAD/NADH), 315 Nicotinia tabacum cv. Xanthi, 217t Niemann-Pick C1 (NPC1), 26, 27t Nitrile hydratase, 316 Nitrogen containing compounds, 146 Nitrogen oxides (NOx), 339 Nitrogen use efficiency, 373 Nitroreductase, 120 Nitzschia, 33 34 NNAlign, 8t Non affinity-based purification, 77 78 Nonalcoholic fatty liver disease (NAFLD), 118 Noninvasive instruments for healthcare, 327 NoroVAXX, 82t Norwalk virus capsid protein, 140 141 Nostoc, 34 Nostoc humifusu, 35 Nostoc muscorum, 34 35 Novozymes, 304, 306, 307f, 319 NP plant soil continuum interaction studies, 221 222 Nuclear magnetic resonance (NMR), 4 5, 58 Nuclease-based mutagenic process, 258 259 Nucleic Acids Research Molecular Biology Database Collection, 10 Nucleoside analogs, 202t Nutraceutical production, 108 by LAB. See Lactic acid bacteria (LAB); nutraceutical production by Nutraceuticals, 42, 108 Nutriproteomics, 56

O Obesity, 293 Oenococcus oeni, 14 Oil crops, 76 Olea europaea L., 217t Oleosin fusion, 78 “Omics-for-industry” policy, 268

OmpR regulon, 289 One-dimensional (1D) PAGE, 13 Open reading frames (ORF), 83t, 303 Open-field and greenhouse production, plants for, 76 oprD, 200 201 Oral tolerance, 116 117 Oral tribology, 329t Organic farming, 189 therapy in, 180 181 Organic pollutants, effect of biochar amendment for remediation of, 376t Organic pollutants remediation, 380 381 Organic solvent extraction, 346 347 Organic xenobiotics, 372 OriginOil, Inc., 347 Oscillatoria sp., 34 35 Osmium tetraoxide, 222 Osmolytes, 96 Osmoprotectants, 96 Oxalate decarboxylase (OxdD), 310 Oxidative stress, 120, 361 Oxidoreductases, 307, 315

P PacBio, 303 PacBio RS/Single-Molecule Real-Time (SMRT), by Pacific Biosciences, 4 Paecilomyces lilacinus, 181 Pan-genome, 6f, 7 Pan-genomics, 7, 303 Paper and pulp industry, 243 245 Particulate matter, 339 Pasteur, Louis, 133, 235 236 Pathogen-annotated Tracking Resource Network (PATRN) system, 293 Penciclovir, 203, 204t Penicillium caseicolum, 283 Penicillium chrysogenum, 186 Penicillium roqueforti, 283 Perfusion batch cultivation, 241 242 Personal digital assistant (PDA), 327 Personalized medicine, 249 250 Pest management practices, 179 Petroselinum crispum, 148t Peyer’s patches, 138 Pfam, 263 264 Phaeodactylum tricornutum, 35 Pharmaceutical proteins derived from plants (PMP), 136t Phenolics, 146 Phenylalanine ammonia lyase (PAL), 164 Phenylylpropanoid biosynthesis, 147 Phormidium fragile, 35

INDEX

Phosphorus, 310, 381 Phosphotungstate, 222 pH-stat assay, 240 241 Phycocolloids, 38 Physcomitrella patens, 77 Phytates, 114, 309 310 Phytic acid, 310 Phytochemicals, 152 153 Pichia pastoris, 308 309 Pigment extraction and production, 44 Pkm2, 14 Plant bioengineering (PB), 213 214 Plant biotechnology (PBT), 213 214 applications of engineered NMs in, 215f Plant breeding program, 5, 90 91 Plant cell suspension cultures, 77 Plant gene transformation, 216 217, 220 Plant genetic modification, 92 Plant metabolic engineering, 145 applications of, 163 165 in agriculture, 165 in food and neutraceuticals, 163 164 in industry, 163 in pharmacy and medicine, 164 approaches and strategies, 147 162 basic methodologies used for, 151f current status and limitations, 165 168 desired diversity, creating, 147 future aspects of, 168 169 management and modulation of metabolic flux, 152 156 by overexpression and silencing of genes, 153 154 by regulation of transcription factors, 154 155 by using cis-regulatory elements, 155 156 identification of key genes, 152 153 metabolites, 146 147 importance of, 146 147 types of, 146 plant systems for, 150f promoters in, 156t systems biology in, 156 162 in silico modeling and simulation of plant metabolism, 160 161 integration of high-throughput omics experiments, 158 160 strategies of systems biology, 157 158 tools and databases for in silico modeling and simulation, 161 162 systems for, 147 152 in vitro cultures, 150 151 microbial cells, 151 152 plant systems, 150 transcription factors in, 155t

399

Plant Molecular Farming (PMF), 73, 75f, 76 Plant nanobionics, 226 227 “Plant phenomics” approaches, 221 222 Plant secondary metabolism, 153 154 Plant theranostics, 222 223 Plant tissue culture, 150 151 NMs as explant sterilants in, 214 216 Plant transformation techniques, 92 93 biological methods, 93 Agrobacterium-mediated plant transformation, 93 in planta transformation, 93 virus-based vectors, 93 physicochemical methods, 92 Plant transgenesis, genetic engineering for, 73 chloroplast genome engineering for pharmaceuticals, 82 84, 85t future directions, 84 85 plant-made pharmaceuticals (PMPs), 78 82 commercial status, 81 82 edible vaccines, 80 81 plantibodies, 80 plants as bioreactors, 74 78 Plantibodies, 80 Plant-made pharmaceuticals (PMPs), 78 82 commercial status, 81 82 edible vaccines, 80 81 future directions, 84 plantibodies, 80 timeline of development events in, 79f Plasmodium falciparum, 140 drug resistance mechanism adapted by, 203t Plasmodium species, 203 Plasmodium yoelli, 140 Pleurotus ostreatus, 64t P-nitrophenoxy analog assay (pNA), 240 241 Podigene, 142 Pollution control, application of omics in, 359 360 Poly (ether-ether-ketone) (PEEK) material, 329 Polyenes, 202t Polyketide synthase, 58 Polymerase chain reaction (PCR), 258, 301 302, 304 degenerate PCR, 25 random PCR, 25 Polymer-based transfection/polyfection, 138 Polymeric NPs, 218 219 Polytetrafluoroethylene, 329 Polyunsaturated fatty acids (PUFAs), 33 34 Porphyridium, 34 35 Portieria hornemannii, 37f Post-genomics, 10 11 Postharvest processing, 284 285 Posttranslational modifications (PTMs), 13, 274 275 Poterioochromonas malhamensis, 34

400 Prebiotics, mechanism of action of, 41 Prefractionation techniques, 278 Primary metabolites, 146 Probiotics, 108, 115 in cancer prevention, 119 122 in healthy host, 122 in intestinal inflammation, 115 122 for obese hosts, 118 119 and reduction of cardiovascular risk, 119 ProdiGene/Sigma, 83t Program packages, 162t Progressive medical care, 336 Promoter/cis-regulatory element, 155 156 ProSAR, 240 PROSITE, 263 264 Protalix, 81 82, 83t Proteases, 309, 313 314 Protegen, 8t Protein arrays, 57 58 Protein engineering, 242 243, 258 259, 264, 304, 311 Protein kinase Cε (PKCε), 27t Proteinases, 275 276 Protein protein interaction (PPI), 11, 274 275 Proteogenomics, 56 Proteome, 14, 56 57, 59, 187, 267 268, 274 275 Proteome based analysis, 159 Proteome mining, 274 275 Proteomic databases, 162t Proteomic techniques, 282 283, 301 302 Proteomics, 13 14, 53, 254 256, 266t, 278 282 in biocontrol technology, 187 classification of, 274f comprehensive data analysis, 282 definition, 273 of food technology, 284 287 cereal and other crops, 286 287 postharvest processing, 284 285 fungal, 56 58 historical perspective, 274 275 of milk and milk products, 282 284, 285t production of edible vaccines using, 134 135 protein extraction, 278 protein identification, 281 282 mass analyzers, 281 282 mass spectrometry, 281 protein separation, 279 281 gel-based proteomic approach, 279 280 gel-free proteomic approach, 280 281 proteomics in assessing, 287 288 quality of foods, 287 288 PRX-12 (oral glucocerebrosidase), 82t PRX-102 (alpha galactosidase), 82t Pseudomonas aeruginosa multidrug resistance in, 200 201 Pseudomonas cepacia, 340 342, 342t

INDEX

Pseudomonas fluorescence F133, 182 Pseudomonas fluorescens, 185 Pseudomonas fluorescens 3551, 184t Pseudomonas putida N1R, 184t Pseudomonas putida WCS 358, 184t Pseudomonas putida WCS358r strain, 182 Pseudomonas solanacearum, 185 PSI-BLAST, 263 264 PSORT, 263 264 Pulse alcometry, 327 Pyrazinamide (PZA), 198 199 Pyrolysis, 246, 368 370, 373 Pyrosequencing, 257

Q QTL (quantitative trait loci), 291 Quality of foods, assessing, 287 288 Quantum dots (Qdots), 218 219 QuikChange method, 239

R Radiolabeling, 279 280 Raffinose, 113 114 Randomly amplified polymorphic DNA (RAPD), 102t Raphidophyceae, 35 Rational protein design, 239 Reactive oxygen species (ROS), 113, 188, 198, 361 Recent trends in biomedical engineering, 325 biofluid mechanics, 330 bioinstrumentation, 326 327 biomaterials, 330 332 biomechanics, 327 328 biorobotic, 333 334 biosensors, 334 335 biotribology, 329 computational biomechanics, 329 330 neuroengineering, 335 336 tissue engineering, 332 Recombinant cell lines, 260 261 Recombinant chymosin, 275 276 Recombinant DNA technology, 145, 150, 214, 306 307 Recombinant enzymes, 276, 304 from fungi, 67t Recombinant gene technology, 242 Recombinant protein production from plants, 74 78 bioreactor-based plant systems, 76 77 increasing heterologous protein accumulation in plants, 77 plant-based expression systems, 76 plants for open-field and greenhouse production, 76 purification of recombinant proteins, 77 78 Red grapes, 148t Red seaweeds, phycocolloids of, 38 47 REDD1, 26, 27t

INDEX

Redox proteomics, 361 362 Regulatory pathways, 11 Rennet, 275 276, 306 307 Resistance, molecular basis of, 196 197 Restriction enzymes, 24, 92, 258 259 Restriction fragment length polymorphism (RFLP), 102t Reverse genetics, 259 260 Reverse translation, 301 302 Reverse vaccinology, 7 Reverse-phase chromatography, 280 281 Rhizoctonia solani, 183 Rhizomucor miehei proteinase, 276 Rhizopus oryzae, 342t Rhizospheric microbes, 180 181 Rhodella, 34 Rhodotorula rubia, 164 Riboflavin deficiency, 111 Ribozyme Pharm, 142 Rice waste based biochar, 371f Rifampin (RMP), 197 198 RNA interference (RNAi) methods, 26 28, 153 154 RNA-induced silencing complex (RISC), 259 260 RNA-Seq, 12, 157 159, 289 290 Robotic capsular endoscope, 333 for examination of gastrointestinal tract, 334f Roche 454 sequencer, 257 Roseoflavin, 109 110 Rosmarinus officinalis, 148t Rotavirus, 141 Rotavirus VP7, 140 141

S Saccharification, 312 Saccharomyces cerevisiae, 12, 61 62, 186, 342 Saccharomyces cerevisiae var. boulardii, 62 Saccharomyces diastaticus ATCC 13007, 186 Safflower (Carthamus tinctorius), 163 Salmonella enteritidis serovar Typhimurium infection, 117 Salmonella typhimurium, 14 Sausages, 308 Scenedesmus almeriensis, 33 Scenedesmus komareckii, 33 Scenedesmus obliquus, 36 37 Schwanniomyces occidentalis ATCC 48086, 186 Sclerotinia sclerotiorum, 188 Scytonema, 34 Seaweed biomass, 47f Seaweeds, 36 47 phycocolloids of red seaweeds, 38 47 bioenergy, 46 biofuel, 46 as biological control against animal and plant pathogens, 43 44 biomineralization, 46 47

401

bionanocrystallization, 47 bioremediation, 44 cosmetics and cosmeceuticals, 42 43 drug delivery, 46 mechanism of action of prebiotics, 41 nutraceuticals, 42 pharmaceuticals, 43 pigment extraction and production, 44 polysaccharides of brown seaweeds, 39 40 polysaccharides of green seaweeds, 40 41 prebiotic potential of polysaccharides, 41 seaweeds tissue culture, 45 46 seaweed phycocolloids, 38 source of bioactive compounds, 44f source of bioactive molecules, 43f Seaweeds tissue culture, 45 46 Secondary metabolites, 58, 63, 146 147, 163 164, 188, 238 239 Second-generation biofuel technologies, 342 344 Secretome, 58 59, 188 189 Secretomics, in biocontrol technology, 188 189 Secretory IgA (s-IgA), 116 117 Seed purity analysis, 101 103 Self-medication practices, 193, 195 Separation systems, 57 Sequence tagged site (STS), 102t Sequencing, 250 Sequencing by Oligo Ligation and Detection, 257 Serene-based metalloenzymes, 200 201 SERS analysis, 223 Short-chain fatty acids (SCFA), 120 Siderophores, 184, 184t SifCosmetics, 83t Silica NPs, 218 219 Silicon-based solid-state fabrication techniques, 221 222 Silver, 279 280 Silver ions, 216 Silver nanoparticles (Ag NPs), 206, 208 application of, 217t as a novel tissue sterilant, 217t Simarouba, 340 342 Simple sequence repeat (SSR), 102t Singletons/strain-specific genome, 6f, 7 Single-walled carbon nanotubes (SWCNTs), 218 219, 225t, 226 227 Site-directed mutagenesis (SDM), 239, 257 258, 317 318 Skin tribology, 329t S-linalool, 163 164 Small RNA (sRNA), 289 SMART, 263 264 SNP (single nucleotide polymorphism) arrays, 277 278 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 279 280

402 Soil metagenomics, 357 358 Soil microbial ecology, application of omics in, 356 359 extracting value from metatranscriptomics, 358 metagenomics and soil function, 357 358 metatranscriptomics and soil function, 358 niche specialization and differentiation, 359 Soil organic carbon (SOC), 368 369, 380 Soil-amendment products, 375 379 Solanum tuberosum, 217t SOLiD, by Applied Biosystems, 4 Solid mechanics, 328 SOLiD sequencer, 257 Solid-state fermentation, 237, 241 242 Sorghum bicolor, 165 Southern corn leaf blight epidemic (1970), 179 Soy-based products, 114, 121 Soybean, 113 114, 142 Specialized plant organs, 150 Spirulina, 31 34, 36 Spirulina platensis, 35 Spodoptera litera, 165 Spontaneous generation of microorganisms, theory of, 235 236 Sports biomechanics, 328 St. thermophilus strains, 110 Stachyose, 113 114 Staphylococcus aureus, 14 Stilbene, 148t, 164 Stilbene synthase, 164 Stoichiometric analysis, 160 161 Strain improvement of biocontrol agents, 185 186 mutagenesis, 185 protoplast fusion, 186 transformation, 186 Stratagene’s QuikChange method, 239 Streptococcus pneumoniae, 186 Streptomyces avermitilis, 6 Streptomyces cinnamoneus, 308 309 Streptomyces mobaraensis, 308 309 Streptomycin/Capreomycin, 199 Strictosidine synthase, 152 153 STRING, 263 264 Structural analysis, 327 328 Structural genomics, 4 11, 56 comparative and pan-genomics, 5 7 immunogenomics, 7 10 post-genomics, 10 11 Structural proteomics, 274 275

INDEX

Subcritical organic solvent, 346 347 Submerged fermentation, 237, 241 Subtractive hybridization, 24 Sulfur dioxide (SO2), 339 Super aging society, 327 Superbugs, 193 195 SuperHapten, 8t Superoxide dismutase (SOD), 113 Surface-enhanced Raman spectroscopy (SERS) studies, 222 Surfactants, 313 315 Surimi, 307 308 Swiss-Prot, 291 SYFPEITHI, 8t Synechocystis sp. PCC 6803, 350 Synthesis motor fuels, 246 Synthetic biology, 261 262 Systemic acquired resistance, 184 185 Systems biology, 17, 17f, 277 278, 291 292 databases and packages for, 162t

T Taenia solium, 140 141 Tandem affinity purification (TAP) based mass spectrometry, 23 Targeting Induced Local Lesions in Genomes (TILLING), 259 260 T-cell epitopes, 7 10 Termamyl, 300 301 Terpenes, 58, 146 Terpenoid biosynthesis, 147 Terpenoid indole alkaloids biosynthesis, 147 Tester and the driver, 24 Thalassiosira pseudonana, 349 Theranostics, 222 223 Thermochemical processes, 343 344 Thermochemical treatment, 343 344 Thermomyces lanuginosus, 304, 313, 340 342, 342t Thermostable enzymes, 300 301 Thiocarbamates, 202t Thioesterase (TE), 163, 348 349 Thiol protein modification process, 361 362 Third-generation biofuel technologies, 342 lipid extraction and biodiesel production, 346 347 microalgae biomass harvesting, 345 346 microalgae cultivation, 344 345 Thymidine kinase (TK), 201 TiO2 NPs, 225t Tissue culture, seaweeds, 45 46

INDEX

Tissue engineering, 332, 332f types of cells used for, 333t TmaDB, 8t Tolypothrix, 34 Topoisomerase II, 199 Topoisomerase IV, 199 Tracking insights of bioremediation, 360f Transcription activator-like effector nucleases (TALENs), 258 259 Transcription and translation boosting, 77 Transcription factors (TFs), 152, 154 155, 289 Transcriptome based analysis, 159 Transcriptomics, 11 12, 55 56, 266t in food safety, 288 291 fungal, 55 56 production of edible vaccines using, 134 Transformation, 135 137, 186 Transformed plants, 152 153, 350 Transgenic crops, 96, 100, 157f, 168 169, 186 187 for commercial use, 97t Transgenic for improved fruit storage, 96 97 Transgenic plants, 76, 137 140 concerns about, 101 Transgenic potato, 140 141 Transglutaminase, 307 309 Transient gene expression system, 76 Translational plant proteomics, 134 135 Tremella fuciformis, 64t Trichloroacetic acid (TCA), 60 Trichoderma, 304 Trichoderma harzianum, 56 57, 183, 184t Trichoderma reesei, 12, 186, 276, 316 317 Trichoderma virens, 182, 183t Trinitrobenzene sulfonic acid (TNBS), 112 113, 115 116 Tropane biosynthetic, 147 Tryptamine, 148t Tryptophan decarboxylase, 152 153 Tuber melanosporum, 64t Tuberculosis (TB), multidrug resistance in, 197 200, 198f Turbinaria conoides, 40 Turbinaria decurrens, 37f 2D/differential-gel electrophoresis, 301 302 Two-dimensional electrophoresis (2-DE), 57, 278 280 Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), 13 Two-dimensional protein gel electrophoresis, 159 Type I diabetes, 139 Tyrosine, 164

403

U Ultra-high-molecular weight polyethylene (UHMWPE), 329 Ulva lactuca, 37f Ulvan, 40 41 Urease, 299 Uronic acid, 40 41 UV mutagenesis, 185

V Vaccination, 133, 138, 140 Valeriana officinalis L., 217t Vancomycin-resistant Staphylococcus aureus, 194 VBASE2, 8t VDJsolver, 8t Vegetable oil methyl esters (VOME), 245 VEN 120, 82t VEN100 (lactoferrin), 82t VEN130, 82t Ventria/InVitria, 83t Verticillium lecanii, 181 Vesicular stomatitis virus (VSV) infection, 26 Vibrio cholera, 140 141, 289 Vinegar, 235 236, 247 248 VIOLIN, 8t Viral biotechnology, omics approaches in, 21 applications, 27 28 cell-based screenings, 27 comparative genome profiling, 28 gain-of-function method, 27 28 loss-of-function method, 28 history of identification of viruses, 22 Viral replication cycle, target proteins in, 27t Virome, 22 Virtual evaluation systems, 59 60 Virus host interactions, advancements in techniques to study, 22 27 hybridization, 24 microarray techniques, 24 subtractive hybridization, 24 metatranscriptomics analysis, 25 26 metagenomics joined with, 26 methods based on PCR, 25 degenerate PCR, 25 random PCR, 25 omics approach for elucidating host and virus interaction, 26 27 viral screening for development of therapeutics, 26 Virus-mediated plant transformation, 93 Vitamins, 31 32, 109 111, 243, 275

404

INDEX

Vitis vinifera, 148t Volatile organic compounds, 339 Volvariella volvacea, 64t, 317

W Waste biomass, 246 247 Water retention (WR), 374 Water use efficiency, enhancing biochar for, 374 Wave mediated transformation, 92, 138 Whole genome-sequencing strategies, 55, 253 254, 264, 319 320 Withaferin A, 148t Withania somnifera, 148t, 150 151 Withanolide A, 148t Withanone, 146 147, 148t Wollea saccata, 35 Wood-based biochar, 376t

X Xanthobacin A, 183t Xanthophyll biosynthesis, 147 X-gal, 240 X-ray crystallography, 4 5, 299 Xylanases, 243 245, 307, 309 310, 312 Xyloglucan endotransglycosylase, 243 244

Z Zeaxanthin epoxidase gene, 145 Zera, 77 78 Zinc finger nucleases (ZFNs), 258 259 Zinc oxide and sulfide NPs, 218 219 Zinc oxide nanoparticles, 207f, 208, 225t as synergic antimicrobials, 207 ZMapp, 81 82, 82t, 136t Zwittermycin A, 183t Zymomonas mobilis, 342