New and Future Developments in Microbial Biotechnology and Bioengineering: Microbial Secondary Metabolites Biochemistry and Applications 0444635041, 9780444635044

Table of contents :
Cover
New and Future Developments in Microbial Biotechnology and Bioengineering: Microbial Secondary Metabolites
Biochemistry and Applications
Copyright
List of Contributors
1 Wild Mushrooms as Functional Foods: The Significance of Inherent Perilous Metabolites
1.1 Introduction
1.2 Mushroom and Its Taxonomy
1.3 The Toxins and Their Perilous Connections
1.4 Prospects and Concerns in Terms of Human Health
1.5 Mushrooms as Dietary Supplements
1.6 Protein Composition
1.7 Lipid Composition
1.8 Carbohydrates and Fiber content
1.9 Mineral Composition
1.10 Conclusion
References
2 Genetic Manipulation of Secondary Metabolites Producers
2.1 Introduction
2.2 Genetic Engineering of the Secondary Metabolic Pathway in Plants
2.2.1 Flavonoids and Anthocyanins: Biosynthesis and Regulatory Genes
2.2.2 Alkaloids
2.2.2.1 Crystalline and Nitrogenous Compounds: Biogenesis and Regulatory Factors
2.2.2.2 Isoquinoline of Nitrogenous Organic Compounds of Plant
2.2.2.3 Tropane Alkaloids and Pyrrolidine Alkaloids
2.2.3 Terpenoids
2.2.3.1 The essential oils of plants and simple derivatives
2.2.3.2 Carotenoids
2.2.4 Carboxybenzene Formatives
2.2.4.1 Phytoanticipins (α-Hydroxynitrile-Type Aglycone and of a Sugar Moiety)
2.3 Secondary Metabolites in Actinomycetes by Metabolic Engineering
2.3.1 Precursor Engineering of Carbohydrate and Fatty Acid Metabolism
2.3.2 Technology Regulating Systems
2.3.3 Engineering Biosynthetic Structural Genes
2.4 The Aspergillus nidulans MAPK Module and Secondary Metabolism
2.4.1 Development Modular Controls
2.4.1.1 MAP Kinase Module for Secondary Metabolite Synthesis
2.5 Conclusions and Future Scope
Acknowledgment
References
3 Role of Rhizobacterial Secondary Metabolites in Crop Protection Against Agricultural Pests and Diseases
3.1 Introduction
3.2 Early Uses of Biocontrol Methods in Agriculture
3.3 Microbial Secondary Metabolites
3.4 Rhizobacterial Secondary Metabolites and Biological Control
3.4.1 Antibiotics
3.4.1.1 Water-Soluble Antibiotics
3.4.1.2 Microbial Volatile Organic Compounds
3.4.1.2.1 Antifungal Activity
3.4.1.2.2 Antibacterial Activity
3.4.1.2.3 Nematicidal Activity
3.4.1.2.4 Insecticidal Activity
3.4.2 Iron Sequestering
3.4.3 Chemical Communication Interference
3.4.4 Priming of Induced Systemic Resistance
3.5 Regulation of Secondary Metabolites’ Production
3.5.1 Regulation by Root Exudates Composition
3.5.2 Regulation by Microbial Signals (Quorum-Sensing Signals)
3.6 Microbial Metabolites and Biopesticides Development
3.7 Concluding Remarks
References
4 Bioengineering of Secondary Metabolites
4.1 Introduction
4.2 Gene Duplication in Idiophase
4.3 Evolution of New Pathways
4.4 Bioengineering of Terpenoids in Plants
4.5 Metabolic Engineering and Microbial Biogenesis of Plant Isoprenoids
4.6 Enzyme Engineering
4.7 Conclusion
Acknowledgment
References
Further Reading
5 Advances in Microbial Technology for Upscaling Sustainable Biofuel Production
5.1 Introduction
5.2 Biomass Feedstocks for Biofuels Production
5.3 Downside of First- and Second-Generation Biofuels
5.4 Metabolic Engineering and Biofuel Production
5.5 Metabolic Pathways for Alcohol-Derived Fuels
5.6 Metabolic Pathways for Isoprenoid-Derived Fuels
5.7 Metabolic Pathways for Fatty Acid-Derived Fuels
5.8 Synthetic Biology and Its Role in Design of Microbial Cell Factories
5.9 Engineering Microbes for Tolerance to Next-Generation Biofuels
5.10 Conclusion
References
6 Bioprospecting Actinobacteria for Bioactive Secondary Metabolites From Untapped Ecoregions of the Northwestern Himalayas
6.1 Introduction to Secondary Metabolites
6.2 Introduction to Actinobacteria
6.3 Distribution of Actinobacteria
6.4 Choice of Actinobacteria as Source of Bioactive Secondary Metabolites
6.5 Actinobacteria From Unusual Environments
6.6 Northwestern Himalayas as Sources of Bioactive Actinobacteria
6.7 Conclusion
Acknowledgment
References
7 Microbial Metabolites: Peptides of Diverse Structure and Function
7.1 Introduction
7.2 Antimicrobial Peptides
7.3 Classification of Microbial AMPs
7.3.1 Class I: Posttranslationally Modified Bacteriocins
7.3.1.1 Class Ia: The Lantibiotics
7.3.1.2 Class Ib: The Labyrinthopeptins
7.3.1.3 Class Ic: The Sactibiotics
7.3.2 Class II: Unmodified Bacteriocins
7.3.2.1 Class IIa: The Pediocin-Like Bacteriocins
7.3.2.2 Class IIb: The Two-Peptide Bacteriocins
7.3.2.3 Class IIc: The Circular Bacteriocins
7.3.2.4 Class IId: The Nonpediocin, Unmodified, Linear Bacteriocins
7.4 Mechanism of Action
7.5 Potential Applications of AMPs
7.6 Conclusion
References
Further Reading
8 Agrobacterium rhizogenes Mediated Hairy Root Cultures: A Promising Approach for Production of Useful Metabolites
8.1 Introduction
8.2 Agrobacterium and Ri T-DNA Genes
8.3 Role of rol Genes
8.4 Secondary Metabolite Production
8.5 Large-Scale Production of Hairy Roots
8.6 Liquid-Phase Bioreactors
8.6.1 Stirred Tank Reactor
8.6.2 Airlift Bioreactors
8.6.3 Bubble Column Reactor
8.6.4 Convective Flow Reactor
8.6.5 Turbine Blade Reactor
8.6.6 Rotating Drum Bioreactor
8.7 Gas Phase Bioreactors
8.8 Hybrid Bioreactors
8.9 Parameters That Affect Productivity
8.10 Conclusion and Future Prospects
References
9 Unleashing Extremophilic Metabolites and Its Industrial Perspectives
9.1 Introduction
9.2 Marine Microbial Metabolites Derived From Benthic Environment
9.3 Marine Sponge—Microbial Symbionts
9.4 Stromatolites: Potential Novel Source for Future Biotechnology
9.5 Polyhydroxyalkanoate-Producing Free-Living Marine Bacteria
9.5.1 Chemical Composition and Material Properties of Polyhydroxyalkanoates
9.6 Stress Acclimatization of PHA-Producing Bacteria
9.7 Production of Polyhydroxyalkanoate by Halophilic Bacteria
9.8 Role of PHA Synthase in Halophiles
9.9 Concluding Remarks
References
Further Reading
10 Hybrid Bioactive Products and Combinatorion Biosynthesis
10.1 Introduction
10.2 Need of Combinatorial Biosynthesis
10.3 Precursor-Directed Combinatorial Biosynthesis
10.4 Enzyme-Level Combinatorial Biosynthesis
10.4.1 Site-Specific Mutagenesis
10.4.2 Directed Evolution
10.5 Pathway-Level Combinatorial Biosynthesis
10.6 Conclusion
Acknowledgment
References
11 Rubromycins: A Class of Telomerase Inhibitor Antibiotics Produced by Streptomyces spp.
11.1 Introduction
11.2 Telomeres, Telomerase, and Cancer
11.3 Rubromycins: A Class of Molecules Telomerase Activity Inhibition
11.4 Mode of Action of Rubromycins Human Telomerase Inhibition
11.5 Streptomyces spp.: The Biofactories for Human Telomerase Inhibitors Production
11.6 Biosynthesis of Rubromycins
11.7 Bioprocess of Rubromycins Production
11.7.1 Future Perspectives
References
12 Regulation by Metal Ions
12.1 Introduction
12.2 Regulatory Mechanisms
12.3 Role of Specific Molecules in Controlling Biosynthetic Pathways
12.3.1 Metal Ions in the Synthesis of Antimicrobial Agents
12.4 Metal Ions in the Synthesis of Organic Acids
12.5 Metal Ions in the Synthesis of Siderophores
12.6 Metal Ions in the Synthesis of Microbial Pigments
12.7 Metal Ions in the Synthesis of Vascular Permeability Factor
12.8 Metal Ions in the Synthesis of Hydrogen Cyanide
12.9 Conclusion
References
13 Citric Acid Cycle Regulation: Back Bone for Secondary Metabolite Production
13.1 History
13.2 Citric Acid Cycle: Process and Regulation
13.2.1 Synthesis of Citrate
13.2.2 Isomerization of Citrate Into Isocitrate
13.2.3 Conversion of Isocitrate Into α-Ketoglutarate
13.2.4 Conversion of α-Ketoglutarate Into Succinyl CoA
13.2.5 Conversion of Succinyl CoA Into Succinate
13.2.6 Conversion of Succinate Into Fumarate
13.2.7 Conversion of Fumarate Into Malate
13.2.8 Conversion of Malate Into Oxaloacetate
13.2.9 Citric Acid Cycle Enzymes as Multienzyme Complex
13.3 Citric Acid Cycle as Biosynthetic Precursors
13.3.1 Anaplerosis and Cataplerosis for Citric Acid Cycle Regulation
13.3.2 Flux Modes of Citric Acid Cycle in Plants
13.3.3 Flux Modes of Citric Acid Cycle in Bacteria
13.4 Example of Synthesis of Metabolites Through Intermediates of Citric Acid Cycle
13.4.1 Enhancement in the Production of Cellulose
13.4.2 Enhancement of Fatty Acid Biosynthesis and Lipstatin Production
13.4.3 Synthesis of Amino Acid
13.4.4 Production of Citric Acid
13.4.5 Production Itaconic Acid
13.4.6 Production of Pyrimidines and Purines
13.4.7 Production of 1,4 Butanediol
13.5 Conclusion
References
14 Resistance in Pathogenic Microorganisms
14.1 Resistance in Bacteria
14.1.1 Biochemistry of Resistance in Bacteria
14.1.1.1 Antibiotic Inactivation
14.1.1.2 Target Modification
14.1.1.3 Alteration in Peptidoglycan Structure
14.1.1.4 Interference in Protein Synthesis
14.1.1.5 DNA Synthesis Interference
14.1.1.6 Efflux Pumps and Permeability of Outer Membrane
14.2 Resistance in Fungi
14.3 Antifungal Resistance From Environmental Origin
14.4 Resistance in Viruses
References
15 Hybrid Approach for Transformation for Betulin (an Anti-HIV Molecule)
15.1 Background of Betulin
15.2 Main Sources of Triterpenes
15.3 Applications
15.4 Value Addition Using the Hybrid Approach
15.5 Key Strategies for Adding Value
15.6 Hybrid Approach to Develop Betulin Derivatives
15.7 Issues in Chemical Synthesis
15.8 Conclusion
Acknowledgments
References
Further Reading
16 Producers of Bioactive Compounds
16.1 Introduction
16.2 Bioactive Compounds
16.3 Major Classes of Bioactive Compounds
16.4 Criteria for the Selection of an Ideal Bioactive Compound
16.5 Diverse Biological Activities of Bioactive Compounds
16.6 Sources of Bioactive Compounds
16.7 Plants as the Sources of Bioactive Compounds
16.8 Invertebrates as the Sources of Bioactive Compounds
16.9 Microbial Producers of Bioactive Compounds
16.10 Bacteria as Producers of Bioactive Compounds
16.11 Fungi as Producers of Bioactive Compounds
16.12 Algae as Producers of Bioactive Compounds
16.13 Conclusion
References
17 Bioremediation of Organic and Inorganic Pollutants Using Microalgae
17.1 Introduction
17.2 Inorganic Pollutants
17.2.1 Heavy Metals
17.2.2 Radioactive Compounds
17.3 Organic Pollutants
17.3.1 Petroleum Hydrocarbons and Polycyclic Aromatic Hydrocarbons
17.3.2 Fertilizers and Pesticides
17.3.3 Dimethyl Phthalate
17.3.4 Tributyltin
17.3.5 Explosives
17.4 Role of Biosurfactants in the Bioremediation
17.5 Emerging Pollutants
17.5.1 Polyfluorinated Compounds
17.5.2 Pharmaceutical Active Compounds
17.6 Conclusion
References
Further Reading
18 Secondary Metabolites From Endophytic Fungi and Their Biological Activities
18.1 Introduction
18.2 Endophytic Fungal Diversity
18.3 Secondary Metabolites
18.3.1 Antibacterials/Antimycobacterials From Endophytic Fungi
18.3.2 Antifungals From Endophytic Fungi
18.3.3 Anticancer, Immunosuppressive, and Antiinflammatory Activities of Endophytic Fungi
18.3.4 Antioxidants From Endophytic Fungi
18.3.5 Industrial Enzymes From Endophytic Fungi
18.4 Conclusions
References
19 Regulation and Role of Metal Ions in Secondary Metabolite Production by Microorganisms
19.1 Introduction
19.2 Manganese
19.3 Copper
19.4 Nickel
19.5 Calcium
19.6 Cadmium
19.7 Zinc
19.8 Cobalt
19.9 Iron
19.10 Rare-Earth Elements
19.11 Other Metals
19.12 Conclusion and Future Prospect
References
Further Reading
20 Metabolic Engineering to Synthetic Biology of Secondary Metabolites Production
20.1 Introduction
20.2 Secondary Metabolites-Producing Microbes
20.3 Discovery of Novel Microbes Producing Secondary Metabolites
20.4 The Functional Genomics of Secondary Metabolites-Producing Microbes
20.5 Biodiversity of Secondary Metabolites-Producing Microbes
20.5.1 Plant-Associated Microbiomes
20.5.2 Microbiomes From Extreme Habitats
20.5.2.1 Psychrophilic Microbes
20.5.2.2 Thermophilic Microbes
20.5.2.3 Halophilic Microbes
20.6 Distributions of Secondary Metabolites-Producing Microbes
20.6.1 Archaea
20.6.2 Bacteria
20.6.3 Fungi
20.7 Synthetic Biology for Secondary Metabolites Production
20.7.1 Anthraquinones
20.7.2 Quinones
20.7.3 Phenolic Compounds
20.7.4 Terpenoids
20.7.5 Polyketides and Lactones
20.7.6 Pyrones and Polyenes
20.7.7 Alkaloids
20.7.8 Immunosuppressive Compounds
20.8 Biotechnological Applications of Secondary Metabolites
20.9 Conclusion and Future Prospects
Acknowledgment
References
21 Microbial Enzymes as Control Agents of Diseases and Pests in Organic Agriculture
21.1 Introduction
21.2 Production of Microbial Enzymes
21.2.1 Phases of a Fermentative Process to Obtain Enzymes
21.2.1.1 Selection and Preparation of Inoculum
21.2.1.2 Fermentation Process
21.2.1.2.1 Solid-state fermentation
21.2.1.2.2 Submerged Fermentation
21.3 Enzyme Purification
21.4 Role of Enzymes in Inducing Plant Resistance to Insect Attack
21.5 Antioxidant Enzymes
21.6 Types of Enzymes and Their Application in Agriculture for Pest Control
21.6.1 β-1,3-Glucanase
21.6.2 Pectinase
21.6.3 Protease
21.6.4 Lipase
21.6.5 Chitinase
21.7 Final Conclusion
References
Further Reading
22 Secondary Metabolites: Metabolomics for Secondary Metabolites
22.1 Introduction
22.2 Secondary Metabolites and Synthetic Biology
22.3 Primary Metabolites
22.4 Secondary Metabolite
22.5 Genome and Genomics
22.5.1 Transcriptome and Transcriptomics
22.6 Proteome and Proteomics
22.6.1 Metabolome and Metabolomics
22.7 Synthetic Biology
22.8 Metabolomics and Synthetic Biology: How to Engineer the Microbes
22.9 Discovery of Secondary Metabolites: How to Discover Secondary Metabolites Through Metabolomics
22.10 Production of Secondary Metabolites
22.11 Role of Metabolomics in Identification of the Bottleneck in Engineered Pathway
22.12 Conclusion and Future Perspectives
Acknowledgments
Conflict of Interest
References
23 Solid-State Fermentation Strategy for Microbial Metabolites Production: An Overview
23.1 Introduction
23.2 History of SSF
23.3 Common Characteristics of Solid-State Fermentation
23.4 Analysis of Substrate Selection for Solid-State Fermentation
23.5 Microorganisms and Growth Kinetics for Solid-State Fermentation
23.6 Physicochemical Parameters for Solid-State Fermentation
23.7 Bioreactor for the Solid-State Fermentation
23.8 Conclusion
Acknowledgement
References
Index
Back Cover

Citation preview

NEW AND FUTURE DEVELOPMENTS IN MICROBIAL BIOTECHNOLOGY AND BIOENGINEERING

NEW AND FUTURE DEVELOPMENTS IN MICROBIAL BIOTECHNOLOGY AND BIOENGINEERING Microbial Secondary Metabolites Biochemistry and Applications Edited by

VIJAI KUMAR GUPTA ERA Chair of Green Chemistry, Department of Chemistry and Biotechnology, School of Science, Tallinn University of Technology, Tallinn, Estonia

ANITA PANDEY Center for Environmental Assessment & Climate Change, G.B. Pant National Institute of Himalayan Environment & Sustainable Development, (An Autonomous Institute of Ministry of Environment, Forest & Climate Change) Kosi-Katarmal, Almora, Uttarakhand, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2019 Elsevier B.V. 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: 978-0-444-63504-4 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Susan Dennis Acquisition Editor: Kostas Marinakis Editorial Project Manager: Kelsey Connors Production Project Manager: Poulouse Joseph Cover Designer: Greg Harris Typeset by MPS Limited, Chennai, India

List of Contributors Mohd. Aamir Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India Department of Life Science, Institute of Information Management & Technology, Aligarh, India

Mohd Musheer Altaf Eliana A. Alves

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Aakriti Bhandari Genetics and Tree Propagation Division, Forest Research Institute, Dehradun, India Microbial Biotech Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, India

Aasif Majeed Bhat

Madhusmita Borthakur Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India Ali Zineddine Boumehira Faculty of Science, University of Algiers, Algiers, Algeria; University of Sciences and Technology Houari Boumediene, FSB, LBCM, Algiers, Algeria Thiarles Brun Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil Vinay Singh Chauhan

Department of Biotechnology, Bundelkhand University, Jhansi, India

Eduardo J. Chica Faculty of Agricultural Sciences, University of Cuenca, Cuenca, Ecuador Balasubramanian Cibichakravarthy Coimbatore, India Ta´ssia C. Confortin

Molecular Microbiology Lab, Department of Biotechnology, Bharathiar University,

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Kashyap Kumar Dubey Bioprocess Engineering Laboratory, Department of Biotechnology, Central University of Haryana, Mahendergarh, Haryana, India Manish Kumar Dubey Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India Gaurav Raj Dwivedi Department & School of Environmental Sciences, Babasaheb Bhim Rao Ambedkar University, Lucknow, India; Microbiology Department, ICMR-Regional Medical Research Centre Bhubaneswar, Bhubaneswar, India Hesham A. El-Enshasy Institute of Bioproduct Development, Universiti Teknologi Malaysia (UTM), Skudai, Malaysia; City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt Vijai Kumar Gupta ERA Chair of Green Chemistry, Department of Chemistry and Biotechnology, School of Science, Tallinn University of Technology, Tallinn, Estonia Hocine Hace`ne Faculty of Science, University of Algiers, Algiers, Algeria; University of Sciences and Technology Houari Boumediene, FSB, LBCM, Algiers, Algeria Gopinath Halder

Department of Chemical Engineering, National Institute of Technology, Durgapur, India

Qazi Parvaiz Hassan Abd El-Latif Hesham Thingujam Indrama

Microbial Biotech Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, India Department of Genetics, Faculty of Agriculture, Assiut University, Assiut, Egypt Department of Biotechnology, Institute of Bioresources and Sustainable Development, Imphal, India

S.R. Joshi Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India Des Raj Kashyap Department of Microbiology and Immunology, Indiana University School of Medicine-NW, Gary, IN, United States Robinka Khajuria Department of Biotechnology, Harlal Institute of Management and Technology, Greater Noida, Uttar Pradesh, India Mohd Sajjad Ahmad Khan Department of Basic Sciences, Biology Unit, Health Track, Imam Abdulrahman Bin Faisal University, Dammam, Kingdom of Saudi Arabia Divjot Kour

Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib, Sirmour, India

Raquel C. Kuhn Anil Kumar India

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Department of Biotechnology, TIFAC-CORE Building, Thapar Institute of Engineering and Technology, Patiala,

ix

x

LIST OF CONTRIBUTORS

Department of Biochemistry, Faculty of Science, Veer Bahadur Singh Purvanchal University, Jaunpur, India

Arvind Kumar

Dhirendra Kumar Microbial Process Development Laboratory, University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana, India; Department of Botany, Chaudhary Bansi Lal University, Bhiwani, Haryana, India Punit Kumar Microbial Process Development Laboratory, University Institute of Engineering and Technology, Maharshi Dayanand University, Rohtak, Haryana, India Luciana Luft

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Marcio A. Mazutti

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Mukesh Meena Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India; Department of Botany, University College of Science, Mohanlal Sukhadia University, Udaipur, Rajasthan, India P.K. Mishra Department of Chemical Engineering and Technology, Indian Institute of Technology (BHU) Varanasi, Varanasi, India Madhumanti Mondal Department of Chemical Engineering, National Institute of Technology, Durgapur, India Ram Naraian Department of Biotechnology, Mushroom Training & Research Centre (MTRC), Faculty of Science, Veer Bahadur Singh Purvanchal University, Jaunpur, India Department of Biotechnology, Institute of Bioresources and Sustainable Development, Imphal, India

Gunapati Oinam

Solai Ramatchandirane Prabagaran Coimbatore, India

Molecular Microbiology Lab, Department of Biotechnology, Bharathiar University,

P.W Ramteke Department of Biological Sciences, Sam Higginbottom University of Agriculture Technology & Sciences (Formerly Allahabad Agricultural Institute), Allahabad, India Kusam Lata Rana

Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib, Sirmour, India

Ali Asghar Rastegari Iran

Department of Molecular and Cell Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan,

Manoj Raturi Genetics and Tree Propagation Division, Forest Research Institute, Dehradun, India Balwant Rawat

Department of Agriculture and Forestry, Graphic Era Hill University, Dehradun, India

Janhvi Mishra Rawat Botany Division, Forest Research Institute, Dehradun, India M. Sudhakara Reddy Department of Biotechnology, TIFAC-CORE Building, Thapar Institute of Engineering and Technology, Patiala, India Aabid Manzoor Shah

Microbial Biotech Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, India

Shachi Shah Environmental Studies, School of Interdisciplinary and Transdisciplinary Studies, Indira Gandhi National Open University, New Delhi, India Shikha

Department & School of Environmental Sciences, Babasaheb Bhim Rao Ambedkar University, Lucknow, India

Saba Siddiqui

Integral University, Lucknow, India

Bhanumati Singh Shalini Singh

Department of Biotechnology, Bundelkhand University, Jhansi, India

School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, India

Brijesh Singh Sisodia Department of Biochemistry, Regional Ayurveda Institute of Fundamental Research, (Under CCRAS, Ministry of AYUSH, Govt. of India), Pune, India; Biochemistry laboratory, Regional Ayurveda Research Institute for Drug Development, Gwalior, Madhya Pradesh, India Stefani S. Spannemberg Manish Srivastava

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Department of Physics & Astrophysics, University of Delhi, New Delhi, India

Neha Srivastava Department of Chemical Engineering and Technology, Indian Institute of Technology (BHU) Varanasi, Varanasi, India Nazanin Tataei Sarshari Isfahan, Iran

Department of Molecular and Cell Biochemistry, Falavarjan Branch, Islamic Azad University,

Onkar Nath Tiwari Centre for Conservation and Utilization of Blue Green Algae, Division of Microbiology, Indian Agricultural Research Institute, New Delhi, India Izelmar Todero

Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

Ram Sanmukh Upadhyay Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India

LIST OF CONTRIBUTORS

M. Vasundhara Patiala, India

xi

Department of Biotechnology, TIFAC-CORE Building, Thapar Institute of Engineering and Technology,

Siddarthan Venkatachalam Marine Natural Products Research Lab, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa; Arctic Division, National Centre for Polar and Ocean Research, Vasco-Da-Gama, Goa, India V. Venkatramanan Environmental Studies, School of Interdisciplinary and Transdisciplinary Studies, Indira Gandhi National Open University, New Delhi, India Ajar Nath Yadav Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib, Sirmour, India Neelam Yadav Gopi Nath P.G. College, VBSP University, Deoli-Salamatpur, Ghazipur, Uttar Pradesh, India Luis Andre´s Yarza´bal Unit of Health and Wellbeing, Catholic University of Cuenca, Cuenca, Ecuador; School of Biology, Faculty of Sciences, University of Los Andes, Me´rida, Venezuela Andleeb Zehra Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India

C H A P T E R

1 Wild Mushrooms as Functional Foods: The Significance of Inherent Perilous Metabolites Madhusmita Borthakur and S.R. Joshi Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya, India O U T L I N E 1.1 Introduction

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1.7 Lipid Composition

8

1.2 Mushroom and Its Taxonomy

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1.8 Carbohydrates and Fiber content

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1.3 The Toxins and Their Perilous Connections

3

1.9 Mineral Composition

9

1.4 Prospects and Concerns in Terms of Human Health

4

1.5 Mushrooms as Dietary Supplements

5

1.6 Protein Composition

7

1.10 Conclusion References

9 10

1.1 INTRODUCTION There is geological evidence of the existence of mushrooms from the fossil record of the lower Cretaceous period about 130 million years ago [1]. Anthropological observations provide evidence of the use of mushrooms as a source of food or medicines by hunters or by food gatherers. Mushrooms were named “the plant of immortality” by the ancient Egyptians, some 4600 years ago, as it was a delicious cuisine favored by the royals. The first ever report of its consumption as a royal dish was written by a Greek philosopher, Theophrastus, during the period 372 287 BC. Mushrooms are widely accepted as a palatable food since ancient time in various countries including Greece, Russia, China, Mexico and Latin America. Mushrooms arrived in India through the northwest via Afghanistan to enter into the famous civilization, the Indus Valley [2]. Aryans throughout the Indus civilization put forward mushrooms as the plants in “Rig Veda” with hallucinogenic properties and they were used in various religious rituals. In ancient era, poisonous mushrooms were known to be as “soma”, where the tribe of the Indus valley was seen in harvesting and selling a poisonous Amanita muscaria for various ritutals [3]. The extract from poisonous mushrooms also known to be as “Somarasa” was used for various traditional rituals to induce the immune system in an immuno-compromised individual and also helped in getting the lost. This was somewhat responsible for the early ruin of the civilization. Mushrooms was used as hallucinogen and also for black magic in the ancient time due to its sudden appearance after rain without proper bud or fruiting body. The word mushroom was named after the cultivation of the common button mushroom, Agaricus bisporus, with a stem (stalk), an umbrella shape cap (pileus), and gills (lamellae) underneath the cap. The term “macrofungi” was first coined for Basidiomycota and Agaricomycota with a defined fruiting body that can be either New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: https://doi.org/10.1016/B978-0-444-63504-4.00001-3

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© 2019 Elsevier B.V. All rights reserved.

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1. WILD MUSHROOMS AS FUNCTIONAL FOODS: THE SIGNIFICANCE OF INHERENT PERILOUS METABOLITES

epigeous or hypogeous and is easily visible to the naked eye [4]. Being diverse, the Chinese refer to mushrooms as the “King of the Planet,” while the Japanese refer to the same as “The Diamond of the Forest.” Fungi has gained its importance in history by the documentation of dessert truffle (Terfezia arnenari) in the Bible as the “Bread of Heaven” and also as the “Manna of the Israelites” [5]. Formitopsis officinalis, a brown rotten fungus, is known as the “Bread of Ghosts” by the indigenous people of the Pacific Northwest and use it to mark the grave of Shamen and are known to treat illnesses caused by supernatural powers. They believe in mushroom sporophore for its spirit catching abilities. Apart from its spiritual beliefs, few mushrooms like Exobasidium vaccinii (Ears of Ghost) are known to infect stem, leaves, and flowers of Ericaceous plant and subsequently form basidia covered gills which are consumed by the indigenous coastal groups of Pacific Northwest who considered them to be berries [6]. Burk [7] reported the spiritual and religious use of puffball mushrooms by the people of North America who believed that they have the ability to ward off ghosts. In Asia the mycophilic societies are associated with the indigenous people of Northeast India, Western Ghats, and Northwest India and China. The local inhabitants collect mushrooms from their neighboring localities, meadows, and forests for consumption and sell them to earn revenue for their family during the monsoon season when other forest nonwood products are unavailable in the market. Among the various species of mushrooms which are commonly consumed by the people are Termitophilous spp., which include T. microcarpus, T. aurantiacus, T. eurhizus, T. clypeatus, and T. Tyleranus [8]. A few indigenous tribes of Northeast India, such as Khasis of Meghalaya, use a traditional technique called “narsuh” for cooking mushrooms, where they heat the tip of a small iron rod and place it in the middle of the bowl containing a cooked mushroom. They believe that the heat released from the tip is responsible for destroying or absorbing the poisonous harmful substances from the mushrooms [9]. The fruiting bodies are washed and boiled with a few cloves of garlic. If the cloves of garlic turn black in color, they believe that the mushrooms are poisonous in nature, otherwise they are safe to consume. With regards to their toxicity, there is a history of relevant practical observations relating to the poisonous scenarios of mushrooms. The perilous nature of mushrooms is observed in various parts of India, most prevalently in the North-Eastern zone where the majority of the population depends on forest products during the monsoon and postmonsoon seasons. There have been tragedies of mushroom poisoning frequently reported and a few instances in 2016 were reported, such as on April 6, 8, and 13 where the death toll rose to more than 10 after the consumption of wild mushrooms in Mawsawa village of Mawsynram and Nongpriang of Sohra, Meghalaya, India [10]. On April 22, 2016, a death toll of six from mushroom poisoning in Rongdong village of Siju in Garo Hill district of Meghalaya, India was reported [11]. The toxic components are known to be present in the fruiting caps of mushrooms [12]. Keeping in mind their poisonous nature, mushroom species are known also to accumulate nonpoisonous diverse secondary metabolites, including polyphenols, alkaloids, terpenes, flavonoid, phenols, and steroids. It has been estimated that there are more than 140,000 mushrooms with so far only 10% being known [4,13]. Being rich in protein content, they can be used as a source of protein supplement to reduce the gap of malnutrition in developing countries and are cultivated worldwide. Europeans are known to have cultivated mushrooms in caves during the 16th and 17th centuries and the Chinese were known to artificially cultivate the mushrooms a few thousand years ago. The cultivation of mushrooms in India was first started in 1940 by Su and Seth [14]. Mushrooms are known to convert the agricultural and forest compost into a useful composting bed and reduce the level of xenobiotics in the environment.

1.2 MUSHROOM AND ITS TAXONOMY The preliminary identification of mushrooms to distinguish different strains is based on both morphological features during field observation and by the microscopic features of their spores, pileus, stipe, volva (if present), and host. But the study of morphological and microscopic features has left many unsolved dilemmas because of the paucity of morphological features together with the absence of phylogenic analysis [15]. Epigenetic factors have made the morphological features unstable both intra- and interspecies making it highly incongruent with the molecular data, thus making the latter the confirmatory identification [16]. The upgrading of various statistical methods along with bioinformatics tools has made the evaluation of the evolutionary clade of a species easier. The molecular approach has been attempted by amplification of specific hypervariable gene loci by polymerase chain reaction (PCR) or by restriction digestion of a specific gene sequence using restriction fragment length polymorphism. The structural gene sequence rRNA is known for its well-conserved regions at either genus or species

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1.3 THE TOXINS AND THEIR PERILOUS CONNECTIONS

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level. So phylogenetic analysis for mushroom is based on the polymerase amplification of a conserved ITS region which consists of ITS1-5.8S-ITS2 located between the rRNA gene of the rDNA cistron [17 20]. Along with the nuclear gene, certain protein genes (tef1, rpb1, rpb2) have also been considered as powerful tools in the study of the evolutionary traits of the fungi [21,22]. The sequence of a few conserved ribosomal DNA (nSSU and nLSUr DNA) and mtDNA (COI) segments are also being used in the analysis of fungal systematic studies. An arbitrary marker-based DNA probe technique is also advantageous to identify mushrooms, whereby the polymerized amplicons are revealed by electrophoresis and are characterized by direct sequencing. But a barcode to identify the poisonous strains from their edible counterparts is yet to be analyzed and developed for rapid identification of the varieties. Few mushrooms might be morphological alike but one might possess the toxic compound while other doesnot. The ethnic communities are often seen with a fallacy to characterise the mushrooms based on their palynological, sporological and morphological trait which are inadequate. But a genetic mushroom barcode is yet to be identified which can be used as a molecular tool to identify the nature of the toxicity of a mushroom. A few mushrooms in the genus Psilocybe, that is, P. semilanceata and P. cubensis, are known to produce hallucinogenic effects while the morphologically similar P. merdaria and P. montana in the same genus do not produce such metabolites [23]. A few of the highly fatal mushrooms from the genus Lepiota are morphologically similar to the edible mushroom Macrolepiota. The mushroom toxins are diverse and are categorized as amanitoxins, phallotoxins, monomethylhydrazine, orellanine, muscarine, ibotenic acid, muscimol, coprine, psilocybin, and psilocin [24]. The nature of the toxicity of a single species also depends on the habitat and various epigenetic factors, the preparation and consumption techniques, or genetic resistivity among the ethnic tribe. For example, Gomphus floccosus, a poisonous mushroom, is well known for causing a severe gastrointestinal disorder in the United States but is known as being edible among the ethnic groups of Meghalaya, India without any such effects. Rapid molecular tools along with the PCR amplification of ITS1 locus would differentiate the hallucinogenic mushrooms Panaeolus and Psilocybe based on their polymorphic length of the region [19,20]. A contradictory observation to the above study was reported by Nugent and Saville [25], where a few hallucinogenic mushrooms of North American species were examined based on PCR amplification of both the ITS region of rDNA (ITS1) and a large ribosomal subunit of the nucleolar RNA (nLSUrRNA). They concluded that the highly polymorphic ITS span does not match with the morphological feature but nLSUrRNA forms clusters and differentiates the hallucinogenic from nonhallucinogenic isolates of distant clades. A highly toxic Amanita mushroom encodes α-amanitin, an amatoxin (AMA1), and phallacidin, a phallotoxin (PHA1), which are known to be synthesized on ribosomes and can be studied by designing region-specific primers [26]. In a study to get a standard barcode for poisonous mushrooms, Qing et al. [27]. reported the use of three markers—the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1 alpha (tef1α)—in a poisonous Amanita sp. of China and concluded that tef1α and nLSU can be proposed as barcodes for the poisonous Amanita, while ITS can be used as a primary barcode for the identification of mushrooms without differentiating the poisonous clade from the edible one.

1.3 THE TOXINS AND THEIR PERILOUS CONNECTIONS Toxicity from mushrooms generally occurs after the ingestion of a poisonous variety which is often misidentified as an edible variety by amateur mushroom hunters. The severity of the poisoning depends on various factors with the concentration of the toxin being the most severe one. Geographical demography, the amount consumed, habitat, growth conditions, and the genetic constituents of the individual, including the age, are mostly the relevant factors for mushroom toxicity. It is often seen that the mushroom ingestion results in higher severity in an older person than that of children or healthy young adults. Apart from these, other factors related to mushroom toxicity are the cooking techniques and method of consumption. Often it is observed that consumption of mushrooms with alcohol causes a higher incidence of poisoning. The most common example is the ink cap mushroom, Coprinopsis atramentaria. When consumed along with alcohol, a poisonous mushroom results in “disulfiram” syndrome and later leads to a myocardial attack. Coprinopsis atramentaria is known to secrete a cyclopropyl glutamine compound, coprine, and an active metabolite, 1-aminocyclopropanol, which blocks the enzyme acetaldehyde dehydrogenase responsible for the breakdown of acetaldehyde, an intermediate of alcohol in the body [28,29]. The toxins in mushrooms are generalized based on the targeted organ specificity, mushroom physiology where geographical habitat is also considered as a variant, the amount of toxin consumed, the season, and the time from

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1. WILD MUSHROOMS AS FUNCTIONAL FOODS: THE SIGNIFICANCE OF INHERENT PERILOUS METABOLITES

ingestion to the onset of symptoms. The most lethal among all the mushroom toxins is a cyclopeptide, the amanitin. There are four amanotoxins, that is, α-amanitin, β-amanitin, γ-amanitin, and ε-amanitin, the most lethal being the α-amanitin found in the death cap of several Amanita sp., Galerina sp., and Lepiota sp. α-Amanitin is known to inhibit RNA polymerase II and protein synthesis. Mostly present in the death cap of Amanitus sp., it gets easily absorbed in the gastrointestinal tract and causes severe gastrointestinal disorders including nausea, vomiting, diarrhea, and hepatocellular disorders. Apart from the common lethal toxin amanitin, gyromitrin (a monomethylhydrazine), orellanine, muscarine, coprin, ibotenic acid, and myotoxin are also considered lethal based on the amount consumed. Gyromitrin is an unstable volatile hydrazine compound which breaks down to form monomethylhydrazine which is a carcinogen. A few of the Gyromitra sp. are edible and can lead to severe fatal symptoms by the inhalation of the toxic compound while cooking [30]. It is also involved in certain hepatocellular damage including hepatic necrosis and jaundice. The next of the mushroom toxins is Orellanine from the Cortinariaceae family. The first epidemiology from Orellanine was observed in Poland during the 1950s where more than 100 people became ill due to the consumption of Cortinarius orellanus [31]. Orellanine mainly interferes with the renal system and causes fatty liver and various other nephritic disorders. Symptoms do not appear immediately after consumption. It depends on the individual genetic resistivity which can last from 2 3 days to 3 weeks and includes symptoms like flu, nausea, vomiting, and headache leading to renal failure [32]. Inocybe and Clitocybe sp. are known to produce a toxic compound muscarine which is also found in trace amounts in Boletus sp., Lactarius sp., and Russula sp. Muscarine is known to mimic acetylcholine and binds to the neurotransmitter acetylcholine receptor and interacts with G protein to inhibit adenylcyclase and decrease cyclic adenosine monophosphate (cAMP). Symptoms of this toxin involve excessive sweating, diarrhea, miosis, abdominal cramps, and salivation which are seen within an hour of consumption and last for up to 24 hours. The muscarine producing mushrooms include Clitocybe dealbata, which is often misidentified with an edible Marasmius oreadus. An agonist to glutamate is the ibotenate or the ibotenic acid which is a common toxin in Amanita sp. It mostly affects the nervous system and effects are observed within 30 180 minutes of consumption. Charcoal treatment, along with the drug atropine, is often administered to the patient after ibotenate consumption [33]. A hallucinogenic compound psilocybin in the genus Psilocybe is converted to psilocin in the body a few hours after consumption and results in the alteration of the mind, including suicidal thoughts, psychosis, and convulsions [34 37]. Apart from these, a few toxins like bolesatine and arabitol (sugar alcohol) are nonlethal and cause less severe abdominal disorders and nausea. These are found in an edible mushroom, Oyster. Its adverse effects depend on the age of and genetic variability among the consumers. Out of 283 species of mushrooms, 100 are known to be highly poisonous and are often accidentally consumed [38]. Ninety-five percent of mushroom poisoning is due to the misidentification of local poisonous ones which morphologically mimic an edible variety. During the onset of the monsoon when the mushroom species tend to produce a fruiting body, accidental mushroom poisoning is often a common phenomenon. Mushrooms are often gathered based on traditional ethnomycological knowledge which is often propounding a myth. There is a need to educate the masses and for research to have a greater emphasis on the local variety of the mushroom to discriminate the toxic ones from the edible ones.

1.4 PROSPECTS AND CONCERNS IN TERMS OF HUMAN HEALTH India is a diverse agroclimatic land with an increasing population and agronomic waste, and thus the demand for a chemical-free functional food with potential health benefits is at a peak. Mushroom production is globally acceptable to consumers, along with environmental sustainability, as it doesn’t compete for farmland since it uses agricultural waste as its bed to grow. It induces diversification of the agronomic farm as it promotes recycling of the agrowaste including periodic industrial waste. Mushroom supplementation to the diet can bridge protein malnutrition and can improve the socioeconomic conditions of the population. Edible mushrooms usually have a significantly higher rate of crude protein, digestible carbohydrate, dietary fibers which act as prebiotics to enhance the growth of beneficiary microbes in the colon, vitamins, and minerals with a trace amount of saturated lipids. Being rich in antioxidant, mushrooms are said to boost immunity, inhibit the growth of tumors, lower the risk of cancer, detoxify the body, and reduce inflammation. The common oyster mushroom is known to decrease the level of cholesterol. Mushrooms such as the button mushroom, A. bisporus, mostly possess ergosterol instead of cholesterol, which in the presence of sunlight breaks apart to form vitamin D2. Mushrooms are considered to be an important source of vitamin C and vitamin B. Mushrooms have been used as folk

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1.5 MUSHROOMS AS DIETARY SUPPLEMENTS

medicines for thousands of years. They are rich in active polysaccharides (β-glucans), which upregulate the immune system and act as scavengers of free radicals. The presence of polysaccharides also makes them a potent anticancerous drug. Phenolics in the forms of flavonoids, phenolic acid, lignans, tannins, and terpenoids are some of the major components of mushroom metabolites exhibiting antioxidant activities. Phenolic compounds are known as free radical inhibitors; oxygen scavengers which inhibit the lipid oxidation in vivo [39]. The presence of a few secondary metabolites in mushrooms, including terpenes, alkaloids, steroids, and quinolones, makes them a target source for novel antimicrobial compounds. The traditional utilization of mushrooms is seen among the ethnic tribes of South Asian countries, through their antineoplastics and immunoregulatory properties and they are considered as minipharmaceutical factories [40]. Fleshy fruiting body mushrooms can be used as a potent source of biopharmaceutical products due to their excessive polysaccharides and triterpenoids. For the developing countries, they can serve as a good substitute for protein malnutrition [41]. A study by Chang [42] reported that 2000 mushrooms are edible, of which 20 are commercially cultivated and four to five are industrially cultivated.

1.5 MUSHROOMS AS DIETARY SUPPLEMENTS Mushrooms have been used as a food item since time immemorial [43]. Mushrooms are considered as the world’s largest untapped resources of dietary supplements (Tables 1.1 and 1.2). Their consumption has increased due to the presence of high amounts of protein content and trace minerals [51,52]. In comparison with other eatables, including fruit, vegetables, and legumes, mushrooms or basidiomycetes can prove to be better as a rich source of certain dietary factors that can be beneficial to human health and growth [53]. They are considered as a healthy food containing essential fatty acids and being low in the number of calories and high in the concentration of protein, fats, and minerals [54]. As wild mushrooms are considered to have greater protein content than commercially available mushrooms, several reports have revealed that they are used in the diet to combat various diseases [1]. For a developing country like India, they can serve as a good substitute for protein malnutrition [41]. Mushrooms with fleshy fruiting bodies can be used as a good source of biopharmaceutical products as they contain polysaccharides and triterpenoids [43]. Various studies have shown that after cooking, the nutritional value was less in dried sample as compared to the freshly collected mushroom samples [55]. In general, mushrooms contain 90% water and 10% dry weight [56]. In a study by Orgundana and Fagade [57], mushrooms were reported to contain about 16.5% dry matter, the approximate composition of the crude protein is 14.6%, crude fiber is 7.4%, and composition of fat and oil is around 4.48%. Though mushrooms are commercially used as a source of nutritional supplement, they have an adverse effect on the health of humans as they are also considered as a source of human poisoning and are associated with various carcinogeneses in animal experiments [53]. Among the various higher species of basidiomycetes, Agaricaceae are widely consumed worldwide. Many polysaccharides and protein-based polysaccharides isolated from Agaricaceae have antitumor activity and specific or nonspecific immune response activity [55,58 60]. The

TABLE 1.1

Essential Amino Acid per 100 g of Proteins From Different Sources

Amino acid

Mycoprotein (g)

Egg (g)

Milk (g)

Wheat (g)

Reference

Histidine

0.35 2.1

0.3 2.1

0.09

0.32

[44]; http://www.mycoprotein.org

Lysine

0.83 5.0

0.9 7.2

0.26

0.30

[44]; http://www.mycoprotein.org

Tryptophan

0.16 0.9

0.16 1.5

0.05

0.18

[44]; http://www.mycoprotein.org

Phenylalanine

0.49 2.0

0.66 6.3

0.16

0.68

[44]; http://www.mycoprotein.org

Methionine

0.21 1.26

0.39 4.1

0.08

0.22

[44]; http://www.mycoprotein.org

Threonine

0.55 4.2

0.6 4.9

0.15

0.37

[44]; http://www.mycoprotein.org

Leucine

0.86 4.4

1.1 9.2

0.32

0.93

[44]; http://www.mycoprotein.org

Isoleucine

0.52 5.8

0.68 8.0

0.20

0.53

[44]; http://www.mycoprotein.org

Valine

0.62 4.65

0.76 7.3

0.22

0.59

[44]; http://www.mycoprotein.org

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1. WILD MUSHROOMS AS FUNCTIONAL FOODS: THE SIGNIFICANCE OF INHERENT PERILOUS METABOLITES

TABLE 1.2 Nutritional Composition in Mushroom Fruiting Body g/100 g (% of Dry Matter) Species

Crude protein

Lipid

Carbohydrates

Fiber

Reference

Agaricus bisporus

26.3 56.3

1.8 2.7

37.5 59.96

[45,46]

Agaricus arvensis

56.3

2.7

37.5

[46]

Amanita rubescens

26 31.9

7.2 27.5

30.6 62.2

[46 48]

Armillaria mellea

21.9

1.8 6.08

70

[46,47]

Auricularia polytrica

7

70.8

87.6

[45]

Boletus aereus

26.9

2.13

4

17.0

[49]

Boletus edulis

26.5 28.7

2.8 4.1

30.6 65.4

15.3

[46,49]

Bolusanthus speciosus

28.1

2.9

28.6

21.0

[49]

Cantharellus cibarius

34.17 53.7

1.40 2.9

31.9 57

Craterellus. aureus

14.1

4.0

61.5

C. cornucopioides

50.10

5.89

34

[47]

Hydnum repandum

34.14

8.80

55

[47]

Lactarius deliciosus

20.2 29.8

2.2 4.0

5.1 25

36.8

[45,46]

Lactarius hatsudake

15.3

1.0

38.2

31.8

[45]

Lactarius volemus

17.6 87.5

73.98 6.7

15 64

40.0

[45,47]

Lentinula edodes

17.1

1.9

30.2

39.4

[45]

Lentinula modes

17.5

8.0

67.5

[49]

Lepista nuda

19.8 59.4

1.8 9

20.3 71.0

[46]

Lycoperdon perlatum

17.2 44.93

0.4 10.5

8 42 50.4

[46,47]

Leccinellum crocipodium

29.3

1.0

12.8

Macrolepiota procera

23.9

2.3

68.4

[46]

Pleurotus ostreatus

10.5

1.6

81.8

[45]

Ramaria botrytis

39.0

1.4

50.8

[46]

Ramaria flava

35 55

5.20

65

[48]

Russula virescens

28.3

1.5

13.4

Sarcodon imbricatus

27.45

8.85

57

[47]

Suillus granulates

16.5

4.0

74.3

[46]

Sarcodon aspratus

12.0

2.8

64.6

Tricholoma flavovirens

18.1

2.0

37.0

Tricholoma portentosum

19.6 30.5

5.5 5.8

34.6

30.1

[50]

Tricholoma terreum

20.1

6.6

31.1

30.1

[50]

Tricholoma matsutake

14.3

5.0

36.7

29.1

[49]

Volvariella volvacea

29.3

5.7

60.0

[46,47] 5.2

37.9

32.8

5.1

[49]

[49]

[49]

[49] [46]

[45]

dietary fiber content of mushrooms is due to the polysaccharides and triterpenoids contained in them. The cell walls of mushroom contain a mixture of fibrillar and matrix components which comprise chitin, the polymer of β 1 4 linkages N-acetyl-glucosamine and polysaccharides of 1 3 linkage of β-D-glucans and mannans, respectively. Mushrooms bear mainly water-insoluble dietary fibers which include both chitin and β-glucans. Under unfavorable environmental conditions, mushrooms regenerate special structures known as sclerotia, which are compact mycelium structures bearing chitin and β-glucans with β 1 3 backbones and 1 6 linked

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1.6 PROTEIN COMPOSITION

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side branches, and due to this they are considered to be a novel source of this dietary component as human enzymes cannot digest them [43].

1.6 PROTEIN COMPOSITION Mushrooms are rich in protein components comprising mostly the essential amino acid components lysine, histidine, arginine, threonine, and tryptophan, which are commonly not present in cereals. The component of lysine has been reported as a most abundant essential amino acid and methionine and tryptophan are the least abundant amino acids. The content of free amino acid is low, comprising only 1% of the dry weight of mushroom, thus limiting the nutritional level but participating in the taste of the mushroom [61]. The protein component of a mushroom depends on its growth substratum, the level of nitrogen, its location, the size of the pileus, and varies among species and with its harvesting time [62]. Immature primordia contain greater amounts of protein than mature primordia, as reported in A. bisporus and Pleurotus spp. [63]. The composition does not change during oven drying of mushrooms to 40 C but a significant reduction in the composition can be visualized upon boiling of fresh mushroom [61]. Protein production efficiency for mushrooms is about twice that of cabbage and asparagus, 4 times that of orange, and 12 times that of apples [42]. On the basis of dry weight, mushrooms contain 65% protein as compared to 25.2% in milk, 20% for pork meat, 4% for beef, 39.1% in soybean, 7.3% in rice, and 13.2% in wheat [42,64]. An early study by Rose [44] compared the presence of all essential amino acids for the growth of mammalian cells, and revealed that the composition of amino acids in mushrooms is adequate except for phenylalanine and methionine. The palatable taste of the mushroom is enhanced by the presence of aspartic and glutamic acid which are monosodium glutamate-like components [65]. It has been reported by Bauer Petrovska [66] that the mean level of protein fractions such as albumins, prolamines, globulins, glutenins, glutenin-like material, and prolamine-like material are likely to be 24.8%, 5.7%, 11.5%, 11.5%, 7.4%, and 5.3%, respectively. Proteins and peptides produced by mushrooms possess interesting biological activities, such as lectin, an antimicrobial protein, laccases, ribonucleases, ribosome inactivating protein, and fungal immunomodulatory protein [67]. A wide range of bioactive compounds has been isolated from mushrooms which include protein—polysaccharide complexes, proteins, polysaccharides, etc. [47,48]. Among these, the most specific is lectin protein, which is a glycoprotein that can bind to cell surface carbohydrates with the ability of cell agglutination. They exhibit antiproliferative activity toward tumor cell lines, specifically to human leukemic T cells, breast cancer MCF7 cell, and hepatoma HepG2 cells, and can recognize human blood group A determinant carbohydrates [67]. The sacred mushroom Reishi (Ganoderma sp.) contains a polysaccharide, β-glucan, which can stimulate or modulate the level of the immune system by activating T cells and macrophages, as well as activating immunoglobin levels which can combat and respond against foreign cells [49]. The crystal structure of lectin protein was first determined by Cioci et al. [50] as a regular seven-bladed beta-propeler fold with an N-terminal region being stuck into the central cavity around a pseudo sevenfold axis. Ribosome inactivating protein enzymes which eliminate one or more adenosine residues from rRNA inactivate ribosomes and inhibit the proliferation of HIV-1 reverse transcriptase activity [68]. Another isolated bioactive compound from mushrooms is laccase. This is a multicopper oxidase which has been implicated in inhibiting HIV-1 reverse transcriptase, and the proliferation of hepatoma HepG2 cells and MCF7 tumor cells [69]. Fungal immunomodulating proteins were isolated from various mushrooms, including Ganoderma sp., Russula paludosa, Pleurotus citrinopileatus, Grifola frondosa, and Antrodia camphorate [70 77], with various immunomodulating, antitumor, and antiviral activities. A novel Se-containing protein Se-GL-P (36 kDa) isolated from Ganoderma lucidum using ammonium sulfate precipitation indicated the incorporation of selenium in the protein in the form of selenocysteine and selenomethionine and had a positive response toward inhibiting the multiplication of tumor cells [70]. Various other proteins have been isolated, including trichogin protein from Tricholoma giganteum with antifungal activity against Mycospaerella arachidicola and Fusarium oxysporum [78]. A report by Chang et al. [79] assessed the utilization of two proteins, Agaricus bisporus lectin from A. bisporus and immunomodulating Agaricus polytricha protein from Auricularia polytricha, that showed good stability after thermal, freezing, acid, alkali, and dehydration treatments, thus indicating the proteins to be stable immunostimulants for food, health, and pharmaceutical applications [79]. The 14-3-3 proteins, a family of conserved regulators (first named in 1967 based on its fraction numbers in DEAE cellulose chromatography and its position after gel electrophoresis with a molecular mass of 28.8 kDa), can act as potential candidates

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1. WILD MUSHROOMS AS FUNCTIONAL FOODS: THE SIGNIFICANCE OF INHERENT PERILOUS METABOLITES

for a phylogenetic relationship because of their highly conserved sequences with similar acidic pIs and can take part in various biological processes with fewer deletions [80]. A few were isolated from mushrooms like Sparassia crispa and Hericium erinaceum.

1.7 LIPID COMPOSITION Fatty acids are considered to play a major role in the function of the immune system and in balancing of hormonal levels [81]. In basidiomycetes, the lipid content ranges from 0.6% to 18.4% (w/w) [82]. The fatty acid profile of mushrooms is relatively higher in basidiomycetes, ranging from saturated palmitic acid (16:0) to oleic acid (18:1, Δ9c) and linoleic acid (18:2, Δ9c, 12c), which are present in the membrane of basidiomycetes [82]. Mushrooms are mostly dominated by the presence of unsaturated fatty acids. However, a low proportion of oleic acid is characterized by the mushroom species Agaricus spp. and Cantharellus cibarius [61]. Boletus species are known for their higher concentrations of monoenic acid. The concentrations of oleic acid are higher in Boletus edulis, Boletus piperatus, Boletus subglabripes, Boletus erythropus, Boletus subtomentosus, and Boletus variipes, as reported by Hanus et al. [81]. From the abovementioned Boletus species, the polar lipids, including phospholipids and betaine, showed diacylglyceryltrimethylhomoserine and phosphatidylcholine as major polar lipids of varying concentrations from 72% to 93% of the total amount of polar lipids. Neutral lipid content ranges from 0.7% to 9.4% and the range of polar lipid content in the case of Agaricus sp. ranges from 2.4% to 11.8% [82]. The number of polar lipids accounts for more than 50% of total lipids. Unsaturated fatty acids account for an average of 74.4% of total fatty acids. The ratios of unsaturated to saturated fatty acid (U:S) ranges from 1.61 for Lycoperdon pyriforme to 5.36 for B. edulis. Davidoff and Kom [83] reported the presence of isomer cis-11 of heptadecenoic acid in the basidiomycetes. Elaidic acid (18:1, D9t), a common fatty acid in milk fat and in the tissues of ruminant animals and occasionally in seed oils are found in a few basidiomycetes as reported by Pfeuffer and Schrezenmeir [84]. From the human nutritional point, long-chain polyunsaturated linoleic acid and α-linolenic acid are important for basal metabolism in humans and mushrooms are known to contain all these nutritional values. Overall, the low value of lipid content in basidiomycetes is due to a low proportion of desirable n-3 fatty acids.

1.8 CARBOHYDRATES AND FIBER CONTENT The polysaccharide of mushrooms is glycogen as opposed to that of starch in plants. Mannitol, glucose, and α,α-trehalose (α-D-glucopyranosyl-(1-1)-α-D-glucopyranoside) are the main representatives of monosaccharides. Carbohydrates usually account for the prevailing component of fruiting bodies. However, the contents of trehalose and glucose in the fruiting body of mushrooms are low, in the order of 100 per gram of dry matter. In A. bisporus the synthesis of mannitol is mediated by NADPH-dependent mannitol dehydrogenase using fructose as its substrate. It functions as an osmolyte, which gets accumulated at a higher concentration in the fruiting body, while after sporulation its amount decreases drastically. Trehalose also serves as a reserve carbohydrate in A. bisporus, getting synthesized in the mycelium and later translocated to the fruiting body [85]. Mannitol concentration also varies widely among basidiomycetes. The concentration is found to be of 1.0%, 6.5%, and 13.7% for dry matter of Tricholoma portentosum, Agaricus arvensis, and Lactarius deliciosus, respectively [86] and 0.8%, 0.2%, 11.7%, and 13.9% of dry matter of Lepista nuda, Lycoperdon perlatum, Ramaria botrytis, and C. cibarius, respectively [61]. The amounts of trehalose and mannitol get reduced considerably while boiling the fresh mushrooms, while freezing and drying result in only limited losses [86]. In C. cibarius, trehalose and arginine are the most important compounds for carbon assimilation [86]. Chitin is a water-insoluble structural polysaccharide that accounts for up to 80% 90% of dry matter in the cell walls of mushroom. The chitin content of dry matter as reported from eight samples of Boletus spp. ranged from 6.8% to 10.2% [55]. Chitin being indigestible for humans apparently decreases the availability of other mushroom components. Information on dietary fiber content in wild growing mushrooms has been very limited. Boletus spp. is reported to contain 4.2% 9.2% and 22.4% 31.2% of dry matter for soluble and insoluble fibers, respectively [43,87]. Cheung [88] determined the hemicelluloses and pectic substances in basidiomycetes [88]. Information on chitin and fiber changes during different preservation and cooking remedy has been lacking. Great attention has recently been given to the fiber content of mushrooms, the β-glucans which act as a health-promoting factor [49].

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9

A recent study reported that two mushroom carbohydrates inhibit breast cancer cell growth by enhancing the immune function [89].

1.9 MINERAL COMPOSITION Wild mushrooms are assumed to accumulate a large amount of both macro- and microminerals. Mineral content varies among different species of basidiomycetes. Research on 11 different mushroom species showed the level of potassium concentration in Tricholoma saponaceum to be around 39.8 mg/g, which is lower in the case of Laccaria laccata and Candida rugosa, being 30.2 and 28.9 mg/g, respectively [90]. Selenium content in the tubes and gills of mushrooms are quite high. Selenium acts as an antioxidant and is also required for the biosynthesis of selenoenzymes, which include glutathione peroxidase, thioredoxin reductases, Selenoprotein W, Selenoprotein P, and iodothyronine 5’-deiodinases. Selenium content in mushrooms ranges from approximately 5 μg/g in Lycoperdon spp. to about 200 μg/g in Albatrellus pes-caprae. King Bolete (B. edulis) is considered to contain an average concentration of approximately 20 μg/g of selenium, whereas Pinewood King Bolete (Boletus pinicola) contains an average of 40 μg/g based on its dry weight. The fruiting body of Lurid Bolete (Boletus luridus), Lepista luscina and Parasol Mushroom (Macrolepiota procera) contained, respectively, 49, 91, and 47 μg/g of selenium in crude mushrooms [91]. Potassium is not distributed evenly within fruiting bodies, the concentration seems to be highest in the cap followed by the stipe, the spore forming part, and finally the spores. It seems to be higher by 20- to 40-folds in the fruiting bodies than the remaining parts [92]. Following potassium, the second most abundant major element in mushroom fruiting bodies is phosphorus, which ranges from 5 to 10 g/kg of dry matter [61]. A report showed a high amount of calcium concentration of 1.600 mg/g in Craterellus tubaeformis followed by 10.50 mg/g in Laccaria laccatta [90]. Sodium concentration is relatively lower in mushroom species, ranging from 100 to 400 mg/kg of dry matter [61]; as a result they have been considered good for patients with hypertension. Few mushrooms including F. officinalis offer chlorine in the form of coumarine, including 6-chloro-4-phenyl2H-chromen-2-one and ethyl 6-chloro-2-oxo-4-phenyl-2H-chromen-3-carboxylate [93]. As per the report of Rudawska and Leski [94], total sulfur content in Amanita rubescens and Xerocomus chrysenteron is found to be between 900 and 4400 mg/kg dry matter, respectively [94]. Mushrooms are considered to contain high amounts of trace elements, including cadmium, mercury, lead, copper, and antimony, in their fruiting bodies [95]. Agaricus spp. accumulates a high level of mercury and cadmium up to a range of 130 mg of cadmium per kg of dry weight of the fruiting body [96]. Other mercury accumulators include L. perlatum, Lepista sp., and Macrolepiota sp. [96]. Chromium and nickel concentrations are higher in L. deliciosus with around 4.51 mg/kg of dry weight, and Tricholoma terreum has 9.9 mg/kg of dry weight [97]. Mushrooms are also known as zinc accumulators. Lactarius sp. is known for its higher concentration of zinc which ranges from 55.7 to 158 mg/kg on the basis of its dry weight. The gills of Clitocybe alexandri are considered to contain a high amount of copper which ranges from 26.3 to 95.9 mg/kg on its dry weight basis. The concentration of iron seems to be slightly higher in Volvariella speciosa, ranging from 220 to 7162 mg/kg [98]. During the Chernobyl accident, mushroom fruiting bodies (radiotrophic fungi) were seen to accumulate huge amounts of toxic heavy metals [99]. It has been reported that the natural radionuclide 40K accumulates greatly in mushrooms and the accumulation factor ranges from 20 to 40 [92]. However, the Boletus group is considered to contain a high amount of selenium content [61]. The heavy metal radiocesium is also found to a greater extent in mushrooms in coniferous forest than in deciduous forest [92].

1.10 CONCLUSION Various wild mushrooms have been traditionally used as a source of food and medicine since the ancient era. However, large sections of the population are still unaware of the positive aspects of mushrooms due to fragmentary and poor information about their bioavailability and toxicity. The identification of the mushrooms can help the mushrooms hunters for easy detection of the toxic one from their edible counterpart. With the excessive nutritional potency of mushrooms, they are considered to be nanofactories and the mushroom industry could be a thriving activity worldwide. But due to lack of knowledge, trained manpower, and inadequate market price support, there have been major hindrances to this activity. Further, a large number of poisonous mushrooms resemble their nontoxic counterparts. So it is of utmost necessity to differentiate their toxic nature which will help in

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the further development of their biological and therapeutic applications and open vistas for their bioprospection and use for human benefits. This is expected to open the scope for their applications in health promotion, dietary feeding, and sustainable income generation. The dearth of information about the nutritional and antioxidant properties of wild mushroom is one of the main reasons holding back the wild mushroom industry, so there is an urgent need to bioprospect their nutritive values worldwide.

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Technol. 219 (2004) 71 74. [96] M. Lodenius, M. Herranen, Influence of a chloralkali plant on the mercury contents of fungi, Chemosphere. 10 (1981) 313 318. [97] J. Vetter, E. Berta, Mercury content of some wild edible mushrooms, Food Res. Technol. 205 (1997) 316 320. [98] J. Vetter, Chromium and nickel content of some common edible mushroom species, Acta Aliment. 26 (1997) 163 170. [99] M. Isiloglu, F. Yilmaz, M. Merdivan, Concentration of trace elements in wild edible mushrooms, Food Chem. 73 (2009) 169 175.

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C H A P T E R

2 Genetic Manipulation of Secondary Metabolites Producers Ali Asghar Rastegari1, Ajar Nath Yadav2 and Neelam Yadav3 1

Department of Molecular and Cell Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib, Sirmour, India 3 Gopi Nath P.G. College, VBSP University, Deoli-Salamatpur, Ghazipur, Uttar Pradesh, India

2

O U T L I N E 2.1 Introduction

13

2.2 Genetic Engineering of the Secondary Metabolic Pathway in Plants 2.2.1 Flavonoids and Anthocyanins: Biosynthesis and Regulatory Genes 2.2.2 Alkaloids 2.2.3 Terpenoids 2.2.4 Carboxybenzene Formatives 2.3 Secondary Metabolites in Actinomycetes by Metabolic Engineering 2.3.1 Precursor Engineering of Carbohydrate and Fatty Acid Metabolism

2.3.2 Technology Regulating Systems 2.3.3 Engineering Biosynthetic Structural Genes

14 15 15 16 17 18

23 24

2.4 The Aspergillus nidulans MAPK Module and Secondary Metabolism 2.4.1 Development Modular Controls

25 25

2.5 Conclusions and Future Scope

27

Acknowledgment

27

References

27

18

2.1 INTRODUCTION The numerous steps involved in the secondary metabolism of plants play as an intermediary between in plant and ecosystem interactions involving insects, plant microorganisms, and herbs [1,2]. The secondary metabolites contain a naturally occurring components, such as phytoproteins and phytolexins, to protect the plants from insects or other animals that may otherwise eat them. Secondary metabolites make a significant portion of human diet (flavor, color, and scent), and herbal colors are also obtained from various plants and flowers. Furthermore, some secondary plant metabolites are used to produce drugs, colors, insecticides, flavors, and perfumes. In addition, secondary metabolism is an important step in plant reproduction. The low development of plant cultivation for the production of secondary metabolites causing using of gene-splicing, and this an encouraging approach in the field [2]. Filamentous fungi produce a variation of small molecules termed secondary metabolites which are used to produce drugs such as penicillin antibiotics, cholesterol-lowering drug lovastatin, and immunosuppressant cyclosporine, as well as robust mycotoxins such as aflatoxin and fumonisin. Secondary metabolites have played a main key environmental role in creating territory, protection, communication, and prevention [3].

New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: https://doi.org/10.1016/B978-0-444-63504-4.00002-5

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© 2019 Elsevier B.V. All rights reserved.

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

There are as many as about 23,000 reported secondary metabolites biologically produced by microorganisms, of which only 150 are used in pharmaceutical, agricultural, or other fields. About 45% of all biologically active microbial metabolites are known, and 80% of these known compounds are used in practical applications. More than 10,000 of these compounds are extracted from actinomycetes. Approximately 7600 compounds are produced by Streptomyces species which falls under actinomycetes [4]. Drugs are produced by chemical synthesis or semiindustrial processes for commercial uses. These microbial strains require sophisticated production methods to separate the compounds through fermentation. Due to these reasons, the production costs involved in the isolation and extraction of these compounds are high, resulting in a limited extraction of secondary metabolites from the naturally available fungi. It clearly shows that the cost cannot be lowered unless the production is increased and it actually increases if a new compound is processed. After the discovery of antibiotics in 1940, the excessive production of pharmaceuticals was the basis for the increase in microorganism fermentation. Processes using the usual “mutate-and-screen” method was advanced for penicillin strains, and the current production of penicillin from Penicillium chrysogenum stands 1000 times more than 70 g/l, which which was earlier only 60 mg/l . The protein production of Pseudomonas denitrificans, 100,000 times the more soluble, of vitamin B2 of through Ashbya gossypii, and this is 40,000 times more soluble than the water-soluble vitamins, and this is another way to heal with the usage of microbe strains [5,6]. Genetic engineering of secondary metabolites is a way to increase or reduce the quantity of a specific compound or a group of target compounds [7
11]. There are more than a few approaches to reduce the production of some unwanted (group of) combination(s). By reducing the level of compatible RNA templates through antisense methods, crystallization or ribonucleic acid interposition procedures, or through the excessive expression of antibodies, contrary to the enzymatic stage in the pathway, can be eliminated. The change of color of flowers has been effectively achieved by antisense gene method [12]. Flux deviation to a competitive pathway or increase in catabolism are the next goals to be achieved. Transferring pathways to other plant species or natural plant species or microorganisms are often the goal to cumulate the efficacy of specific compounds. Moreover, new compounds that are yet to be discovered follow two general approaches to increase the production of these compounds. Initially, one or more genes were used to overcome the specific steps limiting the speed of the pathway, blocking competitive pathways, and reducing catabolism. The second approach attempts to control the multiple biosynthesis genes to alter the expression of regulatory genes. This chapter emphases the recent efforts to classify the genes accountable for the biological production of natural products and to determine the complicated mechanisms of biosynthesis.

2.2 GENETIC ENGINEERING OF THE SECONDARY METABOLIC PATHWAY IN PLANTS The first genetic engineering was used in the flavonoid and anthocyanin synthesis as their biosynthesis pathways were well known and the consequences could be simply detected with variations in the color of the flower [8,9,13]. The various experiments carried out involved excessive expression of diverse pathways of genes, for instance, to produce new flower colors using new plant compounds. Due to their antioxidant nature, external surfaces in food additives and plant pigments have a structure based on or similar to that of flavone which are a significance role in diet. The terpenoid indole alkaloid pathway is the next goal of gene-splicing labors since about 15 of the alkaloids of terpenoid and terpenoid indole alkaloids are of industrial significance, including antitumor alkaloids, vinblastine, vincristine, and camptothecin. An alternative imperative drug group is an important secondary plant metabolite, consisting isoquinoline alkaloids, which include significant drugs such as morphine and codeine. Majority of secondary substances are terpenoids by far. Following the current findings of the 2-Cmethyl-D-erythritol-4-phosphate (MEP) task in plastidial terpenoid biosyntheses, such as red fat-soluble pigments and monoterpenes and diterpenes, numerous genes were duplicated in this pathway [14
17]. Fat-soluble red pigments are significant colorants and antioxidants in mud, foods, and fruits, the most significant of which is vitamin A from β-carotene [18]. Salicylic acid (SA) is a major natural product considered to be produced as a consequence of resistance to the system after stimulating the plant with plant pathogens. Although this is not proven scientifically, it has long been supposed to be composed of phenylalanine. Cyanogenic glucosides are the products of a comprehensive pathway of biosynthesis of secondary metabolites in heterologous plant species by expressing biosynthesis genes of cyanogenic glucoside from sorghum bicolor in Arabidopsis [19]. The extract contains cyanogenetic glycoside dhurrin which is hydrolyzed via β-glucosidase by tissue loss.

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2.2.1 Flavonoids and Anthocyanins: Biosynthesis and Regulatory Genes The red flavonoid pigment found in plants has an antioxidant property, and to synthesize the plant pigments having a structure based on or similar to that of flavone in diet is the next goal. A lot of attempts being made with tomato plant in this regard. Chalcone isomerase (CHI), the primary catalyst of flavonoid, plays an important role in flavonol production [20]. Excessive expression of Petunia CHI gene caused a 78-fold increase in flavonoid in tomato peel. Afterward processing these tomatoes, a 21-fold flavonol increase was gained in tomato paste related to un-transgenic rules. Excluding the flavonoids, neither transgenic herbs nor the transgenic tomato can produce similar results from their individual controls. Isoflavones are found to act as phytoalexins in legumes, which means that the biogenesis from those antimicrobial compositions is produced via microbial infections. These compounds are made into Arabidopsis, tobacco plants and corn, which is generally not able to combine these compounds with excessive expression of isoflavone synthase, the cytochrome P450 catalyst [21,22]. The genesis of isoflavones follows the phenylpropanoid route which results in extra enhancement of isoflavone biosynthesis in heterologous plant species. Instead of controlling numerous pathways for gene expression in single gene pathways, the transcript effects can be used as a substitute. In the corn nucleus, red flavonoid pigment biogenesis is controlled by two transcription agents, R and C1. The R protein is produced by the original helix
loop
helix protein coded from the vertebral proto-oncogene c-MYC, while the C1 protein is homologous to proto-oncogene cMYB. Full protein intake of flavonoids was obtained by overexpressing the transcript effects of R and C1 in in vitro cultured corn cell [23]. In addition to the expression of the transcription factors of corn C1 and R, the chalcone synthase genes triggered the beginning of red flavonoid pigment biogenesis and expanded defiance to fungus in rice [24]. In Arabidopsis, an MYB-type transcript agent (PAP1) was recognized that produced the overexpression to cause plants to have concentrated purple pigments during development [25]. Transcript effects may also act as inhibitors in normal produce buildup. Eliminating the MYB4 genes on the MYB element in Arabidopsis, caused by increased surface of sinapate esters on the leaf, increased the UV-B irradiation tolerance [26]. Similarly, excessive expression of tobacco from the MYB FaMYB1 protein from strawberries primes led to a reduction in flower pigmentation and a decrease in the level of red flavonoid pigment and flavonol compositions, demonstrated in the crop of the strawberry, FaMYB1 was repressed in the flavone pigments route functions [27]. Such consequences show that optimum path technology with transcription controllers needs precise information from the control route.

2.2.2 Alkaloids 2.2.2.1 Crystalline and Nitrogenous Compounds: Biogenesis and Regulatory Factors A large class of organic compounds, including crystalline terpenes and nitrogenous compounds are the candidates for gene-splicing, out of which around 15 crystalline terpenes and nitrogenous compounds are industry critical, such as antitumor alkaloids, (e.g., the cytotoxic compound used in cancer, cisplatin, and camptothecin). Such nitrogenous compounds contribution a path important into reasonable strictosidine, and of which opinion, that numerous paths of plant species producing alkaloids are separated. The coding sequence of tryptophan decarboxylase (TDC), as well as strictosidine synthase (STR), has been widely considered in the cultivation of Catharanthus roseus cells [28]. Excessive expression of TDC instantly causes tryptamine levels to go high, but not to that of alkaloids; high levels of alkaloids have been addressed in STR [29]. TDC and/or STR are also expressed in nonalkaloid- producing plants [30,31]. Nutrition of transgenic tobacco cells with secologanin [31,32] leads to the production of strictosidine, but the glycoalkaloid produced in its place is kept in the vacuole as the indole alkaloid in C. roseus. This shows the importance of the physiological aspects of secondary metabolism along with biosynthesis. Consequently, genes not only generate the enzymes that break down the biosynthetic stages, but also alter the pH and transport routes. Cell culture Weigelia “Styriaca”, formed by biosynthesis of secologanin, excessive expression of TDC and STR genes, produces a small quantity of ajmalicine and serpentine. This shows that the biosynthesis of Indole alkaloids in nonalkaloid- producing derivatives is possible through the expression of one or more heterologous pathway genes [31]. Although both genes were integrated into all 150 analyzed tobacco plants, in 26% of the plants both transgenes were shut down, at both 41% either one of the two genes were extinguished and in both genes, 33% expressed. There is no specific connection among numerous merged proceedings and collected degrees of transcription. Within transgenic weed plants, 24 to 110-fold changes were detected in tryptophan decarboxylase and strictosidine synthase actions. The second transcriptional activators, ORCA2 and ORCA3, exposed numerous regulatory stages in the plant biosynthesis of organic compounds in C. roseus [33
35]. Nevertheless, the excessive expression

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

of ORCA3 did not increase the production of alkaloids, because the G10H gene does not control the coding of an electron transfer agent P450 catalyst which precipitates in the biosynthesis of secologanin. A three times increment in alkaloids production compared to control cells was observed only after feeding the secologanin precursor in loganin [35]. Both ORCA2 and ORCA3 are complicated in terms of the expression of jasmonate in the reaction of the alkaloid biosynthesis genes of terpenoid and alkaloids [33
35]. The fact is they do not rein G10H, although the jasmonate-response is genetic [36], which shows that another jasmonate-responsive transcriptional activator reins a division from path gene. This training piece clearly shows that the regulator gene can increase the state from a series of catalysts to a path, and it is essential to avoid the excessive expression of each gene in a distinct pathway. 2.2.2.2 Isoquinoline of Nitrogenous Organic Compounds of Plant The drug set is the second major substances of a plant, for example, isoquinoline of nitrogenous organic compounds that contain significant narcotic drugs such as opium. Numerous routes of this alkaloid have been explained leading to a method for metabolous technology. Yamada et al. [37] assumed that excessive expression of a catalyst at the site of division must result in expanded flow via a damaged arm. Within this barbarian biogenesis, a catalyst (S)-Scoulerine 9-O-methyltransferase (SMT) takes the control of the coptisine: berberine extra columbamine into Coptis japonica cell [37]. Excessive expression from the gene leads to an increase of 20% in enzyme activity, with total berberine and columbamine from 79% of total alkaloids content in wild cells to 91% in transgenic cells. These explanations demonstrate that the current at a division point can be different by metabolic engineering. Excessive expression of C. japonica-SMT gene in a plant cell of Eschscholzia californica, plant containing this enzyme, has caused the production of columbamine, which was not usually found in this species, which can prove to be a new method to create new compounds in the plant (i.e., combinatorial biochemistry). As a likely way to produce new alkaloids, the Thalictrum tuberosum O-methyltransferase antitoxin was intended to change heterodimeric catalysts by changing substrates of homodimers [38]. 2.2.2.3 Tropane Alkaloids and Pyrrolidine Alkaloids Tropane was the drug studied in detail with regard to this genetic technology of alkaloids [39]. Especially, an alteration from arsenic to scopolamine is very precious, which was the main object of this analysis. An H6-β-hydroxylase (H6H) enzyme catalyzes the alteration. With this excessive expression of that encoded factor, H6H, in the cultivation of genus Hyoscyamus muticus hair roots, can increase the sedative and hypnotic surfaces by about 100 times. On the contrary, to control this, a poisonous compound was present in henbane for the main nitrogenous organic compound of the plant [40]. This surface of hyoscyamine (around 10 times superior to that of preoperative medication in transgenic heritage) was similar in transgenic as well as cellular cells. Many recent attempts were made to increase the flux through biosynthesis pathways [37]. The putrescine N-methyltransferase (PMT) tobacco gene increased significantly in Atropa belladonna and Nicotiana sylvestris, respectively, and the generation from tropane alkaloid, a more pungent liquid made by reduction of nitrogenous organic compounds, increased quantifiably. In other nitrogenous organic compounds organization by methylputrescine takes the primary role in eliminating amine formed from arginine during putrefaction of this pool of metabolite polyamide synthesis. Although the humble increase in PMT action was found to be only 3.3-fold in the A. belladonna strains, no increase in alkaloid levels was observed and except the level of methylputrescine. In some N. sylvestris transgenic plants, the PMT activity rises 4
8 times, while in others it is referred to as cosuppression. Transgenic levels show a 40% increase in nicotine, while in collaboration with co-suppression, the level of nicotine was only two percent of the wild type.

2.2.3 Terpenoids 2.2.3.1 The essential oils of plants and simple derivatives A large class of organic compounds, including terpenes existed via distant that large set from the second metabolite. Looking the latest discovery of the role of the (2 R, 3 R) -2,3,4-trihydroxy-3-methylbutyl dihydrogen phosphate (MEP) in the biosynthesis of plastidial terpenoids, such as the carotenoids and monoterpenes and diterpenes, several genes of this pathway have been cloned [14
44]. Correcting the MEP route may possibly help the users. For example, the cosuppressor sequence of nucleotides, complementary approach, would not eliminate the electron transfer agents P450 catalyst within the nicotine-rich leaf epidermis of a plant gland and will help to increase defiance toward bugs [41]. Cembranoid range changes by a 10-fold increase in unsaturated molecules

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composed of linked isoprene units cembratriene-ol as well as reducing the cembratriene-diol oxidation effect. In another instance, excessive expression of the chimerical isoprenoid biosynthesis factor in sweet wormwood showed that the increase of flow in one synthesis path of cystic boneid results in a two to three times increased antimalarial activity [42]. In tomatoes, the excessive expression of S-linalool synthase transgenesis increased the monoterpenoid flavor compound S-linalool, compared to control plants, although no changes were observed in other levels of terpenoids [43]. Gene-splicing capabilities for fat formation were investigated in spearmint [16]. The excess gene coding for 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) in spearmint caused herbs to have an increased XDR activity by two to four times [15]. This plant has a natural phenotype and showed approximately 50% increase in essential oil (monoterpenoid) production. Growth and production of monoterpenoid decreased in plants found to be resistant to cosuppression. Increasing the level of menthol in mint oil can lead to penetration by cutting one of the competitive branches in the monoterpenoid metabolic network to menthofuran [15]. An attempt is made to avoid a branch that removes the pulegone channel from the pathway of the menthol route using the antisense gene from the menthofuran synthase eruption clause. 2.2.3.2 Carotenoids For various reasons, the pathway for carotenoid biosynthesis is the extremely engaging goal of gene-splicing. The fat-soluble red pigments exist as an essential color in muds, foods, fruits, and inhibits oxidations; moreover, the final and most important retinol is present in β-red plant pigment. The retinol insufficiency is common. Preface from β-red plant pigment synthesis to essential rice, with the excessive expression of prephytoenediphosphate synthase (originating from narcissus), 3,4-didehydrolycopene-forming, and carotenoid beta-end group lyase takes place, therefore a critical success [44]. An introduction of β-carotene biosynthesis into rice is essential, with the excessive expression of phytoene synthase (caused by narcissus), phytoene desaturase and lycopene β-cyclase, so thus an success is critical [45]. In tomatoes, content of β-carotene has been increased by the expression of a 40-carbon intermediate in the biosynthesis of carotenoids into fruit plasticizers. Hence, the whole amount of carotenoids, including a straight result by this lycopene catalyst, is reduced [46]. A number of fat-soluble red pigment catalysts were regulated. Decrease in the number of these pigment levels is likely a result of the restraint of feedback somewhere in the pathway. The demonstration of a 40-carbon intermediate by microbial synthase in the biosynthesis of carotenoids in glossy red fruitage increased two to three times the whole red fat-soluble pigments. Other surfaces by plastidial two too many thousands of isoprene units were not artificial and this activity from different catalysts was not on the particular path. This overexpression of 3,4-didehydrolycopene-forming (βLcy) gene with a specific promoter expanded the direct production of the enzyme, β-carotene, in tomato seven times. Introducing the algae factor that converts β-red plant pigment to an enzyme in both the pentose phosphate pathway in organisms and the Calvin cycle of photosynthesis, produces keto-carotenoid in chromoplasts, especially in nectars. The total level of carotenoids in the flower of transgenic plants is increased [2].

2.2.4 Carboxybenzene Formatives 2-Hydroxybenzoic acid (2HA) is a vital signaling particle in herbs with complicated systematic defiance end exposed to herb pathogenic germs. Although this indication is insufficient, it has long been supposed to be collected of phenylalanine. Lately, it has been revealed that SA is shaped in reply to pathogenic infections is formed from chorismate via conversion to isochorismate by isochorismate synthase (ICS). Microorganisms are produced by SA through chorismate by changing to isochorismate equipment, and after being separated from the pyruvate group, it is produced by the salicylate-forming (SF) or sochorismate pyruvate lyase (IPL). Herbs that demonstrate excessive microbial catalysts (ICS plus SF) in vacuoles show a natural morphological type, yet raise the defense of the virus and fungous bugs [2]. The 2HA or SA level was increased 1000-fold compared with wild-type plants, but growth was not affected. This instant catalyst, SF, looks like a step that limits the creation of the 2HA. The fused protein has been constructed by two bacterial enzymes and was introduced to Arabidopsis [47]. Plants with a cytosolic enzyme had a three times higher 2HA levels but a 20- fold increase after expression in the plastids. These plants obviously displayed a reduction in growth, which may be a result of the absence of isochorismic acid due to creation of phylloquinone. With this activity of weed production, the transgenic producer of SA is limited, therefore ICS produces the excess isochorismate for 2HA and phylloquinone biosynthesis. The 4-hydroxybenzoate (4HB), similar to 2HA, preserve the existing shape through (S) -2-amino-3-phenylpropanoic acid either straight of chorismic acid. Hyde et al. [52] introduced the microbial ubiC gene encoding that

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chorismate pyruvate lyase into Lithospermum erythrorhizon hairy root cultures, which normally produce naphthoquinone shikonin by phenylalanine pathway. Never overproduction in naphtoquinones was observed, even though 4HB was incorporated from both pathways. 2.2.4.1 Phytoanticipins (α-Hydroxynitrile-Type Aglycone and of a Sugar Moiety) The instance by an expression from one comprehensive metabolism, the synthesis path into the heterologous herb kind takes place, providing close to expressing biosynthesis genes of phytoanticipins of great millet inside rockcress [19]. The genus of flowering plants covers phytoanticipins (S)-4-Hydroxymandelnitrile-β-D-glucopyranoside, hydrolyzed through β-D-glucosidase with web harm. This expression of nitrile takes place in defensive pests and insecticides. Dhurrin is produced via tyrosine through the activity of two multifunctional cytochrome P450 enzymes (CYPs) and a precise UDPG-glucosyltransferase. The excessive expression of the first enzyme (CYP79A1) in the Arabidopsis route increases to the formation of p-hydroxybenzoic acid, which is not natural for this plant [49,50]. The rockcress herbs overexpress one or more genus of flowering plant cytochrome P450 genetics produce numerous glycosides that are derived from glucose of 4-hydroxybenzoate, shaped since cyanide decay, yet not (S)-4-Hydroxymandelnitrile-β-D-glucopyranoside. Speciously, nobody by that several Oligo 1, 4 Æ 1, 4 glucan transferase happened into rockcress were capable of produce p-hydroxymandelonitrile glucosylate to method dhurrin. Excessive expression glucosyltransferase of a special genus of flowering plants inside grouping by these second cytochrome P450 genetics caused the generation of origin inside rockcress. The rockcress, a gene that produces dhurrin, release superior grades about nitrile because of web harm, which proposes this (S)-4-Hydroxymandelnitrile-β-D-glucopyranoside takes place hydrolyzed through internal β-D-glucosidase. The sheet tissues of genetic material into which DNA rockcress herbs were denied through the Phyllotreta nemorum caterpillar by this insect protrude root, as well as the eating larvae, died in the genetic material into which DNA leafage. High levels of a foreign metabolite were thus produced in a plant species without negative effects on growth and with positive effects on resistance against pests.

2.3 SECONDARY METABOLITES IN ACTINOMYCETES BY METABOLIC ENGINEERING Microbes exist in numerous narcotics and drugs including additives, anticancer composites, immunosuppressants, antibiotics, medications which are indicated for the treatment of parasitic diseases, and inactive catalyst mixes. Metabolous manufacturing, to increase production, needs to access the metabolically streams, through presenting hereditary variations throughout the genetic engineering, into the method such sustain subordinate metabolites. In addition, the development of modern technologies such as DNA sequencing, transcription profiling, genomics, proteomics, metabolomics, transcriptomics, and metabolite profiling has created new opportunities to engineer microorganisms for the production of natural products in high yields [6]. Instances of the approaches about such advances exist defined below, that summarized in Table 2.1 and as well as a schema in [6] is shown (see Fig. 2.1).

2.3.1 Precursor Engineering of Carbohydrate and Fatty Acid Metabolism The accessibility of biosynthesis forerunners takes place the essential influence in this effectiveness about auxiliary substances. Primary metabolism is the supplier for those precursors that are generally formed through the catabolism of various carbon substrates such as fatty acids, monosaccharides or proteins. The identification and genetic manipulation of key enzymes regulating carbon flux through the metabolic network of the central carbon metabolism can lead to an increase in the availability of a particular precursor as shown in Figs. 2.1 and 2.2. In this carbohydrate metabolism, glycolysis and hexose monophosphate shunt (HMS or PPP) paths exist, presented in Fig. 2.1. An effort was made to genetically manipulate the primary stages of the EmbdenMeyerhof and PPP pathways to increase the generation by β-lactam drug, actinuronidine, as well as (2Z,5Z)-3methoxy-5-pyrrol-2-ylidene-2-[(5-undecyl-1H-pyrrol-2-yl) methylidene] pyrrole. This glycolic path, with a species of gram-positive bacterium notable for producing clavulanic acid, is hereditarily encoded through gap one, and gap two is encoded as GAPDH altered by triose phosphate (TP or G3P) to 1,3-Bisphosphoglyceric acid (1,3-PGA or 1,3-BPG). Meanwhile, TP and (S)-2-Amino-5-guanidinopentanoic acid exist into this synthesis via the β-lactam drug, the TP buildup inside conjugated hiatus alteration increases along with the noteworthy

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2.3 SECONDARY METABOLITES IN ACTINOMYCETES BY METABOLIC ENGINEERING

TABLE 2.1

Increase in Production of Secondary Metabolites Produced by Actinomycetes Achieved by Metabolic Engineering

Compound

Strain

Engineering approach

Increase (fold)

Actinomycin

Streptomyces antibioticus

Ribosome engineering

5.25

Actinorhodin

Streptomyces coelicolor

Fatty acid precursors

6

Upregulation

2.6
40

Ribosome engineering

1.6
180

Carbohydrate metabolism

4
5

Cofactors

4

Upregulation

470

Downregulation

3.5
5

Streptomyces lividans

C-1027

S. globisporus

Biosynthetic structural genes

2
4

Cephamycin C

S. clavuligerus

Biosynthetic structural genes

2
4

Upregulation

2
3

Chromomycin

Streptomyces griseus

Downregulation

3

Clavulanic acid

S. clavuligerus

Carbohydrate metabolism

2.1
3.1

Biosynthetic structural genes

1.6
5

Upregulation

1.5
23.8

Heterologous expression

1

Clorobiocin

S. coelicolor

Daunorubicin

Streptomyces peucetius

Biosynthetic structural genes

8
9

Upregulation

2.4
10

S. coelicolor

Heterologous expression and fatty acid precursors

4

Escherichia coli

Heterologous expression and fatty acid precursors

1.8

Desosaminyl tylactone

Streptomyces venezuelae

Heterologous expression, PKS deletion and upregulation

17.1

Doramectin

Streptomyces avermitilis

Biosynthetic structural genes

4
23

Doxorubicin

S. peucetius

Biosynthetic structural genes

3
74

Upregulation

4

Aeromicrobium erythreum

Fatty acid precursors

2
4

Saccharopolyspora erythraea

Fatty acid precursors

1.25
1.5

Expression of heterologous genes

2
2.5

Plasmid integration

2
5

Expression in industrial strains

50

6-dEB

Erythromycin

Fredericamycin

S. chattanoogensis

Ribosome engineering

26

Formycin

Streptomyces lavendulae

Ribosome engineering

5.2

GE2270

P. rosea

Ribosome engineering

1.8

Hydroxycitric acid

Streptomyces U121

Genome shuffling

5

Kanamycin

Streptomyces kanamyceticus

Self-resistance

3.5

Megalomycin

S. Erythraea

Heterologous expression and 6DOH metabolism

3.4

15-Methyl-6-dEB

S. coelicolor

Heterologous expression and plasmid co-integration

4
25

Mithramycin

Streptomyces argillaceus

Upregulation

2
16

Monensin B

Streptomyces cinnamonensis

Fatty acid precursors

1.76 (Continued)

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

TABLE 2.1 (Continued) Compound

Strain

Engineering approach

Increase (fold)

Nanchangmycin

Streptomyces nanchangensis

Biosynthetic structural genes

3

Neomycin

S. fradiae

Self-resistance

6

Nikkomycin X

Streptomyces ansochromogenes

Biosynthetic structural genes

1.8
2

Upregulation

2

Novclobiocin 122

S. coelicolor

Heterologous expression and 6DOH metabolism

8
26

Novobiocin

S. coelicolor

Heterologous expression

1

S. coelicolor

Heterologous expression and upregulation

3

Streptomyces noursei

Upregulation

3.25

Biosynthetic structural genes

1.6

Nystatin

Oligomycin A

S. avermitilis

Fatty acid precursors

23

Pikromycin

S. venezuelae

Upregulation

1.6
2.6

Pimaricin

Streptomyces natalensis

Upregulation

2.4

Downregulation

1.8

Pristinamycin IIA

Streptomyces pristinaespiralis

Biosynthetic structural genes

1.25

Rapamycin

Streptomyces hygroscopicus

Upregulation

1.2
1.4

ε-Rhodomycinone

S. peucetius

Upregulation

7
100

Salinomycin

Streptomyces albus

Ribosome engineering

1.5
2.3

Shengjimycin

S. spirumyceticus

Biosynthetic structural genes

2

Spinosyn

S. spinosa

Carbohydrate metabolism

3

Tetracenomycin D3

Streptomyces glaucescens

Biosynthetic structural genes

20
30

Tylactone

S. venezuelae

Heterologous expression, PKS deletion and upregulation

2.7

Tylosin

S. frudiae

Upregulation

1.2
4.9

Downregulation

1.5

Genome shuffling

6
8

S. coelicolor

Upregulation

31

S. lividans

Carbohydrate metabolism

4

Downregulation

11
12

Ribosome engineering

1.9
2.9

Undecylprodigiosin

increase in antibiotic creation, with L-arginine increasing to cultures [6]. PPP intends to remove two diverse places: Glucose-6-phosphate 1-dehydrogenase 1 (zwf1) and Glucose-6-phosphate 1-dehydrogenase 2 (zwf2) furthermore 6-phosphogluconolactonase DevB as 6-phosphogluconolactonase in gram-positive, filamentous, soil bacterium. These removals cause flow proliferation throughout glycolysis in place of HMS or PPP that into cause an increase in acetyl coenzyme A, a (2Z, 5Z)-3-methoxy-5-pyrrol-2-ylidene-2-[(5-undecyl-1H-pyrrol-2-yl) methylidene] pyrrole as well as benzoisochromanequinone polyketide antibiotic precursor, and an increase in the production of these antibiotics [51] (Fig. 2.1). The manufacturing of CoASH active glyceride forerunners is known to increase the generation of some of the secondary metabolites, for instance, erythrocin, macrolides created by Streptomyces, polyether antibiotic isolated from Streptomyces cinnamonensis, and benzoisochromanequinone polyketide antibiotic, into a useful product. In the case of erythrocin two of its precursors are malonyl-CoA derived from the carboxylation of acetyl-CoA and methylmalonyl- CoA, which can be synthesized through different pathways such as carboxylation of propionylCoA and rearrangement of succinyl- CoA (Fig. 2.2). Metabolic bulge in the methylmalonyl-CoA manufacturing in

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2.3 SECONDARY METABOLITES IN ACTINOMYCETES BY METABOLIC ENGINEERING

DAUNORUBICIN DOXORUBICIN

Glucose 6PG

DevB

6PGL

Zwf1 Zwf2

G6P

Pgm

G1P

Gtt Gdh

Pgi PPP

21

6DOH

C-1027 SPINOSYN

F6P Arginine G3P Gap1 Gap2 1, 3BPG

Pah Cas2

Clavaminate

Cvm1

ANTIPODAL CLAVAMS

CLAVULANIC ACID CEPHAMYCIN C Pyruvate Valine Cysteine

LAT Lysine

Aspartate

Aspartate β-semialdehyde

Methionine ATP MetK SAM

Propionyl-CoA

UNDECYLPRODIGIOSIN Acetyl-CoA

Oxaloacetate

ACTINORHODIN

Isocitrate

2-oxoglutarate

Malate

Fumarate

Succinate

Glutamate SanU SanV 3-methylaspartate SanO

ACTINORHODIN

NIKKOMYCIN

ERYTHROMYCIN

FIGURE 2.1 Glucose catabolism and the engineering stages that occur in the metabolism of secondary metabolites produced by actinomycetes. 1,3-BPG, 1,3-Diphosphoglycerate; Cas2, clavaminate synthase; Cvm1, an enzyme involved in the biosynthesis of antipodal clavams; DevB, phosphoglucalctonase; 6DOH, 6-deoxyhexose pathways; F6P, fructose 6-phosphate; Gap1 and Gap2, glyceraldehyde-3-phosphate dehydrogenases; Gdh, NDP-glucose dehydratase; G1P, glucose 1-phosphate; G6P, glucose-6-phosphate; G3P, glyceraldehyde-3-phosphate; Gtt, NDP- glucose synthase; LAT, lysine e-aminotransferase; MetK, S-adenosyl-L-methionine synthetase; Pgi, phosphogulose isomerase; 6PG, 6-phosphoglucanate, 6PGL, 6-phosphoglucolactone, Pgm, phosphoglucomatase, palm, proclavaminant amidino hydrolase; PPP, pentose phosphate pathway; SAM, S-adenosyl-L-methionine; SanO, nonribosomal peptide synthase; SanU and SanV; glutamate-mutase; Zwf1 and Zwf2; glucose-6-phosphate dehydrogenase.

the production of erythromycin Saccharopolyspora erythraea and Aeromicrobium erythreum depends on the culture environment utilized. Excessive production by erythrocin was accomplished by deactivating the methylmalonylCoA isomerase (MCI or MCM) mutB gene in further cultures S. erythraea either via A. erythreum per sugar-founded sets, or by reducing the production of erythromycin in the oil-based situation [52,53]. In a carbohydrate-based medium, the MCI reaction acts like a drain on the methylmalonyl- CoA pool, but in an oil-based medium, the same reaction acts to fill the methylmalonyl-CoA pool [53,59]. Moreover, excessive production of erythrocin was achieved through an increase in gene (mutA, mutB, meaB, plus mutR) as well as culture S. erythraea founded on the grease in fermented situation. These experiments led to the conclusion that the carbon flow under oil-based growth conditions was from succinyl-CoA to methylmalonyl-CoA. Antiparasite avermectins and oligomycin inhibited the development of the macrocyclic lactones produced by Streptomyces avermitilis. Throughout the avermectins biosynthesis, two diverse entrant parts were utilized: Polyketide synthase (PKS), as well as II-methyl butyryl-CoASH created a nearby shame about the valine and string isoleucine containing both a carboxyl and amino groups. Inactivating bkdF, the coding of branched-chain α-ketoacid dehydrogenase complex (BCKDC or BCKDH complex) chain primes on the exclusion by the generation of avermectins moreover into the refer to over-production about oligomycin of antibiotics containing a lactone ring nearby starting supplementary parts

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

Glucose UNDECYLPRODIGIOSIN ACC

Acetyl-CoA

Malonyl-CoA

OLIGOMYCIN

ACTINORHODIN Oxaloacetate

Crotonyl-CoA

Isocitrate

CCR 2-oxoglutarate

Malate Butyryl-CoA OLIGOMYCIN

Fumarate

Succinate

ICM Ethylmalonyl-CoA

MONENSIN A

Isobutyryl-CoA

AVERMECTINS

Succinyl-CoA MutB

BkdF

Methylmalonyl-CoA

Valine

MMT PCC

MONENSIN A, B OLIGOMYCIN ERYTHROMYCIN

Propionyl-CoA Leucine

Isovaleryl-CoA IST

SHENGJIMYCIN

FIGURE 2.2 Fatty acid precursors and engineering steps that occur in metabolites of secondary metabolites produced by actinomycetes in biosynthesis. ACC, Acetyl-CoA carboxylase; BkdF, branched-chain a-keto acid dehydrogenase; ICM, isobutyryl-CoA mutase; IST, 400-O-acyltransferase; MMT, methylmalonyl-CoA transcarboxylase; MutB, methylmalonyl-CoA mutase; PPC, propionyl- CoA carboxylase.

(the coenzyme A derivative of the malonic acid, thioester consisting of coenzyme. Inactivation of bkdF encoding a branched-chain α-keto acid dehydrogenase(BCKDC or BCKDH complex) leads to the abolition of avermectins production and in turn to the overproduction of the macrolide oligomycin by enabling additional extender units (malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA) to enter the biosynthetic pathway of oligomycin. In bacterium the genus of Streptomyces produces monensin A, the deactivation of which, by the (2 S)-ethylmalonyl-CoA:NADP1 oxidoreductase/decarboxylating (or CCR), into reducing the (2 S)-ethylmalonyl-CoA toward the coenzyme A-activated form of butyric acid is complicated, resulting in the removal of (S)-ethylmalonyl-CoA manufactured by the coenzyme A-activated form of butyric acid as well as of the buildup polyether antibiotic isolated from S. cinnamonensis in place of Monensic acid. Polyether antibiotic isolated from S. cinnamonensis requires individual thioester consisting of coenzyme A linked to methylmalonic acid, whereas monensic acid needs one of the other thioester consisting of coenzyme A linked to methylmalonic acid as well as (S)-ethylmalonyl-CoA throughout own synthesis. The production of Actinorhodin through soil-dwelling gram-positive bacterium, that belongs to the genus Streptomyces, is an example of increasing the creation from a class of secondary metabolites by altering their own structure. In this case, the overexpression of the genes accA2, accB and accE, coding for the different subunits of the enzyme acetyl-CoA carboxylase (ACC) in S. coelicolor, was sufficient to enhance carbon flux to malonyl-CoA, which is a precursor of actinorhodin together with acetyl-CoA, leading to a six-fold increase in actinorhodin production [57]. Supplementary basics of essential carbon metabolism play a precursor role in the biosynthesis of secondary metabolites, as well as in the advancement of metabolic production. This takes place by a combination of ademetionine (SAMe). It has been shown that the production of actinorhodin in Streptomyces lividans and S. coelicolor increases the expression of the Streptomyces spectabilis metK gene as methionine adenosyltransferase (MAT), increasing

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23

the catalytic SAM synthesis of adenosine triphosphate as well as 2-amino-4-(methylthio) butanoic acid. The same effect was obtained by addition of SAM to the culture medium. The increase in actinorhodin production is the consequence of inducing the expression of pathway-specific transcriptional activator actII-orf4 [57].

2.3.2 Technology Regulating Systems Biosynthesis gene, one minor catabolism paths exist ordered jointly into chromosome clumps, counting their pathway regulator genes. Pathway-specific regulators can have either positive (activators) or negative (repressors) effects on the expression of gene cluster elements. The clumps comprise various other confident-regulative genes that route the pathway of benzoisochromanequinone polyketide antibiotic to the pathway of daunorubicin. Furthermore, several clumps hold one or other accelerators and controllers, for instance, an antibiotic and a bacteriostatic feed additive used in veterinary medicine path which contain second accelerators and second controllercode genetics. Nevertheless, into several habits, regulative gene has never been recognized, except an instance with erythrocin route. In addition, other regulatory genes that are usually exterior a synthesis factor group can have the regulative part into one group, a number of times, showing the properties of pleiotropic created by numerous minor substances. This instance occurs in S. coelicolor which produces some antitoxins [benzoisochromanequinone polyketide antibiotic, calcium-dependent antibiotics(CDA), alkaloid produced by Streptomyces bacteria, and cyclopentanoid antibiotic produced by S. coelicolor A3(2)], further synthesis starts with precise controllers (promoter regions of actII-ORF4, cdaR, redD, and redZ, which encode pathway-specific activators) and is well-ordered. Whereas pleiotropic traits (i.e., afs, abs, and bld) affect antitoxin creation in the morphologic growth of microbe [6]. The vast popularity of pathway accelerators into the bacterium of an order of typically nonmotile filamentous form make a family of Streptomyces antitoxin regulator protein (SARP), which is considered through an attendance by the helix-turn-helix (HTH) pattern to the N-terminus. Overexpression of SARP positive regulators has been reported to increase the production of different secondary metabolites such as actinorhodin and undecylprodigiosin in S. coelicolor by actII-orf4 and redD, undecylprodigiosin in S. lividans and S. parvulus by redD, nikkomycin in S. ansochromogenes by sanG and clavulanic acid in S. clavuligerus by ccaR. The additional second metabolite, for instance, tylocine, cerubidine, and miteramycin have been described to be altered using SARP activating encoding genes or another precise pathway regulators. Tylosin production by Streptomyces fradiae is increased due to the excessive tylS or tylR expressions of the coding of the SARP family transcriptional, with one protein comprising the DNA sequence that can change its position within a genome region. Excessive expression of site dnrR1 genes, coding Streptomyces antitoxin regulator protein controller DnrI, or dnrR2, encode positive controllers DnrN and DnrO nrO comprising LuxR also ArsR the types of HTH patterns, caused by an increase of 10
100 times the production of Ɛ-radomycinone and a 25-fold growth in daunorubicin. A better quality miteramycin can be produced from Streptomyces argillaceous by excessive expression at two diverse regulating gene in wealth small circular DNA strand in the cytoplasm of a bacterium: mtrY encode a protein PadR-like HTH pattern and mtmR encoding a SARP-regulating protein. Furthermore, mtmR can stimulate benzoisochromanequinone polyketide antibiotic synthesis and mutations of ActII-orf4 in S. coelicolor JF1. Further, pathways of secondary metabolite biosynthesis without SARP activators have been improved by means of additional path regulators, for instance, pimM coding as the PAS / LuxR controller, which increases natamycin creation in Streptomyces natalensis, rapH and rapG, proteins by LuxR, and AraC-like the helix-turn-helix (HTH) motif increases the creation of rapamycin in Streptomyces hygroscopicus [6]. Streptomyces antitoxin regulator lineage proteins usually act as path controllers, although may act as pleiotropic regulative proteins in several cases to check the creation of further morphologic diversity by secondary intermediate metabolites. Such is the case of the afsR gene from S. coelicolor able to increase actinorhodin and undecylprodigiosin production through its overexpression in S. lividans. Others SARP pleiotropic homologated toward afsR, for instance, SsmA and afsR-p in Streptomyces noursei plus Streptomyces peucetius, contribute to an increased biosynthesis of nystatin and doxorubicin. Furthermore, the expression of excess afsR-p in S. lividans, Streptomyces clavuligerus, Streptomyces griseus, and Streptomyces venezuelae increases into the production of benzoisochromanequinone polyketide antibiotic, β-lactam drug, streptomycin, and amaromycin, respectively [54,55]. Disabling exact pathway regulators or pleiotropic can also result in excessive production of the secondary metabolites. This is the case of chromomycin that is overproduced when a pathway-specific transcriptional repressor cmmRII is inactivated in S. griseus subsp. griseus [56]. A famous pleiotropic suppressor system for variable phosphate is a secondary production of metabolites, a two-module system of PhoR-phoP. PhoR disturbance or synchronized removal of pair PhoR moreover phoP was lately revealed to increase the creation of pimaricin in

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

S. natalensis. Elimination of the similar systems was reported in S. lividans. It has been described that there were 5 and 12 times increases in the production of actinorhodin and undecylprodigiosin, respectively. In S. coelicolor there is an extra gene which influences two or more seemingly unrelated phenotypic traits, nsdA, which adversely influences the creation of antibiotics, and its disturbance increases the activity of Ca-reliant antibiotics, in addition, cyclopentanoid antibiotic produced by S. coelicolor during its metabolism. In the nsdA mutation, grades at this particular controller of the route acII-orf4 mRNA increase. Reports of other variations, including inactivating the ppk polyphosphate kinase gene, express the actII-orf4 starting the pathway of actinorhodin [6].

2.3.3 Engineering Biosynthetic Structural Genes Generally, genes that are elaborate in biosynthesis of secondary metabolites are targeted at generating new substrates by inactivating or removing them. Furthermore, these mutations can help as hosts for the heterologous genes expression that can help increase the production of new substrates. There are a number of instances that gene expression changes heterologous expressions, and structural expressions of biosynthetic genes have been deployed to increase the Idiophase production [6]. S. pristinaespiralis, a manufacturer of pristinamycin II, is an antibiotic of streptogramin, produced as a combination of two substrates of PIIA and PIIB in the proportion of 80:20. The whole change of the PIIB compound into PIIA includes incorporating an extra copy of the snaA and snaB genes into the chromosome that are deployed to transform heterodimeric monooxygenase which decays this alteration. The creation of snaA / snaB gene does not increase the total amount of pristinamycin, but the quantities of pristinamycin and PIIA, which result in an improved production of this substrate. An analogous technique increases the production of tetracenomycin D3, reasonable in tetracenomycin C biogenesis by Streptomyces glaucescens. In this case, excessive expression of tcmM, encoded for an acyclic carrier protein (ACP) of a type-II polyketide synthase (PKS), to a high copy cut, increases the production of this medium by up to 30 times. The carbon flux of glucose can be deployed to increase the Idiophase production through glucose catabolism as revealed in Section 2.1 and, additionally, by fluctuating the expression of structural genes exactly elaborate in the specific metabolite biogenesis. The genes belonging to the 6DOH pathways are good applicants for this type of manipulation. The case was from the sgcA1 gene, which is a protein of the NDP-glucose synthase complicated in the biosynthesis of 6DOH 4-deoxy-4- (dimethylamino)-5,5-dimethyl-D-ribopyranose, which is used to engineer the antibiotic production of enediyne C-1027 in S. globisporus. The excessive expression of sgcA1 alone would result in the doubling of C-1027 production and quadrupling of the cagA gene for the opaqueness of apoprotein C-1027. It is notable that genes cloned for enzymes involved in the 6DOH metabolism, e.g., sgcA1, are the genes that are generally collected with other metabolic genes involved in aglycone biosynthesis [6]. Nevertheless, there are several instances where genes involved in the 6DOH metabolism are found outside the cluster biosynthesis gene and are common between primary and secondary metabolism. This is connected to the genes in the L-rhamnose biosynthesis, which is generally described outside the cluster in all cases. In order to recover the production of spinosyn in S. spinosa, the duplication of the biosynthetic genes of L-rhamnose gtt and gdh coding of NDP-glucose synthase and NDP-glucose dehydratase has been used. These enzymes are the first activities accountable for the transfer of G1P to the biosynthesis of L-rahmnose and L-dimethyl-forosamine and 6DOH in spinosyn. Other primary metabolites such as G3P, lysine, or 2-exoglutarate can be directed to increase the production of secondary metabolites using specific structural genes of the biosynthesis (Fig. 2.1). Excessive expression or incorporation of clavaminate synthase gene cas2 into chromosome caused a five-fold increase in the production of clavulanic acid in S. clavuligerus. An extra enhancement, up to 23-fold, was gained by simultaneous integration with the activated crossover chromosome ccaR and the clavaminate synthase gene cas2. Furthermore, the creation of the pah2 gene, the encoding of proclavaminate amidino hydrolase, has lately been known to improve the production of clavulanic acid [57]. By encoding a lysine Ɛ-aminotransferase, in a high-copy plasmid in S. clavuligerus, the production of cephamycin C meaningfully increased with the expression of the gene lat [6]. A different method to improve the production of metabolites eliminates the genes that are removed for activities that change metabolites to a metabolite. Such is the case of dnrX and dnrH genes identified in S. peucetius and involved in the transformation of daunorubicin and doxorubicin into their polyglycosylated forms known as baumycins. Deactivation of dnrX and dnrH is 3 and 8.5 times, in the biosynthesis of doxorubicin and daunorubicin, respectively. Furthermore, the disturbance in DNA, complicated by the induction of daunorubicin to 1,3-dihydro daunorubicin, leads to a similar enhancement in the manufacture of doxorubicin. The production of doxorubicin has been enhanced by the disturbance of several genes in the same race, subsequent in a seven time increase in dnrU / dnrX mutation and 26 times in mutant triple dnrU/dnrX/dnrH. An increase of 1.3
2.8 time was found to be

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25

due to the excessive expression of the dnrV and doxA genes at the last stages of biosynthesis of doxorubicin in double or triple mutations. The carbon flux channeling by changing the amino acid precursors to increase the production of clavulanic acid in S. clavuligerus is a race that also produces cephamycin C and other clavams. The production of cephamycin C can be stopped by turning off the lat coding gene for a lysine aminotransferase. This inactivation leads to an increase in the production of clavulanic acid. Similar effects can be accomplished if cholesterol production of nonclavulanic acid is reduced by deactivating cvm1. Both methods, deactivating the lat and cvm1 in the same useful organism (Fig. 2.1), in this case, a developed race of S. clavuligerus, meaningfully gave a better quality production of clavulanic acid, several times advanced than the wild-type of persistent gene deletion, biosynthesis can occasionally result in the construction of secondary metabolites. Such cases include the inactivation of nysF, which increase the production of niesatin in S. noursei. nysF is a reminder of the putative 40 -phosphopantetheinyl transferase encoding that is intended to interfere with post-translational conversion of ACP domains in nystatin type-I, but it was revealed that neystein production is negatively affected. A doramectin (also called CHCB1) is an antihelmintic polyketide composition constructed by a race S. avermitilis, which does not have a biological activity of α-ketoacid dehydrogenase encoded by bkdF. This race generates two relevant compositions, doramectin and CHC-B2. The transformation of CHC-B2 to doramectin is a procedure than is still unclear, but aveC is the gene with unclear action. Efficiency relationships for doramectin can be improved by producing a few revised aveC by site-directed mutagens and error-prone PCR testing for the prime species, and chromosomal insertion of a superproduced refined gene in which the wild-type aveC gene has already been deactivated. In Streptomyces spiramyceticus F21, shengjimycin is manufactured (also called 4v-isovaleryl spiramycin), by placing an improved version of the 4v-O-acyltransferase ist gene on its chromosome from S. mycarofaciens 1748 (Fig. 2.2). On the other side, the efficacy of erythromycin in S. erythraea is amended. By using an integrated chromosome from a Vhb bacterial hemoglobin, it is essentially isolated from the Vitreoscilla spp. The system was previously used to increase cephalosporin C in the Acremonium chrysogenum fungus. Advancement of erythromycin generation in this race was perhaps due to increased salinity of erythromycin biosynthesis as a consequence of an increase in the actuality of an oxygen-related step in the composition of erythromycin, possible in hydroxylation stages [6].

2.4 THE ASPERGILLUS NIDULANS MAPK MODULE AND SECONDARY METABOLISM Many of these molecules comprise heterogeneous fragments produced by microorganisms, in particular bacteria and fungi. It is not amazing that given that these organisms living in compound ecosystems, where they contend and connect with other organisms from bacteria, fungi, and algae to proteins and even metazones, they fix. The evolution of these so-called secondary metabolites over millions of years was most likely accomplished because microorganisms used them as chemical signals for communication, to defend the habitat or to inhibit the growth of competitors [58]. Eukaryotic organisms connect at the cell and nucleus levels to reply to ecosystem signals. The mitogen-activated protein kinase (MAPK) contains a cascade of three protein kinases, a highly protected eukaryotic signaling system for yeast to man. MAP3K phosphorylates is a second kinase, MAP2K, which makes it MAPK phosphorylates. This kinase phosphorylates nucleic target proteins to stimulate gene expression. The sexual pathway from the yeast Saccharomyces cerevisiae characterizes a signal transduction model in eukaryotes. This MAP kinase pathway replies to the pheromones and reasons the procedure of difference that results in the sexual reproduction of the yeast. The essential complexes of MAP3K Ste11, MAP2K Ste7, and MAPK Fus3 collected on the Ste5 protein as a center to preserve these nearby kinases close to the phosphorylation enhancer communicate, thus regulate the current of information [59].

2.4.1 Development Modular Controls Module sexual kinase of AN (Aspergillus nidulans) can substitute their varied yeast functions. The plasmids comprising mkkB (kinase Anste7) along with mpkB (kinase Anfus3) gene, delivered by yeast promoters, were renewed to removestrains ste7 and fus3 species. Response to pheromones through the yeast mutant process have no defect decreased by kinase Anste7 along with kinase Anfus3. Nevertheless, mpkB neutralized the regular defect in pheromone reaction from a dual mutant fus3 kss1, signifying that mpkB is part of the capability of FAP3 / Kss1 to function in the MAP kinase couple. These two organisms specify the fractional overlying of the MAP kinase pathways. A. nidulans MAPK breeding component is considered supplementary by the

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2. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS

documentation of AnSte7 (mkkB) complementing with TAP purification from several developing stages (only vegetative) revealed in Fig. 2.3A and B. Marked AnSte7 does not hire mpkB, although associates SteC (AnSte11) along with SteD (AnSte50) to homologous protein with Baker’s yeast STE50(protein kinase regulator). Yeast S. cerevisiae plays a connector role by YLR362W (standard name: Ste11) membrane removal [60]. Principal elimination An leads to fruiting body defection. In accord with more Mitogen-activated protein kinase (MAPK) mutations, AnSte50 (steD)mutations cannot generate heterokaryotic external the body. Consequently, the AnSte50 adapter is significant for the exact expansion of fungal as more A. nidulans MAP Kinase module steD component parts were AnSte7 supplemental: Wild-type form of TAP, but not in straCΔ strain, shows YLR362W that (AnSte11) interacts with InterSte50-Ste7 (Fig. 2.3A and B). This data characterizes the physical communication of the SteD(AnSte50) as well as a double modular MAP kinase function a complement of SteD- YLR362W
D1525. Reaction associates from SteD(AnSte50) were recognized for total classifying yeast Mitogen-activated protein kinase breeding platform. One employed a staD effective: ctap MAP3K (SteC) and MAPKinase mpkB, apart from MAP2K (MkkB) (Fig. 2.3C and D). Furthermore, to support the A. nidulans MAPK Module AnSte11-Ste50-Ste7-Fus3, it generates an analogous platform Ste50-Ste11-Ste5-Ste7-Fus3, laterally accompanied by Ste5 scaffold mix combination in A. nidulans. 2.4.1.1 MAP Kinase Module for Secondary Metabolite Synthesis MAPK phosphorylation was estimated with a phospho-specific antibody in contrast to MAPK Thr182XTyr184 motif. AnFus3 phosphorylate is permanently obvious in wild-type cultures plant crops (Fig. 2.3E). In divergence, AnFus3 was not changed in mutations that had AnSte11 or AnSte7, whereas the absence of AnSte12 did not alter the level of phosphorylated AnFus3. A dearth of AnSte50, the decrease of phosphorylation of AnFus3,

FIGURE 2.3 Recognition for MkkB (AnSte7) along with SteD (AnSte50) and the MAP Kinase metabolic sequences behavior in Idiophase. (A) Ag (silver) staining of SDS-PAGE (5%
14% SDS polyacrylamide gel) from MkkB (AnSte7): of wild-type form cTAP along with STECΔ (without Ste11 MAP3K homolog SteC) background factors for plants growth (20 hours in 30 C temperature). (B) Considering peptides as protein identifier with wild strains and steCD. SteC(AN2269) [AnSte11] along with SteD (AN7252) [AnSte50] were concluded in the act of MkkB (AnSte7) interacting factor. (C) AnSte50 fusion companion: cTAP fusion of crop plants, immediate sexual cultivation (20 h in 30 C temperature) (M2B; MkkB; MB; MpkB; SD; SteD; SC; SteC). (D) Polypeptides identified from bands. phospho-44/42 MAPK (Thr182/Tyr184) wild-type form of antibodies and pheromone route mutations (24, 48, and 72 h) develop. (E) MpkB phosphorylation status detecting. The veA protein level is loaded as a control. Protein extract was loaded at 80 μg per lane. (F) production of sterigmatocystin (ST) metabolite in pheromone route, Anste11 (steCΔ), Anste50 (steDΔ), Anste7 (mkkBΔ), Anfus3 (mpkBΔ), Anste12 (steAΔ) mutations respectively. Extensible TLC plates show the production of Sts; Sterigmatocystin standard. (G) The amount of ST generation from TLC plates. The wild-type ST acts as a 100% standard. (H) Idiophase definition, in pheromone route mutations. laeA, aflR, stcU for the produce of ST, tdiA and tdiB for the production of terrequinone (TQ) and veA, mkkB, steD, mpkB, steA for development reasons. The strains growth in a culture medium (liquid) for different times (24,48,72 h) followed by entire isolating and blotting RNA (20 μg). The glycolytic gene of glyceraldehyde-3-phosphate dehydrogenase gene (gpdA gene) as well as rRNA were microscopic study colored (stained) with ethidium bromide as a control in experiment (so-called loading control).

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REFERENCES

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characterizes the outstanding AnSte11-Ste7 complex activity. It supports an active A. nidulans MAPK module, which contains AnSte50-Ste11-Ste7-Fus3, which regulates fungal growth. The role of AnSte50-Ste11-Ste7-Fus3 in Idiophase was considered. The Idiophase decreased is defined only for mpkB mutations [61]. The levels of mycotoxin sterigmatocystin (ST) significantly reduced in sterile steC, steD, mkkB, or mpkB mutations, while ST levels in the sterile sterAtAΔ (AnSte12) were like the wild-type (Fig. 2.3F and G). Likewise, the expression of ST (stcU) and terrequinone (tdiA and tdiB) biosynthesis genes, and the expression of laeA and the transcription factorencoding aflR were obligatory for the expression of secondary metabolites genes, in each mutation of the MAPK module (Fig. 2.3H). These data propose that active MAPK AnSte50-Ste11-Ste7-Fus3 is desirable not only for sexual growth, but also for the production of secondary metabolites.

2.5 CONCLUSIONS AND FUTURE SCOPE The future accomplishment of the pharmaceutical industry depends on the identification or expansion of novel compounds with new activities or on more specific aims. Numerous secondary metabolic genes have been overexpressed in the original plants or other organisms. In some cases, the excessive expression controlled the production of the desired creation, whereas in others the overexpression of the straight enzyme product level was simply expressed. Nevertheless, controlling genes are valuable tools for classifying secondary metabolism pathways genes and for creation or down the path or fragment of the route-setting in some organisms, such as bacteria, fungi, and algae of the Fus3 MAPK route from a fungus. It is a correction that controls the regulator of sexual growth and also secondary metabolism, which is a synchronized procedure in tough fungi. Detecting certain functions of Aspergillus Fus3 cells in signal transduction to regulate fungal growth and secondary metabolism is an attractive future. The task will be to assimilate proteomics and metabolomics with genetic data to recognize maps and then metabolic networks. Compound and complex metabolic networks in the cells comprise numerous enzymes. These enzymes can be actual precise or nonspecific, may have positive and negative response control, and may need activation or cofactor. Comprehensive knowledge at the level of these enzymes aids the progress of flow simulation models through metabolic networks.

Acknowledgment The authors are gratefully acknowledge from research council of Falavarjan Branch, Islamic Azad University.

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C H A P T E R

3 Role of Rhizobacterial Secondary Metabolites in Crop Protection Against Agricultural Pests and Diseases Luis Andre´s Yarza´bal1,2 and Eduardo J. Chica3 1

Unit of Health and Wellbeing, Catholic University of Cuenca, Cuenca, Ecuador 2School of Biology, Faculty of Sciences, University of Los Andes, Me´rida, Venezuela 3Faculty of Agricultural Sciences, University of Cuenca, Cuenca, Ecuador O U T L I N E

3.1 Introduction

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3.2 Early Uses of Biocontrol Methods in Agriculture

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3.3 Microbial Secondary Metabolites

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3.4 Rhizobacterial Secondary Metabolites and Biological Control 3.4.1 Antibiotics 3.4.2 Iron Sequestering 3.4.3 Chemical Communication Interference 3.4.4 Priming of Induced Systemic Resistance

34 34 39 40 41

3.5 Regulation of Secondary Metabolites’ Production 3.5.1 Regulation by Root Exudates Composition 3.5.2 Regulation by Microbial Signals (Quorum-Sensing Signals)

42 43 43

3.6 Microbial Metabolites and Biopesticides Development

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3.7 Concluding Remarks

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References

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3.1 INTRODUCTION For more than a century, farmers and scientists have been aware of the existence of the so-called “suppressive soils.” In these soils, the manifestation of soilborne plant diseases—caused by a preexistent or an inoculated pathogen—is either kept to a minimum or completely absent, even if a susceptible plant host is cultivated in there. The reasons explaining this lack of infectivity are diverse: the pathogens may either fail to colonize or persist in these soils; if they are established, they may cause little or no damage to crops; or they may infect crops and cause some disease symptoms at first but, with successive cropping, the disease declines [1,2]. Even though some abiotic factors may account for this suppressiveness (including pH, organic matter, and/or clay content), this intriguing phenomenon is very often the consequence of soil microbial activity [3]. Indeed, when a plant pathogen is introduced into a naturally suppressive soil, the severity of the disease it causes is attenuated or suppressed by the activity of the indigenous microbial communities. This suppression may be either general (i.e., due to the antagonistic activity of the entire microbial community) or specific (i.e., due to the

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activity of a specific group of microorganisms acting against a specific pathogen). Whatever the case, suppressive soils clearly illustrate the natural ability of some soil microorganisms to act as biocontrollers of plant pathogens. Microorganisms proliferate in large numbers in natural soils. These highly heterogeneous environments stand out as one of the environments most densely colonized by microbes, including bacteria, fungi, algae, viruses, and protists. However, the survival and functions of soil microbes depend on the availability of nutrients, a factor that often limits their growth. That is why microorganisms thrive and multiply in huge numbers in the zone surrounding plant roots, the “rhizosphere.” Indeed, this zone is directly influenced by the root exudates (rich in organic molecules like sugars, amino acids, and organic acids), which act as both microbial attractors and nutrients of a myriad of microbial species. Many of these microbes are plant pathogens and, as such, they cause diseases in and even the death of plants. Others, however, are beneficial and able to promote plant growth—these are collectively known as plant growth-promoting microorganisms (or PGPM) [4]. Several mechanisms have been shown to lie beneath the plant growth-promoting ability of PGPM. For instance, some plant growth-promoting rhizobacteria (PGPR) provide or facilitate access to a number of essential nutrients to plants, such as N or P, and are known as biofertilizers. Nitrogen-fixing bacteria (NFB) are the most widely used biofertilizers to enhance crop yield, particularly through the improvement of root nodulation and biological fixation of N2 in legumes [5]. Another group of biofertilizers, namely phosphate-solubilizing bacteria (or PSB), are also used, although less frequently. PSB are a heterogeneous group of PGPR, characterized by their ability to readily and efficiently solubilize mineral forms of inorganic P. On the other hand, organic P mineralizing microorganisms hydrolyze organic forms of P (phosphate esters, phosphonates, and anhydrides) by secreting specific enzymes (mainly phosphatases) [6,7]. This process, called “substrate mineralization” is of fundamental importance in the release of plant available orthophosphate; however, it is often undervalued. In some cases, a single microorganism is able to perform simultaneously inorganic P solubilization and organic P mineralization [8]. Another group of PGPR, namely phytostimulators, are phytohormone-producing microorganisms, which promote plant growth in a direct way by synthesizing and releasing auxins, gibberellins, citokinins, and nitric oxide [9]. These hormones induce important physiological changes in the plant host, including (1) cell elongation in the subapical region of the stem; (2) promotion of germination, stem elongation, flowering, and fruiting; (3) regulation of cell division and differentiation processes in meristematic tissues; and (4) modulation of metabolic, signaling, defense, and developmental pathways in plants. One last group of PGPR, called bioprotectants, biopesticides, or biocontrollers promote plant growth by antagonizing plant pathogens [10,11]. The mechanisms by which biocontrollers exert their mode of action are diverse and have been the subject of several excellent reviews (see, for instance, Refs. [12 16]). Very often these mechanisms of biologic control rely on the production and excretion of microbial secondary metabolites, which either kill, inhibit, diminish, or deceive natural plant pathogens. Even though there are still many uncertainties on how PGPR promote plant growth in different crops and under different environmental conditions, it is becoming increasingly clear that a single microorganism may exhibit several of these plant growth-promoting traits, and that they do not work independently of each other but additively [17]. The rational use of PGPR in the field of agriculture is nowadays a well-established technology that has permitted the significant reduction in the use of agrochemicals, positively affecting the environment by minimizing the risk of deleterious and hazardous environmental effects resulting from the misuse and abuse of agrochemicals, particularly pesticides [18]. In this context, PGPR and their metabolites are of paramount importance for the development of sustainable agriculture practices.

3.2 EARLY USES OF BIOCONTROL METHODS IN AGRICULTURE The use of living organisms and/or their metabolites to control plant diseases and pests can be traced back to the 17th century. For instance, nicotine—an alkaloid found in the tobacco plant—was used in 1690 in France, and later in England, to control insect pests of fruit trees, like aphids or lace bugs [19]. Several nicotine-containing compounds were developed thereafter and used either as insecticides or repellents. However, the first report concerning the role of microorganisms as pathogens of insects (i.e., biocontrollers) is attributed to Agostino Bassi, an Italian entomologist. Beginning in 1807 and for more than 30 years, Bassi conducted an extensive series of experiments which allowed him to demonstrate, among other things, that a microscopic fungus (eventually named Beauveria bassiana in his honor) was the causal agent of the muscardine disease of silkworms, a lethal infection [20]. Beside representing a pioneering demonstration of what later was known as the

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“germ theory of disease,” Bassi’s experiments clearly opened the gates to show that some microorganisms could be used to kill insects and, consequently, to protect any organism attacked by insect pests, plant crops included. Nevertheless, it was still almost a century before the first demonstration of the practical use of microorganisms in the field of agriculture, to control plant diseases. Indeed in 1901 a Japanese biologist, Shigetane Ishiwata, isolated a bacterial strain from a diseased silkworm; ten years later the bacteria was scientifically described and named Bacillus thuringiensis by Ernst Berliner, a German scientist [21]. French scientists began to use B. thuringiensis as a biological insecticide during the early 1920s and the first commercially available Bt product, Sporeine, appeared in France in 1938. As soon as the first reports on the efficacy of Bt-containing products reached the United States in the 1950s, biological control methods began to be applied widely in this country. In fact B. thuringiensis still remains the most widely used biocontrol microorganism to this day. B. thuringiensis kills insects by means of synthesizing two toxic proteins (Cry and Cyt) that are able to lyse midgut epithelial cells by forming pores on their membranes [22]. Incidentally, aside from exhibiting toxicity against insects from different orders (including Lepidoptera, Coleoptera, and Diptera), Cry and Cyt are also active against other plant pests, such as nematodes. These two proteins have been used, in their purified form, in sprays and dusts as insecticides since 1930. Moreover, the genes encoding Cry and Cyt have been successfully cloned in the genomes of several crops to develop transgenic plants. These Bt-transgenic crops express the transgenes on their tissues, and are thus able to defend themselves from herbivore insects. By 2013, 75.87 million hectares (or 187.5 million acres) of Bt-transgenic crops were planted worldwide [23]. Without entering the debate concerning the development and use of GMOs, which is well beyond the scope of this chapter, there is no doubt that the B. thuringiensis model clearly illustrates the potential of microbial-borne compounds to be used as an ecologically friendly alternative to synthetic chemical pesticides. Incidentally, aside from Cry and Cyt toxins, some B. thuringiensis strains also produce and secrete low molecular weight β-toxins, a group of nonproteinaceous and thermostable secondary metabolites toxic not only against a wide range of insects but also against mammals [24]. As previously stated, secondary metabolites produced by a vast group of PGPR have reached the forefront of biocontrol strategies in the agricultural field.

3.3 MICROBIAL SECONDARY METABOLITES Secondary metabolites have been traditionally distinguished from primary metabolites based on a rather ambiguous criterion, that is, their dispensability [25]. In other words, the producer organisms (or microorganisms) can survive and proliferate without synthesizing these metabolites; on the contrary, primary metabolites, almost universally distributed, are vital for the normal functioning of cells. This definition by exclusion, first proposed by Kossel [26] more than a century ago, has been the subject of a strong controversy. Notwithstanding this debate, microbial secondary metabolites are often described as low molecular weight, bioactive compounds, which are usually produced and excreted by microbes during a particular period or phase of their growth cycle [25,27]. For many years a characteristic feature of secondary metabolites was their restricted taxonomic distribution. However, the information gathered thanks to the use of recent technological developments is modifying this perception and increasingly showing that the production of secondary metabolites is more widespread, from a taxonomic standpoint, than previously acknowledged [28]. On the other hand, and even acknowledging their dispensability, the widespread production of microbial secondary metabolites in natural environments, together with the evolutionary conservation of their encoding genes in microbial genomes, suggest that they might have important functions for the survival of the producers. In fact the production and excretion of secondary metabolites by rhizospheric microbes may improve their ecological fitness in various ways: by improving nutrient availability, by providing protection against environmental stressors, by repelling or killing predators, by displacing competitors, by interfering with chemical signals between microbial cells, and/or by favoring the persistence of their plant host either by modulating their defense mechanisms or by killing herbivore insects and plant-parasitic nematodes (PPN). As outlined by Demain and Fang [29], microbial secondary metabolites perform several biological roles, which include (1) chemical weapons used against other organisms; (2) metal transporting agents; (3) agents of symbiosis between microbes and plants, nematodes, insects, and higher animals; (4) sexual hormones; and (5) differentiation effectors. Careful examination of the aforementioned list intuitively suggests that competition is the force driving evolution of the metabolic pathways which lead to the production of secondary metabolites and to the diversification of their chemical structures [27].

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Besides being structurally highly diverse, another characteristic feature of secondary metabolites is that they exert their biological effects at very low concentrations. That is the reason why, beyond their important activity as chemical weapons in microbial warfare, they are also recognized as chemical messengers (semiochemicals) among soil inhabitants [30]. However, despite the importance of the ecological roles played by soil microbes, along with the biological functions performed by their metabolites, most of the studies concerning interspecies communication in nature has been devoted to interactions between plants and insects [31]. Luckily, this situation is starting to change and researchers are increasingly concerned with studying chemically based interactions among microbes. In the particular field of sustainable agricultural practices, the production of microbial secondary metabolites has received increased attention in the last 15 years owing to its potential for practical applications: biological control of plant diseases and pests. Indeed a nonexhaustive search in PubMed database shows that the number of scientific publications dealing with this topic increased from 21 in 2000 to more than 160 in 2016. In the following pages we will review some of the most recent findings and proposals concerning the potential use of biocontrol rhizobacteria and their secondary metabolites for the development of sustainable agricultural practices. Among the modes of action of these bacterial metabolites, we will concentrate on four major mechanisms of antagonism: antibiosis, iron sequestering, chemical communication interference, and induction of plant systemic resistance.

3.4 RHIZOBACTERIAL SECONDARY METABOLITES AND BIOLOGICAL CONTROL 3.4.1 Antibiotics The importance of antibiotic-producing soil microorganisms as biological controllers of plant pathogens, both in vitro and in the field, has been recognized for more than 70 years [32]. Under the generic term of “antibiotics” we can find a chemically heterogeneous group of organic, low molecular weight compounds produced by microorganisms, which—when present at low concentrations—may inhibit the growth of other microorganisms by interfering with their metabolism [33,34]. One particular feature, characteristic of antibiotics produced by soil bacteria, is their broad spectrum of activity, which means they can affect very diverse microorganisms belonging to different domains of life, including other bacteria, fungi, oomycetes, nematodes, protozoa, and insects [12,35 38]. On the other hand, since one particular bacterial strain may produce and excrete several different antibiotics [13,39], a given biocontrol bacterial strain may suppress multiple plant diseases [12]. The number of microbial species able to produce bioactive metabolites identified so far is huge: more than 25,000, including bacteria, actinobacteria, and fungi [40]. Between 60,000 and 80,000 compounds have been reported to be microbial secondary metabolites. Of these around 47% exhibit some kind of biological activity and between 25,000 and 27,000 are antibiotics. In fact more than 23,000 natural products with antibacterial activity are known to be produced by microorganisms [41]. Soil microorganisms are, by far, the most important natural producers of antibiotics. In fact, actino-, cyano-, and myxobacteria, Bacillus, Pseudomonas, Penicillium, and Aspergillus, produce approximately 80% of all known bioactive secondary metabolites [40]. If we consider that (1) the amount of microbial species living on Earth may reach almost one trillion (1012) [42]; (2) the enormous majority of all microbial species (  99.999%) remain to be identified; and (3) we are currently capable of culturing less than 1% of this microbial biodiversity, it becomes evident that natural soils represent a vast reservoir of a still unknown diversity of antibiotics, with a tremendous potential for biotechnological purposes. Mostly, because it is becoming increasingly clear that the majority of these bacteria may in fact be culturable [43]. Notwithstanding the existence of such a vast reservoir of biodiversity, when considering the management of plant pathogens in the context of sustainable agricultural practices, antibiotics have been thoroughly studied almost solely in two bacterial groups, namely Pseudomonads and Bacillus [44]. Therefore the following examples are closely related to these two bacterial genera and are presented to illustrate their biocontrol activities against plant pathogens. We will first focus on water-soluble compounds—highly polar and able to act at short distances—and then we will review some aspects concerning the role of volatile organic compounds (VOCs)—able to diffuse for long distances in the rhizospheric milieu, through air- and water-filled pores.

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3.4.1.1 Water-Soluble Antibiotics Phenazines: these tricyclic aromatic molecules, analogues of flavin coenzymes, are able to inhibit electron transport through respiratory chains and are known to have various pharmacological effects on eukaryotic cells [45]. In the presence of ferripyochelin (a type of siderophore, see Section 3.4.2), phenazines catalyze the formation of hydroxyl radicals, which damage lipids and other macromolecules [46]. Phenazines are mainly produced by pseudomonads, such as Pseudomonas fluorescens and P. clororaphis, which have been shown to effectively antagonize fungal plant pathogens [47]. However, other Gram-negative and Gram-positive bacteria may produce phenazines as well [48 50]. Phenazine-1-carboxylic acid, synthesized by fluorescent pseudomonads, is effective against various fungal and bacterial pathogens such as Gaeumannomyces graminis var. tritici, Pythium sp., Polyporus sp., Rhizoctonia solani, Actinomyces viscosus, Bacillus subtilis, and Erwinia amylovora [51]. Phenazine-1-carboxamide, produced by P. aeruginosa, effectively inhibits Aspergillus niger, Helminthosporium spp., and Fusarium oxysporum [52]. Another well-studied phenazine, pyocyanin, is toxic not only to bacteria and fungi, but also to mammal cells because of the formation of reactive oxygen species [48,53 55]. Pyrrolnitrin: this chlorine-containing phenylpyrrole, which originates from tryptophan [56], is produced by fluorescent pseudomonads such as P. fluorescens [57] and P. aureofaciens [58]. Pyrrolnitron acts as an inhibitor of fungal respiratory chains [59]. Its action also depends on the damage to the cell wall by an exchange reaction with phospholipids [60]. Pyrrolnitrin has wide range antifungal activity; indeed, it is active against deuteromycete, ascomycete, and basidiomycete fungi [61]. Furthermore, pyrrolnitrin was also reported to be active against several Gram-positive bacteria and in particular Streptomyces species [62]. P. fluorescens strains which produce pyrrolnitrin have been used as biocontrol agents against R. solani infection in cotton [63,64]. Also some synthetic analogues of pyrrolnitrin, such as fenpiclonil but mainly fludioxonil, have been used as fungicides for agricultural uses for over 25 years [65]. Lipopeptides (LPs): these compounds, containing a lipid tail with a linear or cyclic oligopeptide, are synthesized by large nonribosomal peptide synthetases with a modular organization [66]. Cationic LPs are said to integrate inside the bacterial membrane and to form a macropore, which depolarizes/disintegrates the membrane and/or facilitates peptide diffusion inside the cell [67]. Some LPs of Bacillus spp. can also chelate cations (e.g., Ca21). Bacillomycin D, an LP produced by Bacillus amyloliquefaciens FZB42—a well-known PGPR—has been shown to be active against F. oxysporum [68]. Mycosubtilisin, another LP, is responsible for the antagonistic properties of some bacilli against various fungal pathogens, such as Botrytis cinerea, F. oxysporum, and Pythium aphanidermatum [69]. Surfactins, the best-studied LPs, protect B. amyloliquefaciens FZB42 against other bacteria and enable biofilm formation during surface colonization [70]. Bais et al. [71] confirmed this protective action and showed that surfactin is essential during the colonization of roots by B. subtilis 6051 strain. In addition, they also demonstrated that Arabidopsis plants colonized by this biocontrol PGPR are protected against Pseudomonas syringae infections. Cyclic lipopeptides (CLPs): include biocontrol active substances as well as toxins of phytopathogenic pseudomonads, which have surfactant properties and are able to insert into membranes and perturb their functions. Other CLPs may bind lypopolisaccharide-bound divalent cations and/or lipids, interfere with cell wall biosynthesis, delocalize essential membrane proteins, or depolarize cell membranes [72]. Altogether these activities result in a broad antibacterial and antifungal spectrum. Polyketides: are a large class of very diverse secondary metabolites, containing multiple β-hydroxyketone or β-hydroxyaldehyde functional groups, which may act as antibiotics, antifungals, cytostatics, and insecticides, among other biological functions [16]. Polyhidroxiphenols such as 2,4-diacetylphloroglucinol (DAPG), produced by Pseudomonas species, belong to this class [73]. DAPG causes membrane damage to Pythium spp. cells, inhibiting mycelial growth and asexual sporulation [74] (Fig. 3.1). The importance of DAPG as an effector of the biocontrol activity exhibited by native strains of P. fluorescens has been clearly demonstrated in the suppression of take-all diseases of wheat (G. graminis var. tritici) [75] and in several other diseases affecting roots and seedlings of a variety of crops [12]. It has been recently shown that the ability to produce DAPG increases the ability of Pseudomonas brassicacearum LBUM300 to form biofilms and to establish in the rhizosphere of tomato ( 5 rhizocompetence) [76]. 3.4.1.2 Microbial Volatile Organic Compounds Microbial volatile organic compounds (MVOCs) are small molecules (100 500 Da) that can evaporate easily owing to their high vapor pressure and low boiling point [77]. These properties make it possible for them to

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FIGURE 3.1 Antagonism between PGPR and pathogenic fungi mediated by soluble compounds. In dual culture assays both microorganisms are grown on top of an agarized medium. The production of soluble inhibitory compounds (antibiotics) is detected by the development of an inhibition halo around bacterial colonies. The asterisk shows the position of Pseudomonas protegens CHA0 colonies. PGPR, Plant growthpromoting rhizobacteria. Source: L.A. Yarza´bal (unpublished).

diffuse very easily, over long distances, through air- and water-filled pores, particularly in the rhizospheric environment. The chemical structure of MVOCs is very diverse and the composition of the blend of VOCs produced by a given species can include dozens of molecules, but the following chemical classes are the most frequent in bacterial volatilomes: alkenes, alcohols, ketones, terpenes, benzenoids, pyrazines, acids, and esters [78]. Even though from a traditional viewpoint MVOCs are seen as side products of the primary and secondary metabolism, this idea is starting to change because many of them exhibit well-established biological activities [16]. Furthermore, thanks to the development of appropriate detection techniques (e.g., Gas Chromatography Mass Spectrometry and the more sensitive Quadrupole Time-of-Flight GC MS), studies addressing the production of MVOCs by microbes in their natural environment and the ecological roles these VOCs might play have increased significantly in the last 15 years. MVOCs have been mostly considered in the context of the antagonistic interactions that take place between the components of complex soil microbial communities. In particular, studies depicting their antifungal and antibacterial activities present MVOCs as tools with a vast potential to develop new and efficient biocontrol strategies in the agricultural field [77 79]. However, MVOCs can perform other biological functions, including growth stimulation of other microbial species, induction of microbial protective responses, attraction of predators, and/or communication between microbes and their host plants. The last three biological activities indicate that very often MVOCs play the role of chemical messages or semiochemicals, between individuals of the same species and even between different species [80]. MVOCs are fundamental in the establishment of intra- and interspecies interactions between different soil inhabiting microorganisms [81]. Besides acting as inhibitors of other microorganisms, their best understood biological role, they can also play other ecological functions, some of which are rather unexpected [79]. As we have stated before, the rhizosphere is densely colonized by microbes that compete with each other for nutrients and space. It is therefore not surprising that the main ecological roles played by MVOCs are related to their function as chemical weapons against other microorganisms. Some examples that emphasize the role played by these volatile metabolites in the context of plant protection against plant diseases and pests follow. 3.4.1.2.1 Antifungal Activity Several studies depicting the antifungal activity of many bacterial VOCs (bVOCs) have been published in recent years [77,82,83]. Among the effects caused by some of these bVOCs, inhibition of mycelium growth and spore germination are the most frequently cited (Fig. 3.2). For instance, VOCs emitted by rhizobacterial isolates of Serratia plymuthica, S. odorifera, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, P. fluorescens, and P. trivialis inhibited the growth of R. solani [84]. Later, the same research group extended their observations to a series of fungal pathogens, including Microdochium bolleyi, Paecilomyces carneus, Phoma betae, Sclerotinia sclerotiorum, Verticillium dahliae, R. solani, Penicillium sp., and Neurospora crassa [85]. The inhibition of mycelial growth sometimes attained 98%. In another study, VOCs

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FIGURE 3.2 Effect of volatiles produced by biocontrol PGPR on the growth of Fusarium oxysporum. In this kind of assay, the volatileproducing bacteria are inoculated on the bottom plate (as a lawn) and the fungus on the top plate. Both plates are sealed and incubated for several days. (A) negative control of inhibition (no bacteria on the bottom late); (B) Pseudomonas protegens CHA0; and (C) unidentified PGPR. PGPR, Plant growth-promoting rhizobacteria. Source: L.A. Yarza´bal (unpublished).

produced by Collimonas spp., S. maltophilia P61, and Serratia plymutica PRI-2C, completely inhibited growth of all fungi tested over eight (saprotrophic and plant pathogenic) fungi [82]. Furthermore, the sulfur containing volatile S-methyl thioacetate, emitted by Pseudomonas donghuensis P482, significantly inhibited R. solani growth [86]. In another illustrative example, Giorgio et al. [87] demonstrated that rhizobacteria able to protect bean plants from bacterial blight caused by Xanthomonas axonopodis pv. phaseoli var. fuscans, did also produce in vitro VOCs which strongly inhibited growth of several pathogenic fungi, including B. cinerea, Fusarium equiseti, F. oxysporum, Macrophomina phaseolina, Phytophtora cactorum, P. nicotianae, Phytium ultimum, R. solani, Rosellinia necatrix, S. sclerotiorum, and V. dahliae. Furthermore, some of the pure VOCs emitted by two Pseudomonas strains were particularly active as inhibitors of mycelium growth and sclerotia germination [88]. Among these, acetic acid and 2-nonanone elicited a strong hemolytic activity against red blood cells, which correlated with ultrastructural alterations of fungal organelles. Among bVOCs, sulfur compounds and alkyl sulfides have been shown to completely inhibit fungal growth [82]. For example, high concentrations of dimethyl disulfide (DMDS), 2 undecanone, dimethyltrisulfide, 4octanone, S-methylmethane thiosulfonate, and 1-phenylpropan-1-one, emitted by Burkholderia ambifaria, significantly inhibited growth of two phytopathogenic fungi, R. solani and Alternaria alternata [89]. Besides, application of DMDS produced by a Bacillus cereus strain significantly protected tobacco (Nicotiana tabacum) and corn (Zea mays) plants against B. cinerea and Cochliobolus heterostrophus, respectively, through activation of the plant’s ISR [90] (see Section 3.4.4). In a surprising discovery, an ectosymbiotic strain of Achromobacter sp. appeared to modulate fungal pathogenicity through the emission of DMDS. Indeed when colonized by a DMDS-producing Achromobacter strain, F. oxysporum MSA35 did not produce any disease in lettuce. However, when the fungus was cured from the bacterial symbiont, it behaved as a pathogenic strain [91]. Incidentally, fungal and oomycetal volatiles can also play important roles in long distance interactions with bacteria. Indeed, it has been shown that fungal VOCs (fVOCs) can lead to specific phenotypical responses in some bacterial species. Among the effects elicited by fVOCs, significant alterations of both swimming (individual cells moving in more liquid environments) and swarming (direct, signal-dependent movement powered by rotating flagella) have been reported [79]. 3.4.1.2.2 Antibacterial Activity In 2011 Romoli and coworkers [92] demonstrated that bacteria, particularly Pseudoalteromonas strains, are able to emit a mixture of VOCs that inhibit the growth of Burkholderia cepacia complex (Bcc). Later on, different studies confirmed the antibacterial activity of diverse Antarctic bacteria against other pathogenic bacteria [93 95]. In the context of rhizospheric inhabitants, the antibacterial activity of some bVOCs have been also demonstrated. For instance, albaflavenone—a sesquiterpene emitted by an odoriferous Streptomyces albidoflavus isolate from corn

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seeds—was shown to be active against B. subtilis [96]. Collimonas spp. are also producers of terpenes able to inhibit bacterial growth. This is the case, for example, of C. pratensis Ter 91 which produces at least four monoterpenes, one of which—namely β-pinene—is able to inhibit Staphylococcus aureus and R. solani [97]. VOCs produced by some strains of P. fluorescens and S. plymuthica inhibited the growth of Agrobacterium tumefaciens and A. vitis strains in vitro. As seen above, it seems that DMDS is one of the most important VOCs involved in this inhibition. Furthermore, this volatile was emitted by tomato plants treated with S. plymuthica [98]. DMDS also suppressed the growth of the cyanobacterium Synechococcus sp. [99]. Finally, DMDS produced by two Serratia strains was shown to kill A. tumefaciens cells in mature biofilms and to suppress their formation [100]. Also VOCs produced by the biocontrol strain B. amyloliquefaciens SQR-9 were active against R. solanacearum, the tomato wilt pathogen, both in vitro and in soil. Among the traits affected are the following: motility, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides [101]. A proteomics analysis showed that many proteins involved in the manifestation of virulence traits of R. solanacearum were downregulated in the presence of these VOCs. Among these proteins were several related to antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding, and translation. Similarly, some virulence traits of A. tumefaciens—such as the ability to form biofilms—were suppressed when cells were exposed to VOCs emitted by Pseudomonas chlororaphis 449, P. fluorescens B-4117, S. plymuthica IC1270, as well as Serratia proteamaculans strain 94 [100]. Among the ketones emitted by the tested Pseudomonas strains, three of them—namely 2-nonanone, 2-heptanone, and 2-undecanone—did not only disrupt biofilm formation but killed individual cells of A. tumefaciens. Recently, Montes Vidal et al. [102] demonstrated that newly discovered longchain aliphatic nitriles, volatiles emitted by both Gram-positive Micromonospora echinospora and Gram-negative Pseudomonas veronii bacteria, showed antibacterial activity against B. subtilis, Micrococcus luteus, and especially against S. aureus, including resistant strains. It is not clear how these MVOCs act on their targets. However, it has been proposed that their hydrophobicity might enable them to partition the lipid bilayer of biological membranes, increasing their permeability. This has been shown to occur, for instance, in the case of monoterpenes, which are able to disrupt the integrity of the plasma membrane of S. aureus and E. coli, leading to leakage of intracellular materials [103]. Whatever the case, and considering that MVOCs can spread over a long distance, it has been proposed that they can create a bacteriostatic microenvironment around the antagonist communities of producers, keeping pathogens away from the rhizosphere of plants. This effect would rely not only on a reduction of the growing rate of the pathogens, but also in a restriction of their movements to invade plant roots and a significant inhibition of other important virulence traits [101]. 3.4.1.2.3 Nematicidal Activity Among rhizospheric soil inhabitants, PPN are one of the most prevalent. These helminths, of which more than 4100 species have been recognized [104], cause serious damages to plants and crops and are responsible for financial losses estimated well above US$ 80 billion per year [105]. Several microbial secondary metabolites have been shown to act as effectors of antagonistic relationships between PPN and other microbial colonizers of the rhizosphere. The particular role played by mVOCs in this respect deserves to be mentioned in some detail. Hydrogen cyanide (HCN), produced by some species of Pseudomonas and particularly by P. fluorescens CHA0 strain, inhibits mitochondrial cytochrome oxidase and has been shown to effectively kill juvenile specimens of Meloidogyne javanica in vitro and to inhibit egg hatch [106]. By doing this, P. fluorescens CHA0 effectively protects tomato seedlings from root-knot disease caused by M. javanica. Besides, strain CHA0 exhibits a biocide action against nematodes and inhibits galling in tomato roots grown in soil inoculated with eggs or juveniles. Hydrogen sulfide (H2S) is another VOC produced by several bacterial species. Pioneer works demonstrated that, when applied exogenously in flooded rice fields at concentrations found under normal conditions, H2S killed 100% of nematodes in a few days [107]. The nematicidal effect of H2S, in combination with other bacterial metabolites, was also shown to be effective against Xiphinema index [108]. Surprisingly, Miller and Roth [109] recorded some positive effects of H2S on Caenorhabditis elegans, at lower concentrations though, including an increased thermotolerance and an extended life span. Other MVOCs such as alcohols, aldehydes, ketones, alkenes, and ethers, produced by soil bacterial isolates, were shown to be lethal to Panagrellus redivivus and Bursaphelenchus xylophilus [110]. Nine pure volatiles, extracted and purified from these blends and including phenol, 2-octanol, benzaldehyde, benzeneacetaldehyde, decanal, 2-nonanone, 2-undecanone, cyclohexene, and DMDS, displayed 100% nematicidal activity to both model nematodes. Similarly, volatile compounds emitted by Bacillus megaterium YFM3.25 were active against juveniles and

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eggs of M. incognita. Among these VOCs, 2-nonanone and 2-undecanone exhibited strong nematicidal activities ( . 80%) to both juveniles and eggs [111]. 3.4.1.2.4 Insecticidal Activity The ability to readily and effectively colonize insects and to cause lethal infections has been shown to be a feature exhibited by many strains throughout the P. fluorescens group. This lethal effect has been observed not only when bacteria belonging to strains of the so-called P. chlororaphis subgroup [112,113] are injected, but also when ingested by insect larvae [38,114]. However, the mechanism which lies beneath this insecticidal ability remains largely unknown. To date only one bVOC has been unequivocally related to insect killing by rhizobacteria: HCN. Indeed, when exposed to HCN produced by P. fluorescens CHA0 strain, termites are killed as a result of cytochrome oxidase inhibition [115]. Cyanide was also found to be responsible for Drosophila melanogaster mortality, when infected by P. aeruginosa PAO1 [116]. More recently, Flury et al. [38]. showed that HCN-impaired mutants of Pseudomonas protegens CHA0 and P. chlororaphis PCL1391 exhibited a lesser lethality when injected into the hemocoel of Galleria mellonella and Plutella xylostella larvae. The results are in line with the proposed role of HCN as one of the most important volatile effectors of biocontrol elicited by some rhizobacterial strains against insects.

3.4.2 Iron Sequestering Since iron is an essential nutrient for all living organisms, any event that limits its availability in natural environments can therefore be considered as a biocontrol event. Thus when PGPR compete with other rhizospheric microorganisms for this limited vital chemical resource, they are jeopardizing the possibility of other species to proliferate in this habitat [117,118]. Under iron-limiting conditions, many bacteria and fungi produce and excrete low molecular weight, ironchelating compounds called siderophores, which have a very high affinity for ferric ions [119]. When the siderophores are produced by PGPR, they can control well-known fungal phytopathogens, since bacterial siderophores have a considerably higher affinity for Fe than fungal siderophores [120]. This is the case for pyoverdine, for example, a siderophore produced by some Pseudomonas species, which has been shown to effectively control Pythium, Fusarium, and Gaeumannomyces species and to protect crops such as tomato, potato, wheat, barley, peanuts, and maize from fungal infections [120 123]. In fact beside producing a wide variety of siderophores, pseudomonads mainly produce over 50 different pyoverdines [124]. Another siderophore produced by Pseudomonas sp., namely piochelin, also protects tomato plants from Pythium infection [125]. B. subtilis strains also synthesize siderophores that are active against F. oxysporum, the causal agent of the Fusarium wilt of pepper [126]. This is also the case for siderophores produced by endophytic Streptomyces strains of Azadirachta indica, which also had a high affinity to chelate Fe(III) from soil and, thereby, inhibit growth of several fungal pathogens [127]. For the abovementioned reasons, it has been proposed that siderophores and/or siderophore-producing microorganisms might be an environmentally friendly alternative to hazardous pesticides [128]. In addition to their role in the biocontrol of phytopathogens, it has been also shown that microbial siderophores may also help plants to capture iron cations, mostly when their bioavailability is low [129]. For example, endophytic Actinomycetes from A. indica produce ferrioxamines which contribute to plant Fe nutrition and promote the root and shoot growth, even though the exact mechanism remains poorly understood [127,130]. Concerning the potential role of siderophores in plant protection against pathogens, some PGPR strains go one step further: siderophores induce the plant systemic resistance. This is the case, for example, of pseudobactin—a fluorescent siderophore Pseudomonas putida WCS358—which induces ISR in tomato and bean, allowing them to resist B. cinerea and Collectotrichum lindemuthianum infections [131] (see Section 3.4.4). Among several noncanonic roles played by siderophores it is important to mention another one: their function as antibiotics [132]. In fact, this was the first biological function documented for this kind of iron-binding molecule [133]. Sideromycins, for instance, are a class of molecules in which the bactericidal moiety is bound to a siderophore [134]. If a given microbial strain or species expresses the appropriate machinery, it will capture the sideromycin and transport it—including the antibiotic moiety—into the cytoplasm. This “Trojan horse” strategy increases the antibiotic diffusion through the bacterial membrane by several orders of magnitude and enhances significantly the biological activity of the antibiotic toward target cells [135].

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Albomycin, produced by strains of Actinomyces subtropicus, was the first natural sideromycin antibiotic to be characterized [136]. In fact members of this particular group of Gram-positive, aerobic, mycelial, soil bacteria, collectively known as Actinobacteria, are natural producers of several well-known sideromycins, like ferrimycin and salmycin [137]. Members of the Streptomyces genus, which are frequent colonizers of the rhizosphere of plants, have been shown to antagonize pathogenic fungi or oomycetes like Rhizoctonia sp., Pythium sp., Colletotrichum sp., Phytophthora sp., and Fusarium sp., among others [138]. Therefore it has been proposed that sideromycin production, among many other bioactive compounds, might be involved in crop protection against pathogenic fungi.

3.4.3 Chemical Communication Interference Bacteria colonizing natural environments communicate with each other through a complex array of chemical messengers, a communication mechanism called quorum-sensing (QS) [139]. In this kind of communication, bacterial cells synthesize and excrete diffusible signal molecules, which gradually accumulate in the local environment and are detected by related kins, promoting a synchronous expression of genes by an entire population when a threshold concentration is surpassed. This collective behavior is characteristic of many pathogenic bacteria of plants and animals, which act in a coordinate manner to colonize (invade) their natural hosts, causing the symptoms of the corresponding disease and, eventually, the plant death [140]. Among the chemical messengers synthesized by Gram-negative bacteria, N-acyl homoserine lactones (AHLs) are the most studied. These AHLs diffuse freely across the cell envelope and bind to their cytoplasmic receptors, a family of diffusible transcriptional regulators which, once activated through this molecular interaction, modulate target gene expression [141]. Alternatively, some AHLs are detected by membrane-bound sensor kinases, a family of transmembrane proteins which, following binding, translocate the signal to a cytoplasmic transcriptional regulator through a phosphorelay of signaling cascades. The genes whose expression is modulated through this QS mechanism in pathogenic bacteria are very often related with the manifestation of virulence. Therefore any interference with QS would, theoretically, alter the severity of the corresponding disease these pathogens cause. Interference with QS, known as quorum-quenching (QQ), is very frequent in natural environments and can be achieved in many different ways. By doing so, the phenotypic output of the QS mechanism interfered with is either attenuated or completely inhibited. It would not come as a surprise therefore, that many PGPR may exert their protective action over their plant hosts by interfering with QS of bacterial phytopathogens by any of the aforementioned mechanisms. All the steps involved in QS may be targeted by QQ: at the genetic level, expression of QS genes can be repressed in the emitting cell; the chemical messenger itself may be degraded; and, finally, detection of the signal molecule by the cognate receptor and/or by the regulatory protein may be blocked. QQ has been proposed as a very interesting alternative to be used as an alternative strategy of biocontrol [142]. For example, many microorganisms naturally produce enzymes that degrade AHLs, mainly AHL lactonases or AHL acylases. Both kinds of enzymes are hydrolases that cleave the AHL molecule and are produced by a number of soil bacteria belonging to the Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes [143]. The genes encoding lactonases have been cloned and expressed in plants to fight against bacterially caused diseases with encouraging results [144 146]. However, due to strong opposition against the use and consumption of GMOs in the modern world, other strategies making use of QQ mechanisms are being developed. One of these approaches is inoculation of natural environments (e.g., soil, plant tissues) with microbial strains naturally producing QQ enzymes [147 153]. In a different experimental approach targeting the same biological mechanism, other researchers are experimenting with a biocontrol strategy based on selectively stimulating resident QQ microbes able to degrade AHLs [154,155]. Another unexpected QQ mechanism was discovered when studying some marine algae and their bacterial epiphytes. In a surprising discovery, McDougald et al. [156] demonstrated that certain eukaryotic secondary metabolites, mainly brominated furanones, produced by algae can bind bacterial AHL receptors competitively. This QQ strategy, also called QS signal mimicry, has been found to target both Gram-negative and Gram-positive bacteria and to exert an antimicrobial activity, affecting bacterial QS-dependent behaviors [157]. According to some authors, there is increasing evidence that it is the microbes living endophytically in higher plants that are responsible for producing the mimic compounds and other related metabolites [158].

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Even though there are still no particular examples of AHL mimicry among soil bacteria, it has been shown that some PGPR, such as P. putida WCS358, can indeed produce small molecules (e.g., cyclic dipeptides) that may interact with AHL receptors of diverse origin [159]. Such kinds of cyclic dipeptides were also shown to affect S. liquefaciens swarming [160]. Microbial volatile organic compounds (MVOCs, see Section 3.4.1.2) produced by certain PGPR have been also shown to quench the signal molecules of plant pathogens. For instance, Chernin et al. [161] found that P. fluorescens and S. plymuthica VOCs can interfere with cell-to-cell communication in pathogens belonging to the genera Agrobacterium, Chromobacterium, Pectobacterium, and Pseudomonas. Exposure to these VOCs significantly reduced the amount of AHLs produced by these bacteria, negatively affecting the expression of QS regulated genes. In a follow-up to this work, Plyuta et al. [162] demonstrated that the QQ phenomenon depending on MVOCs might be more widespread than previously acknowledged. Indeed, using three Escherichia coli lux-reporter strains, frequently used as biosensors for AHLs, the authors demonstrated that natural volatile ketones—usually found in bVOCs—are able to modulate the QS response and, to a lesser extent, affect the viability of biosensor cells. According to the authors, this QQ mechanism might be important for displacing bacterial competitors and could be another way to protect plants from their pathogens. In a striking example of evolutionary adaptation to pathogens, it has been shown that plants may respond not only to AHLs released by phytopathogenic bacteria (i.e., Pseudomonas aeruginosa), but also to the ones synthesized by beneficial bacteria. For example, it has been shown that Medicago truncatula, a model legume, responds in a global manner to the presence of AHLs synthesized by the symbiotic, N-fixing Sinorhizobium meliloti by inducing the expression of some genes and, consequently, accumulating at least 150 proteins, many of which are related to plant defense mechanisms [163]. In another example of this interkingdom communication, Schuhegger et al. [164]. further demonstrated that when exposed to AHLs produced by two rhizobacteria, Serratia liquefaciens MG1 and P. putida IsoF, tomato plants increased their systemic resistance against the fungal foliar pathogen A. alternata. It has been speculated that this kind of communication between rhizobacteria and plants may benefit the latter by allowing them to sense the presence of bacteria and to “prepare for the attack” by activating plant defenses. A number of studies have shown indeed that AHLs—whether they are of pathogenic or symbiotic origin—may trigger the expression of genes involved in plant defense [165]. AHL sensing by plants might also promote plant development. For example, exposure of Arabidopsis thaliana to rhizobacterial AHLs was shown to alter its root architecture [166 169]. Similar results were observed in mung beans [170]. Recently Veliz-Vallejos et al. [171] demonstrated that S. meliloti AHLs also stimulate nodulation in M. truncatula.

3.4.4 Priming of Induced Systemic Resistance Interactions between PGPR and plant roots in the soil can result in the priming of ISR in the plant. ISR is a milder form of immune response in plants similar to the one observed after the attack of a pathogen (systemic acquired resistance, SAR). Since plants do not have an adaptive immune system or specialized mobile defense cells, they rely on their innate immunity to respond to a pathogen infection or to signals originating at infection sites [172]. During a pathogen attack, plants can trigger both generic and pathogen-specific responses depending on the type of signals received from the pathogen. Generic responses, termed pattern-triggered immunity (PTI), are launched after recognition of pathogen-associated molecular patterns (PAMPs), such as flagellin, or from damage-associated molecular patterns (DAMPs), such as systemin or oligogalacturonides [173]. Pathogen-specific responses instead are triggered by specific effector molecules (effector-triggered immunity; ETI) generated by the pathogen, whose recognition is highly dependent on the coevolutionary history of the pathogen host pair [172]. Interestingly, based on their molecular identity, PAMPs are not only produced by pathogens, but they can also be generated by nonpathogenic microbes interacting with the plant, a reason why now they are preferentially called microbe-associated molecular patterns (MAMPs) [174,175] to better imply their generic nature. Consequently, given that they can produced common signals, both pathogenic and nonpathogenic microbes are capable of stimulating the receptors that prime PTI [176]. Although nonpathogenic microbes (in the case of this chapter, PGPR) can stimulate the same receptors targeted by PAMPs during infection, the magnitude and characteristics of the PTI responses are not identical. For instance, following a pathogen attack, complex signaling cascades cause a massive transcriptional reprogramming in the host cell that results in the production of proteins whose functions allow a “counterattack” against

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the pathogen (e.g., glucanases, defensins), the reinforcement of protective structures (mainly cell walls), and the prevention of further pathogen cells gaining access to the plant (stomatal closure) [175,177]. In contrast, responses induced by interactions with nonpathogenic microorganisms cause minor, often nondetectable, transcriptional reprogramming in the host without the activation of defense proteins [178]. Also at the plant level, systemic immune responses derived from pathogen attacks are mostly regulated by salicylic acid (SA), whereas ISR responses are regulated mainly by jasmonic acid (JA) and ethylene [179]. Although responses induced by nonpathogen organisms are subtle in comparison with those induced by pathogens, when inoculated with PGPR the plant becomes permanently sensitized to MAMPs and can trigger an accelerated and amplified response in the event of a pathogen attack [178,180]. This sensitization is not only local but also systemic, and since it is derived from PTI, it is also generic. Thus this kind of activation results in an enhanced systemic response to future attacks of a wide variety of pathogens. Consequently, the use of PGPR to induce the plant’s systemic response has attracted increased interest because of its potential for agricultural purposes [181]. The chemical nature of PGPR MAMPs is highly diverse [181]. In the case of PGPR, fragments of cell components such as flagellin, together with some secondary metabolites (e.g., antibiotics, siderophores, LPs/CLPs, and MVOCs) can induce ISR in plants. For instance, the siderophore pseudobactin 358 has been shown to prime ISR in Arabidopsis, bean, and tomato against P. syringae, Colletotrichum lindemuthianum, and B. cynerea, respectively [131]. Likewise, polyketide antibiotics such as DAPG also primed ISR in Arabidopsis challenged with Peronospora parasitica [182]. Also, LPs/CLPs, such as surfactin, fengycin and massetolide A, generated a similar ISR-priming response in beans and tomato plants, allowing for a reduction of disease symptoms after challenge with B. cynerea and Phytophtora infestans [183,184]. Among MVOCs, compounds such as 2,3-butanediol, DMDS, acetoin, and C10 C13 alkanes produced by B. subtilis have been shown to elicit ISR responses in Arabidopsis [90,185 187]. The plant-protecting effects of the aforementioned compounds can be accumulative and synergistic since, as reviewed in previous sections, antibiotics, siderophores, LPs/CLPs, and MVOCs can also directly limit pathogen growth by microbe microbe interactions in addition to their ISR-inducing effects. However, some other bacterial metabolites, such as N-alkylated benzylamine, can induce ISR without any known antagonist effect on pathogen growth [188,189]. Further, it has been recently proposed that most of the protective effect of B. subtilis GB03 VOCs is related to its ISR-priming effect rather than to its pathogen antagonist effects [190,191]. Due to its generic nature, PGPR-induced ISR responses are not specific and could be an effective way to improve the plant’s defense response against different pathogens. For instance, reduced symptoms of diseases caused by F. oxysporum, Colletotrichum orbiculare, P. syringae, and Erwinia tracheiphila have been reported in response to inoculation with the same strain of the PGPR P. putida 89B-27 [192]. Furthermore, the positive effects of P. putida 89B-27 inoculation on cucumber plants were also related to reduced populations of two insect pests, Diabrotica undecimpunctata and Acalymna vittatum, apparently due to a reduction in the concentrations of cucurbitacin, a plant metabolite that acts as a feeding stimulant for these insects [193,194]. Although promising, the feasibility of using PGPR to prime ISR in commercial crops growing under field conditions remains to be determined. Some constraints to the practical applications that need to be overcome are the likely already induced states of crop plants under field conditions and the trade-off of diverting carbon and other nutrients from growth to ISR responses. Since PTI responses are generic and induced by many different organisms, it is likely that as soon as the crops start growing in the field, they will begin interacting with the soil indigenous microorganisms, thus potentially priming ISR and yielding the plant uncapable of developing further induction [195]. These responses can also be induced by environmental factors, because DAMPs generated by abiotic factors such as heat or drought could also induce PTI [173]. On the other hand, activation of ISR associated metabolic pathways represents a diversion of carbon and nutrients needed for growth and development, resulting in reduced fitness at the organism level [196,197]; although in the presence of pathogens, active defense mechanisms will promote fitness [198]. It is noteworthy, however, that to date, growth-defense trade-offs have been evaluated mostly using synthetic molecules and not in vivo PGPR-produced secondary metabolites, and consequently, expectations remain high about the practical development of microbe-derived ISR under field conditions [181].

3.5 REGULATION OF SECONDARY METABOLITES’ PRODUCTION As said in the introductory section of this chapter, one single PGPR strain may exert different beneficial effects on its plant host through the simultaneous expression of several growth-promoting traits [8]. An increasing

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amount of evidence seems to show that these mechanisms do not work independently of each other but additively [17]. On the other hand, since several different PGPR may coexist in the rhizosphere of a given plant, they may act in either an independent or coordinate manner to influence positively their host. Irrespective of the aforementioned facts, the expression of PGPR’s plant beneficial genes is tightly regulated at the genetic level. In the particular case of protective secondary metabolites, their production depends on both abiotic factors (like pH, oxygen concentration, clay content, temperature, etc.) and biotic factors [199]. Among the latter, root exudates and QS signals emerge as the most important modulators of microbial secondary metabolite production.

3.5.1 Regulation by Root Exudates Composition Root exudates are composed mainly of sugars, amino acids, organic acids, vitamins, and high molecular weight polymers, thus becoming a rich source of nutrients for the rhizospheric microbes [200]. Aside from representing a strategy to recruit and shape the rhizospheric bacteriome [201], modulation of the composition and abundance of root exudates also influences the expression of a wide group of microbial genes, particularly those related to plant-beneficial traits. A good example of this regulation was revealed by Duffy and De´fago [202], by showing that specific components of root exudates affect the production of antibiotics, such as DAPG and pyrrolnitrin, in biocontrol strains of P. fluorescens. This observation received further support from Notz et al. [203] and Jousset et al. [204]. In both reports, expression of phlA—the gene encoding DAPG in P. protegens CHA0—was shown to depend on the type of exudate to which the bacteria were exposed. Aside from affecting phlA expression, other plant compounds (like flavonoids, phenolic acids, phytohormones, or salicylate) may also modulate the expression of pltA, one of the genes involved in the synthesis of pyoluteorin, a strong antifungal compound [205,206]. Since the composition of root exudates depends on the developmental stage and on the physiology of the plants [207], it is not surprising that changes in environmental conditions (such as those related to shifts in altitude, climate, or season) also affect rhizospheric microbial communities. This effect can be seen at the taxonomic but also at the functional levels [208]. Even though the latter is often neglected in studies concerning the functioning of soil microbial communities, some recent observations clearly indicate that this is the case [209].

3.5.2 Regulation by Microbial Signals (Quorum-Sensing Signals) As discussed before, microbes colonizing the rhizosphere communicate with each other by exchanging chemical messengers. The best example of such chemical communication is QS, a mechanism allowing bacteria to monitor the environment and to detect the presence of related kins, in order to coordinate gene expression when a cell density threshold is attained (see Section 3.4.3). As shown before, this kind of chemical communication can be achieved among members of the same bacterial species with a high degree of specificity, even though some examples of cross talk between species have been recorded in natural habitats [210 212]. On the opposite side, QS between related bacterial cells is also subjected to interference and hindrance by chemical signals emitted by different bacterial—and even fungal—species [213]. Strikingly, some plant-derived molecules may also modulate the expression of plant-beneficial traits, by producing compounds that mimic bacterial chemical signals and bind to a different group (subfamily) of bacterial receptors, closely related to canonic QS-receptors [214 218]. This kind of complex interdomain communication appears to be more frequent than previously thought [201]. In a remarkable example of autoregulation, some antibiotics—like DAPG—may enhance their own production, acting thus as chemical signals as well [205,219]. Plants in turn increase root exudation in response to metabolites such as DAPG, resulting in a feedback loop that improves biocontrol of fungal pathogens by rhizobacteria [204,220]. The importance of the intricate chemical dialog established between rhizospheric microbial species is receiving increasing attention by researchers. In fact microbial species do not act alone under field conditions; on the contrary, rhizospheric microbiomes are large, dense, and complex. That is why studies focusing on the use of combined inoculations of different plant growth-promoting microorganisms, including not only bacteria but also fungi, are increasingly growing. The results obtained clearly show that mixed inoculation of different plant growth-promoting species, and even of beneficial consortia of microorganisms, allow better results to be obtained than for single inoculations [221,222].

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3.6 MICROBIAL METABOLITES AND BIOPESTICIDES DEVELOPMENT In the previous pages we have seen how microbial secondary metabolites act directly as effectors of antagonistic relationships between PGPR and other inhabitants of the rhizosphere. When plant pathogens are affected by such metabolites they are either killed, inhibited, diminished, or deceived; therefore some PGPR strains also behave as biocontrollers of plant diseases and/or pests, playing a dual ecological role in the rhizosphere (Fig. 3.3). This dual behavior explains why many commercial biofertilizers already marketed worldwide, containing one or several microbial strains, exhibit a strong biopesticide effect in addition to the plant growth-promoting effect [223]. The efficacy of such commercial biopesticides does not rely on a unique mechanism. Instead, it relies on several complementary mechanisms among which we can mention hyperparasitism, competition, antibiosis, secretion of lytic enzymes, and induction of a plant’s systemic resistance [224]. This means that one single microorganism is able to synthesize and excrete multiple secondary metabolites of different natures and functions; by doing so their antagonistic effect may be increased because of the synergy between these metabolites. This also explains why one single microorganism may suppress more than one pathogen at a time. For example, two well-known biocontrol agents, namely B. cereus UW85 and P. protegens CHA0 (formerly P. fluorescens CHA0), are active against different pathogenic fungi, owing to the production of multiple antibiotics, both soluble and volatile [225 227]. According to Marrone [228], approximately 1500 biopesticide products were marketed and sold ten years ago. But the increasing trend of bioprospection and innovation in this field during the past decade has promoted an unprecedented number of registrations of similar products. In fact the market is currently shared between large enterprises, such as Syngenta, Bayer, and Monsanto, and numerous small enterprises, including government and

FIGURE 3.3 Interactions between biocontrol plant growth-promoting rhizobacteria (PGPR) and plant pathogens. Negative interactions are depicted as red lines, whereas positive interactions are depicted as green lines. PRB, Pathogenic bacteria; PF, pathogenic fungi; PN, pathogenic nematodes; QS, quorum-sensing semiochemicals; QQ, quorum-quenching mechanisms; MAMPs, microbe-associated molecular patterns (ISR elicitors); ISR, induced systemic resistance; bVOCs, bacterial volatile organic compounds; SA, soluble antibiotics (Copyright: Gabriel Yarza´bal Buela, with permission).

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nongovernmental agencies. Although information about the biopesticide market is scattered and fragmented, its growth rate has been estimated at 20%, with a current value of US$ 2.5 billion and a projected gain of US$ 7 billion by 2019 [229,230]. Where do microbial metabolites stand in this figure? Well, the answer is that, even though there are many microbial metabolites useful to develop environmentally friendly agrochemicals (as many as 23,000, according to Katz and Baltz [41]), only one has been successfully deployed in the global market: DMDS. As seen in Section 3.4.1.2.1, DMDS is a volatile sulfur compound present as a major component in the volatilome of several microbial species. Owing to its ability to control plant pathogenic fungi [231,232], nematodes [233], weeds [234], and even to induce a plant’s systemic resistance [90], an effective soil fumigant based on DMDS was developed and registered in 2010 by Arkema and is currently marketed under the name of Paladin. Approximately 250 field trials were conducted by Arkema in 15 countries from 2004 to 2010 in order to verify the efficacy and crop safety of the new biofumigant. At present, Paladin is marketed worldwide and used to fumigate fields before the planting of crops such as berries (blueberry and strawberry), cucurbits (cucumber, squash, and melon), solanum vegetables (tomato, pepper, and eggplant), field-grown ornamental, and forest tree nursery crops [235]. This biofumigation strategy reduces the incidence of fungal/oomycetal pathogens like Verticillium spp., Fusarium spp., Pythium spp., Sclerotinia spp., and Rhizoctonia spp.; it has also been reported to control nematodes causing root knot (southern, northern, and Colombian) and stubby root. Additionally, Paladin also acts as an herbicide, inhibiting growth of weeds such as nutsedge (purple and yellow), chickweed, lambsquarters, purslane, and several grasses. It was recently demonstrated that even at low doses, DMDS fumigation could drastically reduce the population of the major soilborne tomato and lettuce pathogens F. oxysporum and R. solani during the whole cultivation season [236]. It has been shown that the impact of soil fumigation with DMDS on the activity of beneficial microbes is low [237]. Nevertheless, the use of DMDS as a fumigant is not free of some detrimental effects. In fact DMDS is moderately toxic to mammals and birds, even though at the recommended application rates anticipated exposures are not estimated to rise above levels of concern. However, due to its volatility, strong odor (similar to that of garlic and decaying fish), and low toxicity when inhaled by humans, Paladin has some limitations for applications near densely populated areas and strict safety measures should be followed to reduce the odor. Aside from DMDS-related products, progress in developing new biopesticdes from pure microbial metabolites is slow. Though our understanding on the mode of action of many of these metabolites has improved significantly in the last decade, going “from the lab to the farm”—as stated by Parnell et al. [238]—is a difficult task, still far from being achieved for these molecules. Luckily, some recent field assays show that other MVOCs can perform successfully under open field conditions against both plant pathogens and herbivores [239]. For instance, Song and Ryu [240] demonstrated that 3-pentanol and 2-butanone—produced by soil microbes—were effective at protecting cucumber plants against a bacterial pathogen (P. syringae pv. lachrymans) and a sucking insect aphid (Myzus persicae) in assays conducted under natural field conditions. Similarly, application of (2R,3R)-butanediol—produced by some bacteria—under field conditions was shown to reduce anthracnose symptons in Nicotiana bethamiana seedlings, a disease caused by the hemibiotrophic fungus, C. orbiculare [241]. Further, application of the same compound reduced by 20% 40% the severity of the diseases caused to Agrostis stolonifera (creeping bentgrass) by three fungal pathogens, namely Microdochium nivale, R. solani, or Sclerotinia homoeocarpa [242]. In the aforementioned examples, the priming of the plant’s ISR by MVOCs appeared to be the mechanism involved in plant protection. The abovementioned examples show that in fact pure microbial volatiles or their combinations may ultimately lead to a better control of agricultural pests and diseases.

3.7 CONCLUDING REMARKS As the human population grows, by 2050 food production will need to be increased by 60% relative to 2005/2007 levels [243]. Current technologies have been proven to be unsustainable to produce food given the present and future environmental constraints. Consequently, current agriculture will need to be further intensified, although using a different set of technologies and practices in order to keep this intensification process sustainable. The paradigm shift and development process of these new technologies has been called by Swaminathan the Evergreen Revolution [244], and microbe-based technologies, that take advantage of natural ecological relationships among microorganisms, are part of this revolution.

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Microbes are fundamental players of this Evergreen Revolution, and their rational use to develop bioinoculants with plant growth promotion and biocontrol abilities is crucial. As we have seen, a huge potential lies in rhizospheric microbes, and particularly in their bioactive secondary metabolites. In the past a number of antimicrobial agents have been developed from microbial metabolites into drugs for therapeutic use like streptomycin (Streptomyces griseus), penicillin (Penicillium chrysogenum), bacitracin (B. subtilis), and lovastatin (Monascus ruber). Many of us believe that the same can be achieved in the case of biopesticides. Alas notwithstanding the enormous potential of these metabolites, their reduced toxicity, and increased efficacy, as well as their rapid turnover in natural environments, biopesticides still represent a minute fraction of all the agrochemicals used worldwide (less than 4%), whereas chemical pesticides account for 15% [245 247]. Furthermore, owing to the complexity of natural interactions among microbes and other organisms, their discovery characterization formulation field evaluation cycle is relatively long and slow. In spite of these challenges, increased efforts should be devoted to developing ecofriendly and effective bioinoculants in order to guarantee sustainable agricultural practices to meet the future needs of humanity. The reward is worth the effort.

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Available from: https://doi.org/10.1017/CBO9780511626463.014. [229] Global Industry Analysts, Global biopesticides market to reach US$2.8 billion by 2015, according to a new report by Global Industry Analysts, Inc. Available from: , http://prweb.com/printer/8041130.htm . , 2015. [230] Transparency Market Research, Biofertilizers (Nitrogen Fixing, Phosphate Solubilizing and Others) Market for Seed Treatment and Soil Treatment Applications—global Industry Analysis, Size, Share, Growth, Trends and Forecast, 2013 2019, Transparency Market Research, Albany, NY, 2014. [231] J. Fritsch, Dimethyl disulfide as a new chemical potential alternative to methyl bromide in soil disinfestation in France, Acta Hortic. 698 (2005) 71 76. [232] M. Kai, M. Haustein, F. Molina, A. Petri, B. Scholz, B. Piechulla, Bacterial volatiles and their action potential, Appl. Microbiol. Biotechnol. 81 (2009) 1001 1012. Available from: https://doi.org/10.1007/s00253-008-1760-3. [233] J. Coosemans, Dimetyl disulphide (DMDS): a potential novel nematicide and soil disinfectant, Acta Hortic. 689 (2005) 57 64. [234] J. Freeman, S. Rideout, A. Wimer, Dimetyl disulphide use for bacterial wilt management and weed control in Virginia tomatoes, Hort. Sci. 44 (2009) 571. [235] Environmental Protection Agency (EPA) Pesticide Fact Sheet. Available from: , https://www3.epa.gov/pesticides/. . ./ fs_PC-029088_09-Jul-10.pdf . , 2010. [236] C. Papazlatani, C. Rousidou, A. Katsoula, et al., Assessment of the impact of the fumigant dimethyl disulfide on the dynamics of major fungal plant pathogens in greenhouse soils, Eur. J. Plant Pathol. 146 (2016) 391. Available from: https://doi.org/10.1007/s10658-0160926-6. [237] S.R. Dangi, R. Tirado-Corbala, J.A. Cabrera, D. Wang, J. Gerik, Soil biotic and abiotic responses to dimethyl disulfide spot drip fumigation in established grape vines, Soil Sci. Soc. America J. 78 (2014) 520 530. [238] J.J. Parnell, R. Berka, H.A. Young, J.M. Sturino, Y. Kang, D.M. Barnhart, et al., From the lab to the farm: an industrial perspective of plant beneficial microorganisms, Front. Plant Sci. 7 (2016) 1110. Available from: https://doi.org/10.3389/fpls.2016.01110. [239] C.N. Kanchiswamy, M. Malnoy, M.E. Maffei, Bioprospecting bacterial and fungal volatiles for sustainable agriculture, Trends Plant Sci. 20 (2015) 206 211. Available from: https://doi.org/10.1016/j.tplants.2015.01.004. [240] G.C. Song, C.M. Ryu, Two volatile organic compounds trigger plant self-defense against a bacterial pathogen and a sucking insect in cucumber under open field conditions, Int. J. Mol. Sci. 14 (2013) 9803 9819. Available from: https://doi.org/10.3390/ijms14059803. [241] A. Cortes-Barco, P. Goodwin, T. Hsiang, Comparison of induced resistance activated by benzothiadiazole, (2R,3R)-butanediol and an isoparaffin mixture against anthracnose of Nicotiana benthamiana, Plant Pathol. 59 (2010) 643 653. [242] A. Cortes-Barco, T. Hsiang, P. Goodwin, Induced systemic resistance against three foliar diseases of Agrostis stolonifera by (2R,3R)-butanediol or an isoparaffin mixture, Ann. Appl. Biol. 157 (2010) 179 189. [243] N. Alexandratos, J. Bruinsma, World agriculture towards 2030/2050: the 2012 revision. ESA Working Paper 12-03. Retrieved from: , http://www.fao.org/docrep/016/ap106e/ap106e.pdf . , 2012. [244] M.S. Swaminathan, An evergreen revolution, Crop Sci. 46 (2006) 2293 2303. [245] D.R. Fravel, Commercialization and implementation of bio control, Ann. Rev. Phytopathol. 43 (2005) 337 359. [246] T. Glare, et al., Have biopesticides come of age? Trends Biotechnol. 30 (2012) 250 258. [247] K. Wilson, et al., Pest control: biopesticides’ potential, Science 342 (2013) 799.

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4 Bioengineering of Secondary Metabolites Ali Asghar Rastegari1, Ajar Nath Yadav2, Neelam Yadav3 and Nazanin Tataei Sarshari1 1

2

Department of Molecular and Cell Biochemistry, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib, Sirmour, India 3Gopi Nath P.G. College, VBSP University, Deoli-Salamatpur, Ghazipur, Uttar Pradesh, India O U T L I N E 4.1 Introduction

55

4.6 Enzyme Engineering

65

4.2 Gene Duplication in Idiophase

57

4.7 Conclusion

66

4.3 Evolution of New Pathways

58

Acknowledgment

67

4.4 Bioengineering of Terpenoids in Plants

59

References

67

4.5 Metabolic Engineering and Microbial Biogenesis of Plant Isoprenoids

62

Further Reading

68

4.1 INTRODUCTION Plants produce a remarkable combination of low-molecular weight compounds. Though structures of close to 50,000 of these have been elucidated, there possibly exist hundreds of thousands of these compounds. These compounds constitute a significant part of the primary metabolic pathways common to all organisms and are called secondary metabolites. This term is old and originally related to inequality, but now the secondary metabolite is a distinct combination whose biosynthesis is incomplete in defining plant collections. The capability to synthesize secondary compounds throughout the evolutionary period is selected in dissimilarity of plants when such compounds actualize precise supplies Fig. 4.1. The extracts contain floral roots and the released pigments are pollinated by absorbers, and if they are consumed, they increase the fertilization rate [1 3]. The capability to synthesize toxic chemicals is progressive in supplementing pathogens and herbivores (from bacteria and fungi to insects and mammals) or in preventing plant growth. Most chemical compounds in fruits decrease the accidental degeneration and features such as color, aroma, and flavor signaling the availability of potential rewards like sugar, vitamins, and amino acids to attract Frugivorous animals and ultimately allow seed dispersion. Other chemicals serve cellular functions that are unique to the particular plant in which they occur (e.g. resistance to salt or drought) [4 10]. The enhancement of cellular phenotypes through the outline of genetic controls is an essential subject in metabolic engineering—that is to overproduce metabolic. For instance, metabolite overproduction can be

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4. BIOENGINEERING OF SECONDARY METABOLITES

FIGURE 4.1 Secondary plant metabolites with future functions in plants from which they were remote. (A) The substance Rutin is extracted from Forsythia Intermedia and acts as a visual pollinator. (B) Rotenone is extracted from Derris elliptica and acts as an insecticidal agent. (C) Linalool is extracted from Clarkia breweri and acts as an olfactory pollinator. (D) Berberine is extracted from Berberis wilsoniae and acts as a defensive detoxification. (E) 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) is extracted from Zea mays and is assumed to act as a defensive detoxifier. (F) Brassilexin is extracted from Brassica spp. and acts as an antifungal toxin.

noted. Therefore metabolic engineering activities are involved in the characteristics of the general metabolic network and are involved in conflicting differences in the single-gene examination that represents the common genetic engineering demand. This is a difficult task due to the fact that molecular genetic relations are complicated, nonlinear, and uncertain. As a result of incomplete knowledge concerning molecular interactions and their kinetics, the analysis, assessment, and recognition of perfect metabolic pathways is of highest concern in metabolic engineering. A significant elimination to the absence of kinetic information is the limits performed upon metabolic function by the stoichiometry of the reaction network [11,12]. Consequentially, rarer requests increased from reduced complete rich systems, or from gene manipulation not directly associated with the product synthesizing pathway. These lesser understood systems have traditionally been handled by sequential approaches. Whereby a single gene is found (usually from a combinatorial search) to affect significant phenotype, and consequent study is prepared in a genetic-linked resolute by the removal or overexpression of the abovementioned gene. In spite of developments using this view, there is no indication such that combined research, or that after the sequential modification, global phenotypes will be optimized. In addition, it is not known how to determine the gene targets approving the entire network [12,13]. The courses that may obtain the most secondary compounds have not yet been discovered as there are possibly hundreds of thousands of dissimilar enzymes participating in the secondary metabolism in plants. There are many common substances in secondary metabolism, in which the synthesis of multiple productions through a single enzyme, in addition to other infrastructure, hardly, from the same substrate, is activated [14 16]. In the available examples so far, the enzymes in a plant’s secondary metabolism are unique to an expected substrate and yield a single output. Plant genomes containing a range of 20,000 60,000 genes constitute 15% 25% of these enzymesencoding genes for the secondary metabolism [17,18]. The genome of a presumed plant species only encodes a small segment of all the enzymes essential in synthesizing the entire set of secondary metabolites. The focus in this chapter are the molecular evolutionary mechanisms responsible for the emerging massive variety of plant secondary metabolites [10]. Pursuing constant studies in the pharmaceutical field for new molecules, primarily

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4.2 GENE DUPLICATION IN IDIOPHASE

due to the expansion of contemporary drugs to combat new diseases, is essential. And, in the case of infectious diseases, studies on the increasing resistance of microbes to various antibiotics are crucial [19,20].

4.2 GENE DUPLICATION IN IDIOPHASE It is hypothesized that, in primary metabolism, new genes nearly can occur the importance of gene duplication and diverged in sequence [21]. This, in turn, yields a single gene organism that preserves the original function together with its second copy, which is called natural selection. This is the second version that can sometimes be mutated, as long as new functions that may be retained by the public are increased [22]. This raises the question whether gene-rating duplication and divergence lead to generating genes for secondary metabolism? How often does the simple allelic deviation increase the genes? To respond to these questions, one must adapt behavior proportional analyses of orthologous loci from linked groups that constitute the recognition of gene functionality, but this is yet to be shown. If the normal gene has the required behavior, its duplicate representing is a must. In theory, it may be determined for the main allele of a plant genetic location if it can encode the power of producing a different protective agent, while the former alleles are synthesizing an additional protective agent no longer active in threatening the enemies of the plant. Accordingly, in secondary metabolism, there exists a potential for a new generation of genes in order to change the duplication process. There is a possibility that the orthologous genes in linked groups would encode proteins through dissimilar activities. A new enzyme-encoding gene can be described separately from its ancestral status. A gene can be defined as new and different from its ancestral gene when (1) it is able to encode an enzyme that catalyzes a similar reaction chemically, but on a diverse substratum than the stimulant is encoded on by its ancestor gene, or (2) the encoded stimulant is subject to a particular chemical reaction on a similar substratum. It is less possible, alteration to have a single step involving substrate and the type of reactions. The rate of new idiophase genes evolving from both other idiophase genes and from trophophase genes is the raised question here. The latest product in wholegenome sequencing EST databases contains essential data while no clear solutions are found yet. In general, the procedures in which different trophophase genes are founded can be assumed based on their intention level (i.e., their level of sequence identity). Various sequencing projects have discovered many gene “complexes,” the nature and volume of which is presented in Table 4.1. The above mentioned biological clusters are known by their “motifs” in the encoded proteins (consisting of the active site and/or domain acts as the binding site of substrate and cofactors). Because the real behavior of most elements of plant gene complexes are unidentified, the above subjects are not addressed. The large plant gene complex of cytochrome-p450s-dependent oxygenases consists of solely a few members said to be linked to the main metabolisms, like in steroid and phenylpropanoid biosynthesis [23]. This large gene complex consists of elements which were previously recognized as being complicated in idiophases (e.g., menthol and carvone TABLE 4.1

Designated Plant Gene Families With at Any Rate Some Members Complicated in the Secondary Metabolism of Plants

Enzyme gene family

Examples from secondary metabolisma

Number of copies in Arabidopsis

2-Oxoglutarate-dependent dioxygenases

Flavone synthase

.10

Acyl transferases

Acetyl-CoA:benzylalcohol acetyl transferase

.70

Carboxymethyl methyltransferases

S-adenosylmethionine:salicylic acid methyl transferase

.20

Cytochromes p450

DIBOA hydroxylase

.100

Glutathione-S-transferases

Petunia An9 gene

.20

Methylene bridge-forming enzymes

Berberine bridge enzyme

.10

NADPH-dependent dehydrogenases

Isoflavone reductase

.50

O-Methyl transferases

(Iso)eugenol O-methyltransferase

.20

Polyketide synthases

Stillbene synthase

.10

Terpene synthases

Linalool synthase

.20

a

Not from Arabidopsis.

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formations) [24]. Similarly exists in the complex of genes in encoding O-methyltransferase enzymes, connected in trophophase (e.g., the formation of lignin), also in idiophases (e.g., phenylpropene and alkaloid biosynthesis) [1,25]. The supplementary instance is an idiophases biological group of glycosyltransferases that consisting of the gene for carboxypeptidase of primary metabolism [26]. On the contrary, several large biological groups of genes solely containing certain elements with a quantified behavior all dealing in idiophases, we recognized here. For instance, big gene biological groups, counting 70 elements in Arabidopsis, stimulant into coded form for acyltransferases connected in the composite of sundry, stain, and protection substances, modify. Part of the unknown elements of this gene complex could be complicated in trophophase [10].

4.3 EVOLUTION OF NEW PATHWAYS The advances in gene expression are significantly effective on self-development along with opening. These changes can generally be mingled with the source of an original gene. Alike, C. breweri synthesizes Linalool in its petals, while the Clarkia concinna qualified cannot do the same, even if C. concinna has contained a similar stimulant in Linalool Synthase (LIS), to organize C. breweri. In general, C. concinna LIS is generated solely in the stigma with less closeness to expression of C. breweri [2,27]. Consequently, if an idiophase is synthesized in a specific organ of a plant species and its relations do not generate this substance in the same organ, it is important to confirm whether these families yield such a substance outside the plant. If they do this, perhaps it means that no new biosynthetic genes have progressed and the expression pattern of the current biosynthetic genes vary (e.g., through an altered promoter or transcription factor). Biochemical processes do not follow the same path, while here they are exactly involved in the role of the reaction network, and one should not forget the fact that the source of new enzymes enhances the development of new products. Although a new reaction in secondary metabolism often increases the final product, this is more than the amount of the main metabolism that is overexploited by the plant. The process of “flash” can be created in the form of another stimulus in the middle of the process that becomes the middle of a different process. Essentially, the compounds that their other stimulant can be medium in the real process are not the only final product. In the preparation of early plant metabolism, an example of this method was recently presented as aluminum condiment (Liquidambar styraciflua), coniferyl aldehyde 5-hydroxylase (CAld5H), and the combination of 5-hydroxyconiferyl (OMT, COMT isoform). The change in 4-hydroxy-3-methoxycinnamaldehyde occurs in synaptic organic compounds via C10H12O4 and suggests that the CAd5H-COMT binding process to synaptic acid may be possible in certain plants [28]. This phenomenon concurs with the regularly known path of sinapic acid transfer from ferulic acid through 5-hydroxyferulic acid. Considering two similar sets (or partial sets) of the stimulus, along with two distinct processes that show at least midway in Fig. 4.2, without any previous function, the new process can, without creating a new stimulus, to make. The only target in the plant groups associated with the actual biological processes can be set to the regulating initial (based on parsimony analysis), which provides a comprehensive review of the occurrence or nonresponse of specific reactions which shows the need for future studies. The two instances determine gene frequency and divergence. The initial process for combining the

FIGURE 4.2 Two methods that can create new biochemical pathways: (A) through the formation of a new enzyme that binds the two earlier pathways; and (B) through coexpression in the same section of the enzymes designated in two ways which divide the normal.

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protective materials below DIMBOA (drive from Zea mays, expected to be resistant to poison attack) is four consecutive hydroxylated responses by the same POR [29]. If such a process progresses in time, it is necessary to discuss some of its adaptive advantages; otherwise, the actual monooxygenase of the 2,4-dihydroxy-7-methoxy-1,4benzoxazin-3, the one may initially be able to expedite all (or several) of the four hydroxylation responses, that with the high composition in the available reactants is found [10,29]. The new processes maybe completed through successive gene courses’ replication and separation. The second instance is considered the flavonoid biosynthetic pathway. In this pathway two analogous stimulants (flavone synthase and anthocyanidin synthase) are core enzymes in two notable processes that disunited from F3H production. These stimulants consist of 2(C5H12N2O2). C5H6O5 connected dioxygenases and have portion of the same high sequence [30]. Anthocyanins are formed in the unique process, and the flavone production creates a shunt-driven stimulus for the production of flavonol glucosides in its place. By multiple replications and following the separation of the F3H or its precursor gene, the multiple different stimulants and two processes are changed.

4.4 BIOENGINEERING OF TERPENOIDS IN PLANTS The basic idea of biosynthesis and adaptable compounds for any bioengineering is very significant. The proper insight into their first making and production allows them to transfer these results to the biological engineering of natural herbs or heterologous hosts. In spite of their extensive structural difference, terpenoids have a common biochemical basis and follow pathways similar to synthesis. Altogether terpenoids originate from the overlap of isoprene (C5H8) and the number of isoprene units controls their organization. In higher plants, biosynthesis with the production of isopentanyl pyrophosphate (IPP) is accepted out through mevalonate (MVA)/3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) or 2-C-methyl-D-erythritol 4-phosphate (MEP)/1-deoxy-D-xylulose 5-phosphate (DOXP)/non MVA route. IPP isomerized to dimethylpyridine phosphate (DMAPP). The sequential condensation of the principles of the IPP and DMAPP units in the formation of premade pyrophosphates is the preceding precedent of several classes of terpenoid (Fig. 4.3). These compaction reactions are catalyzed by specific prenyltransferases produced as said by the product. The specific therapeutic syntheses then adapt these precursors to the terpenoid skeletons, which are then decorated with various enzymatic changes to produce the structural and functional variety of the terpenoid. Plants also demonstrate a clear partition for the production of IPP and the synthesis of terpenoids [31 33] (Fig. 4.3). Isopentanyl pyrophosphate (IPP) for biosynthesis of triterpenoid is produced through the cytosolic protein, peroxisome, and current MVA pathway of the ER. The head-to-head density of the two IPP units produces a DMAPP, C15 farnesyl pyrophosphate (FPP) unit, two of which ultimately ferrite to head-to-head, producing a high C30 triglyceride scalene. This compound is transformed into epoxy 2,3-oxidosqualene [34], which, in turn, is specifically changed by oxidosqualene cyclases (OSCs) to tetra or pentacyclic. For example, structures, dammarenes, tirucallanes, phytosterols or oleananes, ursanes, lupans, and taraxasteranes are converted [35]. In some herbaceous species, 2, 3-oxidosqualene can be renewed into the mono- and tricyclic of triterpenoid into the backbones [36] (Fig. 4.4). The cyclical 2, 3-oxidosqualene module systematizes the primary branch of the main and secondary triterpenoid metabolisms. Cycloartenol, consisting of cycloartenol synthase (CAS) with an emphsae on cyclization of 2, 3-oxidosqualene, is an essential precursor for phytosterol biosynthesis. In advanced plants the composition of joint sterols of cycloartenol can be collected in a free pathway, for example, esters or glycosides [37]. In its place, phytosterols cholesterol, campesterol, and sitosterol are important hormones of the C27, C28, and C29 brassinosteroid, individually [19,38]. Moreover, cholesterol can also produce a set of oxygenates along with glycosylates for generating idiophase, while saponins consist of steroidal [39]. More cyclic productions of 2, 3-oxidosqualene generate idiophase predecessor for biogenesis; see Fig. 4.4. In order to produce C30H50O5, the sodium cycle produced by the above is oxidized through one or more of the cytochrome P450s (CytP450s). The biosynthesis of terpenoids is tightly controlled in plants because they cause countless plant growth, evolution, and environments as well as feedback from environmental factors [33,40,41]. Terpenoid synthesis occurs in certain tissues during the plant’s progressive stages [41]. Triterpenoids saponin glycyrrhizin accrues independently in the underground plant organs, runners, and licorice roots (Glycyrrhiza glabra) [19]. Avenacins, active bioactive saponins in Avena sativa (oat straw), collect root cuticles in which resistance against phytopathogenic fungi is accrued [42]. Such terpenoid-specific synthesis is mostly controlled at transcription process grade. The

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4. BIOENGINEERING OF SECONDARY METABOLITES

Cytoplasm

Pyruvate

Plastid

Mitochondria and plastid

PDC

Mitochondria

Acetyl-CoA

Ubiquinone

Peroxisome

AACT

1-Deoxy-D-xylulose 5-phosphate

DXR

Acetoacetyl-CoA

5-Phospho mevalonate

HMGS

PMK

3-Hydroxy-3-methyl glutaryl-CoA

5-Diphospho mevalonate

FPPS + 2x IPP

HMGR

PMD

DMAPP

Mevalonic acid

IPP

IDI

ER

MVK

FPP

2-C-methyl-D-erythritol 2,4-cyclodiphosphate

HDS (E)-4-hydroxy-3-methylbut-2-enyl diphosphate

IPP

IPP Terpenoid indole alkaloids

IDI

DMAPP

Hemiterpenes

GPPS + 1x IPP

GGPPS + 3x IPP

FPP Sesquiterpenes

Squalene Squalene Triterpenes

Prenylation of proteins Cytokinin

2-Phospho-4-(cytidine 5’-diphospho)2-C-methyl-D-erythritol

HDR

ER SQS + FPP

GGPP

4-(Cytidine 5’-diphospho)2-C-methyl-D-erythritol

MDS

IPP

FPP

FPP

CMK

DMAPP FPPS + 2x IPP

DMAPP

2-C-methyl-D-erythritol 4-phosphate

CMS

IDI

5-Phosphomevalonate

GGPPS + 3x IPP

Pyruvate + glyceraldehyde-3-phosphate

DXS

Geraniol

GPP

GGPP

Monoterpenes Phytosterols

Saponins

Brassinosteroids

Chlorophylls tocopherols Gibberellins

Carotenoids Apocarotenoids

diterpenes

PSY + GGPP

phytoene

tetraterpenes

isoprene

polyterpenes

FIGURE 4.3 Terpenoid biogenesis in plants. There are two distinct pathways for synthesis of global IPP and DMAPP(C5H12O7P2) precursors in plants. Cytoplasm-, peroxisome-, mitochondria-, plastid- and endoplasmic reticulum (ER) -localized mevalonic acid (MVA) route (purple in online version) and the plastid localization nonmevalonate (MEP) route (blue in online version). Prenyltransferases (orange in online version) is the production of precursors for various Terpenoid (green in online version). Spotted arrows show multiple reactions. The spotted (gray) boxes indicate the subcellular localization of the pathway. Gray arrows represent the metabolites transmitted between the subcellular portions. AACT, acetoacetyl-CoA thiolase; CMK, 4-diphosphocytidyl-methylerythritol; CMS, 4-diphosphocytidyl-methylerythritol synthase; DMAPP, dimethylallyl pyrophosphate; DXR, deoxyxylulose 5-phosphate reductoisomerase; DXS, deoxyxylulose 5-phosphate synthase; FPP, farnesyl pyrophosphate; FPPS, FPP synthase; GGPP, geranylgeranyl pyrophosphate; GGPPS, GGPP synthase; GPP, geranyl pyrophosphate; GPPS, GPP synthase; HDR, hydroxymethylbuthenyl 4-diphosphate reductase; HDS, hydroxymethylbuthenyl 4-diphosphate synthase; HMGR, 3-Hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl pyrophosphate; MDS, methylerythritol 2,4-cyclodiphosphate synthase; MVK, mevalonate kinase; PDC, pyruvate dehydrogenase complex; PMD, 5-diphosphomevalonate decarboxylase; PMK, 5-phosphomevalonate kinase; PSY, phytoene synthase; SQS, squalene synthase.

biogenesis of avenacin can be produced entirely in the root epidermis, in which the avenacins are collected [43,44]. In addition to this spatiotemporal regulation, induced terpenoid biosynthesis is often observed in response to herbivore feeding, pathogen attack or various abiotic stresses [33,41]. Larval function decreases when the affected leaves are no longer [19]. An increase in gathering or dispersion of terpenoids in response to numerous pressures is often considered by the enlarged transcriptional action of terpenoid biosynthetic pathway genes [40,41]. This transcription response is managed by an integrated signaling stream, where jaw hormones (JAs) are very helpful. The functional culture of plants with JAs, as well as cells, often leads to the development of

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acetyl-CoA

steroidal saponins and brassinosteroids

mevalonate pathway

SHC

hopane

HO

Cholesterol synthase

Cholesterol

cucurbitane

HO

O

squalene

lanosterol HO

SCs

cucurbitane synthase

taraxasterol LUP

SQE

LAS

marneral

MRN

tirucallane synthase

OSCs

CAS

sterois

O HO

DDS aAS

OH

dammarenediol CYP716A47

cycloartenol

LUP

bAS

HO

HO

THAS HO

2,3-oxidosqualene

tirucallane

HO

HO

α-amyrin CYP716A12 CYP716A15 CYP716AL1

β-amyrin

HO

thalianol

lupeol CYP716A12 CYP716A15 CYP716AL1

CYP708A2

OH OH

OH COOH

COOH

HO

HO

HO

ursolic acid

protopanaxadiol CYP716A53v2

CYP88D6

CYP51H10

HO

thalian-diol

betulinic acid

OH

CYP93E1 CYP93E2 CYP93E3

OH

CYP72A154 CYP72A63 HO H2C

CYP716A12 CYP716A15 CYP716A17 CYP716AL1 OH

O

HO

HO

HO

OH

11-oxo-β-amyrin

protopanaxatriol

CYP705A5

CytP450s

HO

desaturated thalian-diol

30-hydroxy-β-amyrin

O COOH OH HO

12, 13-epoxy dihydroxy oleanane

HO HOH2C

HO

oleanolic acid CYP72A68v2

24-hydroxy-β-amyrin CYP72A61v2

OH COOH HO HOH2C

HO

soyasapogenol B UGT71G1 UGT74M1 UGT91H4 SAPONINS

COOH

gypsogenic acid UGT73C11 UGT73F3 UGT73K1 UGT73P2

FIGURE 4.4

UGTs

A simple plan of biosynthesis of triterpenoid saponin as expressed in Saccharomyces cerevisiae. Arrow dotted shows different moves. Outstanding enzymes (red) and compounds (blue) were expressed and identified, respectively. aAS, α-amyrin synthase; bAS, β-amyrin synthase; CAS, cycloartenol synthase; CytP450s, cytochrome P450s; DDS, dammarenediol synthase; LAS, lansetrol synthase; LUP, lupeol synthase; MRN, marneral synthase; OSCs, oxidosqualene cyclases; SCs, squalene cyclases; SHC, squalene-hopane cyclase; SQE, squalene epoxidase; THAS, thalianol synthase; UGTs, UDP-dependent glycosyltransferases.

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transcriptional and metabolic processes that are similar to pathogenic or herbal attacks. Plants’ functional culture with JAs as well as cells frequently lead to transcription and metabolic process developments that are analogous to pathogenic or vegetarian attacks. Exposure to Medicago truncatula sensitivity to methyl jasmine (MeJA) increases the strength of saponine due to transcriptional genes from saponine biosynthesis [45]. The activation of the present transcription of entirely secondary metabolism pathways is preserved by JAs throughout the plant kingdom. In any way, in the lower solar reaction current and the main signaling opening, there is not a precise transcription apparatus that generates certain transcriptional activity [19]. There are no recognized transcription factors obtained through JA signaling cascade which activates the biosynthetic terpenoid (sesqui) genes, moreover as if now there exist no C30H48O7S. It is well known that JAs are not the only regulator of terpenoid metabolism in plants [46]. The strict regulation, most terpenoids are produced in very small amounts in their natural sources. Low yield is extractive, which eventually reproduces its market worth. Consequently, there exists an extensive gap between usage and source of terpenoids that disturbs their enormous applications. The classical approach to ensure a constant or improved yield is the selection and propagation of high producing cultivars or the production and/or elicitation of (transgenic) plant (cell) cultures [19]. Growing concern on terpenoid biosynthesis, with functional developing genomics and system biology tools, has expanded the whole metabolism of plants as to growth, efficiency, and alteration of delivery of terpenoid in the field [19,47]. The terpenoid biogenesis along with accurate transcription as renew efficiency is highly managed. Despite the classification of transcription factors that produce biological terpenoids, the excessive expression of transcription factor only increases the production of the mixture. The amplification of more complex signaling cascades, which primes to an increase in the buildup of terpenoids, forming the expansion of large-scale metabolism of transgenic terpenoid by means of transcriptional principals. As yet, the absence of information around the mechanisms leading to gene expression has been reduced in the field of triterpenoid farm engineering [48]. Hence, a challenge for future triterpenoid research will be to identify the transcription or other regulatory factors that steer their biosynthesis. The second technique to increase the efficiency is used to complete the excessive expression of the speed-reducing stimulant in the metabolism pathway. Exceeding the expression genetic code used in plant tissue culture enzymes like HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), DXS (deoxy xylulose 5-phosphate synthase), and a class of prenyltransferases enzymes in order to promote isoprenoid directions. The production growth of terpenoid is also because of the alteration in the place of the enzyme cells, maybe by reason of the conclusion of biosynthesis and regulation [19]. A single study has reported an attempt to engineer triterpenoid synthesis in tobacco (Nicotiana tabacum) by the heterologous expression of an avian FPP synthase (FPPS) and a yeast squalene synthase (SQS) gene targeted to the cytoplasm or plastid. As a result, no changes are made of squalene increase in the precise target of the enzyme. Nevertheless, among the stimulants are concentrated in the trichomes, a promoter of a “trichome-specific” of C30H50 (30-carbon organic compound), which with passive materials on plant development collected and physiology developed. It is notable that these extra properties are not experimental as the similar genetic code are declared as active controlled promoters [49]. However, this study suggests the ability of the triterpenoids of the plants with the effect of the pathway of biosynthesis and increasing flux, and future studies in this class encourage the use of terpenoid.

4.5 METABOLIC ENGINEERING AND MICROBIAL BIOGENESIS OF PLANT ISOPRENOIDS With respect to plant production systems, microorganisms are attractive alternatives as heterologous hosts due to fast doubling times, reliability in process settings, ease of scalability, and simplicity of product purification due to the lack of competitive and cost-effective contaminations. All of this is not economical due to the presence of competitive contaminants. The efficacy of the adaptation of inexpensive supplies into appreciated compounds [50]. The choice of a suitable host (or ‘chassis’) is critical and should be based on multiple factors, including the chemical nature and complexity of the product to be synthesized, the genetic amenability of the host, the intrinsic availability of precursors for product biosynthesis, the codon usage bias of the host, the need for post-translational modifications and the feasibility to metabolically engineer the host to boost productivity [51]. The microbial synthesis of any plant production is accomplished through a synthetic precursor synthesis, when a current host pathway is available to arrange a pathological or “de novo synthesis” and changes in order to avoid rules of the response in the new pathways of biosynthesis in the host occur. To run heterologous synthesis, it is essential to

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produce the host metabolism for enhancing production and gaining rates [19]. The colloquial hosts Escherichia coli and Saccharomyces cerevisiae have been employed for both precursor-mediated and de novo synthesis of mono-, di-, sesqui-, tri- and tetraterpenoids [52], with artemisinic acid, the precursor of the antimalarial drug artemisinin, as the showcase for plant-derived terpenoids [53]. The prokaryotic E. coli is an intrinsic pathway of 2-C-methyl-D-erythritol 4-phosphate (MEP), and S. cerevisiae as the eukaryotic is a mevalonate pathway (MVA) for the production of isopentenyl pyrophosphate (IPP) and is isomer of dimethylallyl pyrophosphate (DMAPP). By hypothetically, terpenoid biosynthesis can be expressed by expressing genes in the host, but due to the incomplete IPP pool, the cellular internal construction can be weak. The levels of prevention of IPP (and afterward) by metabolic engineering are accomplished: (1) the pathway for MVA in E. coli; (2) the pathway for MEP and prenyltransferases in E. coli; (3) the MVA pathway by HMGR poor response in S. cerevisiae; (4) the MVA pathway by reducing the downstream enzymes for the precursor gathering in S. cerevisiae; (5) the global transcription factor adaptable the biosynthesis of sterol; and (6) protein skeletons for the MVA pathway in S. cerevisiae [19] (Fig. 4.5). (A)

(B)

MEP genes

Prenyl transferase

S. cerevisiae MVA pathway

E. coli MEP pathway

MVA pathway in E. coli

(C)

(D) tHMGR

ER

Sesquiterpene IPP

HMGR

Triterpene

FPP Squalene

Nucleus

Triterpene

2,3-Oxidosqualene

ER localized HMGR

Ergosterol

Cytosolic truncated HMGR

(E)

(F) Sterol Upc2p UPC2

HMGS Scaffold

Upc2-1p upc2-1

ERG ERG genes activated upon low sterol levels

ERG

ERG genes always activated

Normal enzyme ratio

Modulated enzyme ratio

AACT tHMGR

FIGURE 4.5 Strategies adopted in increasing the production of isopentenyl pyrophosphate (IPP) and terpenoid in Escherichia coli and Saccharomyces cerevisiae. (A) Expression of the pathway of S. cerevisiae mevalonate (MVA) in E. coli. (B) Express the frequency of 2-C-methyl-derythritol 4-phosphate (MEP) enzymes in E. coli. (C) Expression of a short form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in S. cerevisiae. ER, endoplasmic reticulum. (D) Setup of endogenous sterol biosynthesis for the collection of terpenoid precursors in S. cerevisiae. FPP, farnesyl pyrophosphate. (E) Expression of a mutated version (upc2-1) of the universal transcription part (UPC2) regulates the expression of natural sterol biogenesis genes in S. cerevisiae. (F) Protein scaffolds in preventing the restriction of S. cerevisiae by the spatial regulation of biosynthetic sterol stimulant that limits the rate at a modulation ratio. AACT, acetoacetyl-CoA thiolase; HMGR, 3-hydroxy-3-methylglutarylCoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase.

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In the face of targeted engineering, global approaches to the cultivation of the terpenoid element are located in microbial hosts. The policy of making chromosomal promoters in expressing certain endogenous MEP from a potent bacteriophage T5 promoter in E. coli gene using β-carotene biosynthesis genes, which leads to an increase in β-carotene production in relation to the parental strain. Similarly, engineering of global transcriptional devices on rpoD gene encoding σ70, the main factor of sigma, increases lycopene in E. coli production. Likewise, engineering of global transcriptional apparatuses on rpoD gene encoding σ70, the main sigma factor, increases the of lycopene in E. coli production [19,54]. The oxidosqualene, dissimilar OSCs, and CytP450s are expressed in enhanced engineering strains to accumulate oxidiosqualene, particularly for their presentation features [34] (Fig. 4.2). Engineering endoners are incomplete in β-amyrin production. Through the conservative engineering method, a final 6 mg21 test is expressed in a suspension of S. cerevisiae and a β-amyrin synthase (bAS) from Artemisia annua [55]. After attaching the genotype to the graft phenotype, the development of β-amyrin with 500% expression of natural genes, ERG8, ERG9, and HFA1 in suspension of S. cerevisiae expression from Pisum sativum bAS is forming. The last test was 3.93 mg21 [56]. The levels of β-amyrin produced by parental in the above reports indicate the cyclic efficiency of the consumed enzymes. Therefore, by employing a more efficient bAS (or any other saponin biosynthetic gene), followed by targeted and/or global engineering, it should be possible to further enhance β-amyrin (or triterpenoid) levels. The strains of β-amyrin S. cerevisiae are expressed in vivo, and they are one of the newest CytP450s. The simultaneous coexpression of CytP450 with CPR resulting from the plant mains to the production of yeast strains that produce dissimilar sapogenins. Expression of M. truncatula CYP716A12 with Lotus japonicus bAS and L. japonicus CPR leads to oleanolic acid during yeast production [57] (Fig. 4.2). β-Amyrin has altered usual and rare triterpenoids, joining numerous CytP450s in yeast. The expression of M. truncatula CYP72A68v2 and CYP93E2 in acid-making oleanolic acid produces gypsogenic acid and 4-epi-hederagenin in a separate manner. For β-amine producing strains, α-amyrin, lupeol, and dammarenediol yeasts have been used to describe CytP450s. M. truncatula CYP716A12 oxidizes C-28 from α-amyrin to ursolic acid and lupeol to betulinic acid in yeast [57]. Likewise, hydroxylation of C-6 and C-12 dammarenediol is revealed by CYP716A53v2 and CYP716A47, in yeast in a separate manner [19]. In making metabolism, the emphasis is placed on the use of the latest formulation, with less attention to moderate performance. In contrast, the bottom-up biologic method allows separating energy release paths into independent sections that have been improved for precise host expressions, and thereafter there is a reason to create modules. Reservoirs of functional mechanisms (promoters, ribosomal binding sites, protein domains, terminators, etc.), developed in synthetic biology creativities, enable the meeting of metabolism pathways. Two reservoirs with improved codon names for making paths in E. coli (the registry of accepted organic sectors, parts registry.org/Main_Page) are named with the isoprenoids organization in S. cerevisiae. Synthetic biology promotes the expression of flexible biology genes to prevent metabolic constraints. The collection of strong artificial proteins, including a distinct promoter, allows the expression of a modular gene in bacteria and yeast. Adjustable synthetic regions, which include secondary structures of mRNA and RNase discovery places, are used to check the differences in mRNA segments that encode multiple enzymes in the operons system. Synthetic protein systems that are precisely produced to overcome the speed-limiting steps are developed to enhance flux through pathways of metabolism by binding enzymes [19]. Reconstruction metabolic pathways are a valuable benchmark for generating new and popular products that can be modulated by partial synthesis. In the simplest system, the compound of biosynthesis, the method of producing multiple molecules, through the structure, is simply connected by collecting many of the genes of the organism in a single host (Fig. 4.6A). Excessive expression of CYP88D6 and CytP450 from the roots of Glycyrrhiza uralensis, which produces glycyrrhizin, in M. truncatula can lead to the formation of a compound with natural saponins. The combination of biosynthesis of high levels of triterpenoid saponins is high because of numerous biological activities. Bardoxolone methyl, resulting from the chemical synthesis of pentacyclic triterpene, is assessed for action chronic kidney failure (CKD). The composition of bardoxolone methyl is based on the variation of synthetic composition on the three mechanisms of the C30H48O3 dynamics, which is stronger than the starter molecule. In addition, the enzyme has additional features to the backbone of the triterpenoid through hybrid biosynthesis that can increase the number of sites used in artificial changes. The main drawback of making new molecules of the plant is the difficulty in extracting plant metabolites and the difficulty in cleaning up a vital compound in the context of large molecule pools, counting substrates with similar contractions and physiochemical drugs. Biosynthesis is a combination of secondary metabolites of the plant in microorganisms that do not produce compounds with target combination (Fig. 4.6B). In section (B) biogenesis through a combination of saponins in a sterol-reduced S. cerevisiae by expression of heterologous genes of saphenous biosynthesis from M. truncatula and G. uralensis and the evolution of conduct stimulant affects mutagenesis and the allocation of the appropriate enzymes. New carotenoid structures can be introduced by the cumulative antioxidant activity of E. coli via the expression of a grouping of bacterial and plant genes. Recently, there are rare triterpenoids in S. cerevisiae [58]. A NEW AND FUTURE DEVELOPMENTS IN MICROBIAL BIOTECHNOLOGY AND BIOENGINEERING

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FIGURE 4.6 Production policies for new triterpenoid for saponin inoculation. (A) combined biosynthesis in the Medicago truncatula bacon model that produces 3-Glc-28-Glc-medicagenic acid endogenously. (B) Biosynthesis a combination of saponins in a sterol-reduced S. cerevisiae strain by the heterologous expression of saponin biosynthetic genes from M. truncatula and G. uralensis. (C) The process of directed enzyme evolution involves mutagenesis and selection for desired enzyme properties. Here, the evolution of a multifunctional enzyme with an increased reaction specificity is depicted. Glc, glucose; GlcUA, glucuronic acid; IPP, isopentenyl pyrophosphate; MtbAS, M. truncatula β-amyrin synthase; MtCytP450s, M. truncatula cytochrome P450 monooxygenases; UGT, UDP-glucosyltransferase.

current obstacle to the wider utilization of combinatorial biosynthesis for plant-derived compounds is the limited availability of plant genes encoding the enzymes that catalyze the biosynthetic reactions. Finally, these blocks can be resolved by detecting genes in plants (or medicinal plants) or, otherwise, by developing the expansion of enzymes to new goals reached [19].

4.6 ENZYME ENGINEERING Small molecular drugs, as the main molecules, are generally associated with a heavy chemical problem with different species along with the study of chemical molecules [59]. In their usual basis, these molecules are frequently NEW AND FUTURE DEVELOPMENTS IN MICROBIAL BIOTECHNOLOGY AND BIOENGINEERING

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created by stimulants with high region and stereoselectivity, catalysis amount, and locomotion belongings. Simple enzymes have often inappropriate for supplying production chemicals about substrate tolerance, efficiency, and economic life [60] (Fig. 4.6C). In section (C) evolution of the directed enzyme involves mutagenesis and the selection of the desired enzymes. Here, the evolution of a multipurpose enzyme with an enhanced property is being reacted. Glc Glucose; GlcUA, Glucuronic acid; IPP, isopentenyl pyrophosphate; MtbAS, M. truncatula β-amine synthase; MtCytP450s, M. truncatula cytochrome P450 monooxygenases; UGT, UDP-glucosyltransferase. The development of the enzyme has become more advanced and now it is possible to produce enzymes to obtain abnormal concentrations and recurrence of specific regional and stereo reactions with similar functions to the common enzymes [61]. The promiscuous nature of proteins gives them an inherent ability to generate novel or altered functions with a small number of amino acid substitutions, and computational methods, such as catalytic active site prediction (CLASP) and directed evolution using CLASP: an automated flow [65, 66], utilize virtual screening for spatial, electrostatic and scaffold matching to identify target progenitor proteins. Catalytic enzymes are indirect active responses in multibranch pathways, where the infrastructure varies in several products. Moreover, evolving enzymes exhibit many outstanding mutations and are known as a common motif character on the diversity of structural infrastructure [19]. Oxidosqualene, an immediate prediction of triterpenoid biosynthesis, is a multipurpose molecule that has been converted into several different products through several OSCs. Most OSCs carry out individual tasks and complete products [67], which are important in predicting and developing the enzyme. Through direct progress, the OSC’s multipurpose cyclization of a key product can be distinguished as a precise or new product. For the extra terpene synthases, this multipurpose OSC is strained. Subsequently, site saturation mutagenesis, the property of carotenoid synthase, changes to indirect products of the C45 and C50 in E. coli [64]. The product specificity of a γ-humulene synthase from Abies grandis that cyclizes FPP to 52 different sesquiterpenoids was evolved by site-saturation mutagenesis to generate independent synthases, each producing one or a few products derived from a predominant reaction pathway [65]. This evolutionary technique can be extended to biosynthesis enzymes, in particular, CytP450s. The backbones of triterpenoid saponin consist of 30 carbons, 20 of which can be used for CytP450 variations, as is observed from saponins [66]. Many functional groups can be generated in altered carbons, representing the attendance of sure CytP450s that catalyze these special chemical transformations., which exactly recognizes the α-hydroxylation catalyst. Up to now, only a few families of CytP450 are known in triterpenoid alteration (Fig. 4.4). Such approaches are effective in carotenoid desaturases, which are changed randomly, that to have become possible the unique C35 carotenoid in E. coli [19]. Protein engineering, based on molecular evolution, is a helping agent to provide an enzyme or to remove enzyme responses. Through developmental adaptation, in vivo HMGR reduces yeast supplements for optimal action in E. coli while increasing the production of final product c.1000 times [67]. It is important that the evolutionary study takes serious attention from the sequence to the function of binding a protein. The triterpenoid cyclase’s (TTCED) [68] and CytP450s (CYPED) [69] integrated databases simplify the identification of the causes of the function and selectivity of amino acids in a protein element group through extensive sequencing and searching. Protein engineering can increase the fabricated biology (synthetic biology) in the C30H48O7S manufacturing (triterpenoid engineering) field.

4.7 CONCLUSION The sequences of enzymes present in the stream are followed by subsequent sequences and mass evolution of EST. It is essential to increase the actual system to test the catalytic activities, because, in idiophase (secondary metabolism), solely the exhibition of stimulus action (enzymatic activity) may exclusively classify the protein function. This is a must in the progress of an effective system to test as a whole the catalytic activities. It is evident that the development of plant secondary metabolism plant is stabilized for main advances with applying totally the original things appropriate to biotechnology (molecular biology) together with enzymology (biochemistry) in this context. A great number of functional genomic techniques, with improved simplicity along with ordering (genome), RNA molecules set (transcriptome), protein (proteome), interaction, as well as metabolism analysis, provide the list of all possible fundamentals complicated upcoming compound of isoprenoids in plant. A metabolic connection mainly composed of investigation of a transcriptome sign and parenchyma. It is a major foundation of gene involving in biosynthesis classification, carriers, and evidence features.

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Acknowledgment The authors gratefully acknowledge financial assistance from the research council of Falavarjan Branch, Islamic Azad University.

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[51] J.D. Keasling, Manufacturing molecules through metabolic engineering, Science 330 (2010) 1355 1358. [52] N. Misawa, Pathway engineering for functional isoprenoids, Curr. Opin. Biotech. 22 (2011) 627 633. [53] J.D. Keasling, Synthetic biology and the development of tools for metabolic engineering, Metab. Eng. 14 (2012) 189 195. [54] H. Alper, G. Stephanopoulos, Global transcription machinery engineering: a new approach for improving cellular phenotype, Metab. Eng. 9 (2007) 258 267. [55] J. Kirby, D.W. Romanini, E.M. Paradise, J.D. Keasling, Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin synthase from Artemisia annua, FEBS J 275 (2008) 1852 1859. [56] K.M. Madsen, G.D. Udatha, S. Semba, J.M. Otero, P. Koetter, J. Nielsen, et al., Linking genotype and phenotype of Saccharomyces cerevisiae strains reveals metabolic engineering targets and leads to triterpene hyper-producers, PLoS ONE 6 (2011) e14763. [57] E.O. Fukushima, H. Seki, K. Ohyama, E. Ono, N. Umemoto, M. Mizutani, et al., CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis, Plant Cell Physiol. 52 (2011) 2050 2061. [58] E.O. Fukushima, H. Seki, S. Sawai, M. Suzuki, K. Ohyama, K. Saito, et al., Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast, Plant Cell Physiol. (2013). Available from: https://doi.org/10.1093/pcp/pct015. [59] D.P. Nannemann, W.R. Birmingham, R.A. Scism, B.O. Bachmann, Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis, Future Med. Chem. 3 (2011) 809 819. [60] P.A. Dalby, Strategy and success for the directed evolution of enzymes, Curr. Opin. Struct. Biol. 21 (2011) 473 480. [61] M. Goldsmith, D.S. Tawfik, Directed enzyme evolution: beyond the low-hanging fruit, Curr. Opin. Struct. Biol. 22 (2012) 1 7. [62] S. Chakraborty, An automated flow for directed evolution based on detection of promiscuous scaffolds using spatial and electrostatic properties of catalytic residues, PLoS One 7 (2012) e40408. [63] S. Chakraborty, R. Minda, L. Salaye, S.K. Bhattacharjee, B.J. Rao, Active site detection by spatial conformity and electrostatic analysis unravelling a proteolytic function in shrimp alkaline phosphatase, PLoS One 6 (2011) e28470. [64] D. Umeno, F.H. Arnold, Evolution of a pathway to novel long-chain carotenoids, J. Bacteriol. 186 (2004) 1531 1536. [65] Y. Yoshikuni, T.E. Ferrin, J.D. Keasling, Designed divergent evolution of enzyme function, Nature 440 (2006) 1078 1082. [66] B. Dinda, S. Debnath, B.C. Mohanta, Y. Harigaya, Naturally occurring triterpenoid saponins, Chem. Biodiversity 7 (2010) 2327 2580. [67] Y. Yoshikuni, J.A. Dietrich, F.F. Nowroozi, P.C. Babbitt, J.D. Keasling, Redesigning enzymes based on adaptive evolution for optimal function in synthetic metabolic pathways, Chem. Biol. 15 (2008) 607 618. [68] S. Racolta, P.B. Juhl, D. Sirim, J. Pleiss, The triterpene cyclase protein family: a systematic analysis, Proteins 80 (2012) 2009 2019. [69] D. Sirim, F. Wagner, A. Lisitsa, J. Pleiss, The cytochrome P450 engineering database: integration of biochemical properties, BMC Biochem. 10 (2009) 27.

Further Reading B.M. Lange, G.W. Turner, Terpenoid biosynthesis in trichomes current status and future opportunities, Plant Biotech. J. 11 (2013) 2 22.

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5 Advances in Microbial Technology for Upscaling Sustainable Biofuel Production Shachi Shah and V. Venkatramanan Environmental Studies, School of Interdisciplinary and Transdisciplinary Studies, Indira Gandhi National Open University, New Delhi, India O U T L I N E 5.1 Introduction

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5.1 INTRODUCTION Energy is quintessential for most activities of modern society. The development of human society is symptomatic of energy use coupled with the discovery of energy sources, mining, and innovative uses. The everincreasing demand for energy and the cataclysmic environmental impact of fossil fuel use impel us to find alternative renewable energy sources. Bioenergy is indeed a promising form of alternative energy. As a matter of fact, the carbohydrate economy is not new to human society. Until the early 19th century, our economy was primarily based on biomass. The paradigm shift to a fossil fuel-based economy occurred on account of the availability of inexpensive fossil fuel energy sources, lower purchasing costs, and the portability and transportability of fossil fuels. Bioenergy is a generic term for energy derived from materials like plant waste, straw, wood, or animal waste. The materials of biological origin can either be directly burned as solids (biomass) to produce heat energy or converted into liquid biofuels, which find extensive use in the transportation sector. Global climate change and the dependency on nonrenewable sources of energy are causes for great concern, because the use of fossil fuel has pumped into the atmosphere enormous amounts of heat-trapping greenhouse gases. In this regard there is an urgent need to factor in new renewable resources for biofuel production using microorganisms [1]. Biofuels have emerged as one of the important sustainable fuel sources and are considered as a promising way to minimize greenhouse gas emissions and to provide new energy sources. Biofuels are regarded as a New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: https://doi.org/10.1016/B978-0-444-63504-4.00005-0

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commercially viable alternative to fossil fuels as well as a lifestyle and “business as usual” option for the future of the mobility sector [2]. Socioeconomic, political, and environmental factors play a sterling role in the production and utilization of biofuels [3]. Pragmatic and goal oriented public policies on biomass production can deliver tangible benefits like energy security, foreign exchange savings, local employment generation, reduced urban air pollution, and the avoidance of CO2 emissions [4]. Biofuels are nonfossil fuels produced by organisms that tap solar energy and store it in the form of high-energy organic compounds. Through the photosynthetic process, the atmospheric carbon dioxide is converted into sugars. The sugars are converted into fuel by engineered microorganisms and the combustion of the fuel in automobile engines releases the carbon dioxide back into the atmosphere. This closed carbon dioxide cycle is a carbon-neutral process as the use of biofuels does not present any net increase in atmospheric carbon dioxide [5]. Improvements in biofuel production are destined to develop the carbon-neutral economy through the sustainable production of biofuels [6]. The first-generation biofuels like ethanol are contested due to their physical and chemical properties and due to the fears for food security and the high lifecycle greenhouse gas emissions [7]. The first-generation biofuel feedstocks require huge quantities of fertilizers, pesticides, and water, which make the first-generation biofuels nonsustainable in the long run [5]. Unlike bioethanol, alcohols with long carbon chains like n-propanol, n-butanol, isopropanol, and isobutanol are suitable candidates for biofuel production as they possess high energy intensity and are amenable to storage and transportation infrastructure. Nevertheless, the low volumetric productivity of biologically-derived hydrocarbons questions the sustainability of biofuel production and its potential to meet the rising demand for transportation fuel. Alternate biofuels like biodiesel, propanol, and other hydrocarbons are being contemplated for research, development, and economic and ecological sustainability. Biofuel production from biomass through microbial action is considered as one of the sustainable ways of biomass utilization. Fortunately, scientific advancements in the domain of microbiology, biotechnology, metabolic and process engineering, and systems biology have provided us with a ray of hope to understand the cellular processes and metabolism, to design new metabolic pathways in a cell, and to optimize the biofuel production through systems biology approaches. This chapter discusses the metabolic engineering approaches for biofuel production and emphasizes the need to integrate synthetic biology and systems biology in order to optimize host organisms and overcome biosynthetic pathway bottlenecks.

5.2 BIOMASS FEEDSTOCKS FOR BIOFUELS PRODUCTION Biomass encompasses all the living matter present on the Earth. Biomass has been used since time immemorial. Trees have been widely used as a fuel for more than 5000 years [8]. Biomass energy resources include woody plants, food crops, agricultural crop residues, forest wastes, etc. Biomass is the fourth largest source of energy in the world after coal, petroleum, and natural gas, providing about 14% of the world’s primary energy consumption [9]. Biomass is a repository of solar energy on Earth. The plants, through the process of photosynthesis, harvest solar energy to produce carbohydrates which form the bulk of biomass. Globally about 150 billion tons of biomass are generated per annum [10]. Biomass generally comprises carbon, hydrogen, oxygen, and nitrogen. The use of biomass for biofuel production reduces the dependency on fossil fuels. Since biomass is considered a carbon-neutral resource during its lifecycle, it occupies an important position in climate change mitigation and adaptation. Biological materials that are rich in carbon are suitable for biofuel production. Generally materials of biological origin like plants, microorganisms, and animal waste are amenable for microbial conversion into biofuels. In effect, the photosynthetic process of harvesting the sun’s energy forms the bedrock of biomass generation. The biofuels are categorized into different generations of biofuels based on the type of biomass and the technology options for biofuel generation. Recently fourth-generation biofuels have been produced by using innovative tools from synthetic biology [6]. Due to the constant effort and urge to replace the use of fossil fuels with renewable fuels, biofuels have attained primacy among the renewable fuels. Biofuel feedstocks is the term used to refer to the crops or their by-products, such as vegetable oil, that can be converted to biofuels. First-generation feedstocks basically include the crops that are widely grown food crops. They include sugar crops (sugar cane, sugar beet, sweet sorghum), starchy crops (cassava, corn, sorghum, sweet potato), oil seed crops (coconut, oil palm, jatropha), and waste feedstock (citrus peels) [8]. Plant biomass that is rich in carbohydrates is mainly used for the synthesis of first-generation biofuels [11]. Ethanol, a first-generation biofuel, is produced from maize (United States) or from sugar crops like sugarcane in Brazil. In fact in the United States during the 1990s, ethanol was used as an additive to gasoline, as ethanol can oxygenate gasoline. Since the

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energy crisis of the 1970s, some countries, in particular the United States and Brazil, have embarked on investment in innovative technologies that enable the generation of ethanol from biomass. As regards biofuel production and consumption, the positions of the United States and Brazil among the biofuel-producing countries are remarkable [12]. Currently fuel ethanol accounts for around two-thirds of the world’s ethanol production. Since the first-generation feedstocks include mainly food crops, a conflict of interest arises between the use of crops either for food or fuel. Pertinent issues like food security, limited agricultural land resources, and rising energy demand has impelled the search for alternative feedstocks such as cellulosic feedstocks. Nonconsumable parts of agricultural food crops, wastes from agricultural activity, etc., are used as feedstocks for second-generation biofuels. Nongrain- and nonfood-based cellulosic feedstocks have high potential for biofuel production [8]. Second-generation feedstocks unlike the first-generation feedstocks avoid the competition with food and feed products. However, further advances in technological development were needed to unlock the hidden sugars present in the crop residues or woody plant materials. The best known carbohydrate-rich renewable resource is lignocellulosic biomass. Lignocellulosic biomass is composed of lignin (25%) and carbohydrate (75%). Hydrolytic products of lignocellulosic biomass are hexoses and pentoses. However, the lower yield of such feedstocks presents a challenge for the commercialization of biofuel production from the second-generation biofuel feedstocks. About 99% of global biofuel production is contributed by first- and second-generation biofuels. The third-generation biofuels are of recent development. They include algal biofuels. Algal biofuel is advantageous on account of factors like the possibility of algal cultivation in varied environments involving freshwater, saltwater, and wastewater. Algae can be grown in open ponds, closed-loop systems, and photobioreactors. Algae grows much faster and also requires a comparatively lesser land area. Algae has two important properties. The triglycerides produced by the algae are converted into biodiesel in a refinery [12]. Secondly, algae can be genetically manipulated to produce a wide variety of products like ethanol, butanol, and diesel. However, on the downside, the growth of algae requires large amounts of water, nitrogen, and phosphorus. The huge cost of nitrogen and phosphorus required for algal growth challenges the sustainability of algal biofuel production. Irrespective of alternative biofuel feedstocks, microbe-mediated biomass conversion into biofuels is construed as a primary approach of biofuel production. Microbes play a pertinent role in the sustainable production of biofuels. The microbial diversity and the metabolic diversity of microbes provide the impetus for utilizing diverse substrates for biofuel generation and open the floodgates for generating the next-generation biofuels.

5.3 DOWNSIDE OF FIRST- AND SECOND-GENERATION BIOFUELS Growing concerns about the increasing energy demand, the mismatch between energy supply and demand, and global environmental change have forced the scientific fraternity to search for alternative fuels that are renewable, sustainable, and have the potential to replace fossil fuels in the mobility sector. Biofuels found a place on the list of alternative fuels. The first-generation biofuels like ethanol and biodiesel found use in countries like the United States and Brazil. The ethanol was generated from maize and sugarcane. Basically the feedstocks used in the generation of first-generation biofuels are sugar and starchy food crops. On account of biofuel feedstocks being food crops, contentious issues emerged, like the diversion of agricultural land for biofuel production, food insecurity, and food price spikes. Although ethanol has a high energy value, reflected by its octane number of 116, its inherent properties, such as its corrosive and hygroscopic natures, render it inharmonious with the present day infrastructure for the distribution and transportation of petroleum products [13]. Adding to these problems, the energy- and cost-intensive distillation process raised questions regarding the sustainability and economic feasibility of ethanol generation from first-generation biofuel feedstocks. Subsequently, lignocellulose was considered as the next best option for biofuel production for obvious reasons including the abundance of lignocellulosic material on Earth, the potential for utilizing agricultural waste and by-products for biofuel production, and the reduction of the pressure on food crops and agricultural land for biofuel production. The production of energy-dense advanced biofuels is indeed a challenge. The advanced biofuels must possess properties similar to the transportation fuels in current use. These fuels must be economically produced and be compatible with the storage and transportation infrastructure [13]. In order to achieve this goal of finding biosynthetic alternatives for transportation fuels, certain factors pertaining to engine and fuel characteristics must be considered while designing the biosynthetic pathway [14]. The transportation fuels currently in major use are gasoline, diesel, and jet fuel (Table 5.1), and there is a dire need to find biosynthetic alternatives for these transportation fuels by using the advancements in microbial engineering and systems and synthetic biology.

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TABLE 5.1 Transportation Fuels, Properties and Potential Biosynthetic Alternatives. Transportation S. No. fuel type 1

Gasoline

Biosynthetic alternatives (biofuels)

Properties of potential biofuels

C4 C12 hydrocarbons

Short-chain alcohols and alkanes

Energy content must be comparable to 32 MJ/L

Linear, branched, and cyclic alkanes (40% 60% of the fuel mixture)

Butanol, isobutanol

Octane number (87 91)

Fatty Acid Methyl Esters (FAMEs), fatty alcohols, alkanes, and linear or cyclic isoprenoids

Freezing temperature (29.5 C)

Engine type

Fuel composition

Spark ignition engines

Aromatics (20% 40% of the fuel mixture) 2

3

Diesel

Jet fuel

Compression engines

Gas turbines

C9 C23 hydrocarbons. Average carbon length is C 16

Linear, branched, and cyclic alkanes (75% of the fuel mixture)

Vapor pressure (0.009 psi at 21 C)

Aromatics (25% of the fuel mixture)

Cetane number (50 60)

C8 C16 hydrocarbons

Linear, branched, cyclic aromatics

Fatty acid- and isoprenoidbased biofuels

Possess net heat of combustion. Low freezing temperature (240 C) High energy density (53.4 MJ/L)

Aromatics (25% of the fuel mixture) Adapted from P. Peralta-Yahya, J. Keasling, Advanced biofuel production in microbes. Biotechnol. J. (2010) 5 (2) 147 162 [13]; P. Peralta-Yahya, F. Zhang, S. del Cardayre, J. Keasling, Microbial engineering for the production of advanced biofuels. Nature (2012) 488 (7411) 320 328 [14]; P. Xu, M. Koffas, Metabolic engineering of Escherichia coli for biofuel production. Biofuels (2010) 1 (3) 493 504 [15].

5.4 METABOLIC ENGINEERING AND BIOFUEL PRODUCTION Of late, the cellular phenotypes of microorganisms have been greatly improved through metabolic engineering. Metabolic engineering can be defined as the “design of native or entirely new metabolic pathways in a cell.” It provides a platform to change the metabolic pathway with the aim of augmenting biofuel production [15]. The metabolic activities of living cells are realized by a regulated, highly coupled network of about 1000 enzymecatalyzed reactions and selective membrane transport systems. Nevertheless, metabolic networks that have evolved in natural environments are not optimized genetically to undertake practical applications [16]. So the metabolic processes can be augmented by the genetic modification of the cells. Metabolic engineering is construed as the improvement of cellular activities by the manipulation of enzymatic, regulatory, and transport functions of the cell by the use of recombinant DNA technology. Metabolic engineering differs from the traditional genetic approaches from the point of view of the incorporation of heterologous genes and regulatory elements. Metabolic engineering has immense potential to produce a diverse group of chemicals from simple, inexpensive starting materials. Microbial production of natural products can be achieved by transferring product-specific enzymes or entire metabolic pathways from rare or genetically intractable organisms to those organisms that can be readily engineered [17]. The engineered metabolism can be applied for the generation of transportation fuels from inexpensive, easily available carbon sources like biomass. In fact microorganisms have been used to produce alcohols. Of late, engineered microbes like yeasts and bacteria have been reported to be used for alcohol production. The engineering of microbial biosynthetic pathways enables the production of advanced biofuels like alcohols, esters, and alkanes. The scientific developments in industrial fermentation processes have aided in the production of biofuels in bioreactors [13].

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5.5 METABOLIC PATHWAYS FOR ALCOHOL-DERIVED FUELS The first-generation biofuels like bioethanol dominate the biofuel landscape. However, the first-generation biofuels are contested due to the concern for economic and ecological sustainability, food security, and their higher lifecycle greenhouse gases emissions [7]. There are other short-chain alcohols which are sustainable and can be produced by microorganisms through metabolic engineering [5]. The metabolic pathway can be appropriately exploited for the production of advanced biofuels. Short-chain or higher alcohols like butanol are potential advanced biofuels that can be added to gasoline as oxygenates [14]. Further, unlike bioethanol, three-carbon isopropyl alcohol (C3H7OH) and four-carbon butyl alcohol (C4H9OH) possess higher “energy intensity” and lower “hygroscopicity.” Clostridium spp. is found naturally to produce isopropyl alcohol and butyl alcohol from acetylCoA. In the fermentative pathway, CoA from acetoacetyl-CoA is transferred to acetate to form acetoacetate and acetyl-CoA. The removal of carbon dioxide from acetoacetate leads to the formation of acetone, which subsequently forms isopropyl alcohol on reduction. The enzymes involved in this biosynthetic scheme are “Thl, CtfAB, and ADC” (Clostridium acetobutylicum); “ADH” (Clostridium beijerinckii); and “AtoAD” (Escherichia coli) [5]. Although Clostridium spp. produce valuable products like isopropanol and butanol, the slow growth rate and the requirement for anaerobic growth conditions hinder the production of higher concentrations of alcohol. The isopropyl alcohol biosynthetic pathway found in Clostridium sp. was reconstructed in the bacteria E. coli for the synthesis of isopropanol [18,19]. The reasons for choosing E. coli for metabolic engineering are its fast growth rate and tractable genetic system [15]. The isopropyl alcohol production achieved by Jojima and coworkers [19] was 13.6 g/L, which was about 51% of the maximum expected theoretical yield [5,13]. Isopropanol properties like high energy density (23.9 MJ/L) and low water solubility enable it to be chosen as a preferred additive to petroleum products. Butanol, a C4 compound, is considered as an alternative of biological origin to gasoline due to its high energy density (29.2 MJ/L) and octane number of 87. The hydrophobicity of butanol allows for the use of the existing storage and transportation infrastructure. The 1-butantol biosynthesis occurring naturally in C. acetobutylicum involves the formation of acetoacetyl-CoA through the condensation of two acetyl-CoA molecules. Subsequently, acetoacetyl-CoA undergoes the processes of reduction and dehydration resulting in the formation of butyryl-CoA, which eventually due to dehydrogenation results in 1-butanol [5]. Atsumi and coworkers [20], Inui and coworkers [21], and Nielsen and coworkers [22] successfully constructed the “Clostridium butanol biosynthetic pathway” in E. coli. The butanol synthesis function occurring naturally in Clostridium was inserted into E. coli by inserting “atoB, hbd, crt, bcd, etfAB, and adhE2” genes and deleting “ldhA, adhE, frdBC, pta, and fnr” genes [23]. On the other hand, Steen and coworkers [24] constructed the Clostridium butanol biosynthesis pathway in Saccharomyces cerevisiae. The butanol production varied between 13.9 mg/L and 1.2 g/L. However, there is a need to improve the butanol tolerance in E. coli as it cannot withstand more than 1.5% butanol by volume. S. cerevisiae is reported to exhibit tolerance to ethyl alcohol and butyl alcohol. Lactobacillus brevis also shows tolerance to butyl alcohol [14]. Microorganisms possess various mechanisms to overcome toxicity. For instance, while Pseudomonas putida overcomes toxicity through efflux pumps, Bacillus subtilis overcomes toxicity through alterations in its cell wall composition [14]. Higher alcohols can also be produced from amino acid biosynthetic pathway intermediates. Yeast uses the Ehrlich pathway or 2-keto-acid pathway to produce alcohols. The pathway enables alcohol formation through step-wise keto acid decarboxylation and subsequent reduction. The Ehrlich pathway is found to be endogenous to a few yeasts, wherein the fusel alcohols are the fermentation by-products. Isobutanol can also be produced from valine using this pathway [14].

5.6 METABOLIC PATHWAYS FOR ISOPRENOID-DERIVED FUELS Isoprenoids are the oldest known biomolecules and the largest group of contemporary natural products, encompassing over 30,000 known compounds. Structurally and functionally, isoprenoids are a varied group of organic compounds with immense diversity. Isoprenoids are formed through the condensation of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). There are two distinct and independent biosynthetic routes to IPP. The pathway to IPP in mammals and yeast starts from acetyl-CoA and proceeds through the intermediate mevalonic acid. In algae and higher plants, IPP is synthesized by the condensation of pyruvate and glyceraldehyde-3-phosphate, via 1-deoxyxylulose 5-phosphate (DXP) as the first intermediate [25]. IPP and DMAPP precursors are combined in the presence of prenyltransferases to form intermediates like “geranyl pyrophosphate” (GPP, C10), “farnesyl pyrophosphate” (FPP, C15), or “geranylgeranyl pyrophosphate”

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(GGPP, C20). These intermediates are converted by the enzyme terpene synthases into C10 monoterpenes, C15 sesquiterpenes, and C20 diterpenes. Shorter, branched isoprenoid molecules and larger, cyclic isoprenoids are suitable candidates for biofuels production [5]. The former are gasoline substitutes and the latter are diesel and jet fuel substitutes. The structure of isoprenoids, which includes branches and rings, enables them to be potential candidates for biofuel production. The structure provides properties like high octane number, reduced premature ignition, etc. [14]. In order to increase the quantity of isoprenoids and related biofuels, the deoxyxylulose-5phosphate and mevalonate pathways can be engineered. In yeast, C15 farnesol was obtained from C15 farnesyl pyrophosphate through adopting strategies like the augmented expression of MEV pathway enzymes, the repression of the endogenous squalene synthase, and the use of a native pyrophosphatase. Similarly, E. coli that was engineered to coexpress the “heterologous MEV pathway and FPP synthase” produced farnesol [5].

5.7 METABOLIC PATHWAYS FOR FATTY ACID-DERIVED FUELS Fatty acids are found in cell membranes, vegetable oils, and animal fats. They occur normally as phosphoglycerides and triglycerides. Fatty acids for biofuel production need to be converted into nonionic, hydrophobic compounds, such as fatty alcohols, fatty acid alkyl esters, etc. Fatty acid derivatives on account of their high energy density and low water solubility are construed as viable biofuels. The fatty acid biosynthesis pathway can be engineered for the production of long-chain alkanes and esters. Fatty acids can be reduced step-wise to form fatty alcohol. Fatty acids form diesel through esterification with small alcohols. For instance, fatty acid methyl esters and fatty acid ethyl esters are formed as a result of the esterification of fatty acids with CH3OH and C2H5OH, respectively. Nevertheless, biodiesel production is constrained and challenged by crop geography and the seasonal constraints of growing oil-yielding crops [13].

5.8 SYNTHETIC BIOLOGY AND ITS ROLE IN DESIGN OF MICROBIAL CELL FACTORIES Metabolic engineering through granular metabolic analyses aims at identifying appropriate targets for manipulation and directed genetic modification using recombinant DNA technology for the improvement and/or design of cells. The cells may be improved or designed to widen the substrate range, to increase productivity and cellular robustness, and for the production of novel and useful products. On the other hand, synthetic biology emerged as an advanced field of study aiming to reconstruct artificial biological systems and to synthesize DNA and complete chromosomes. Synthetic biology offers striking opportunities to create cell factories custom-made for efficient synthesis of value-added chemicals and biofuel on a scale and quantum that is sustainable [26,27]. Synthetic biology has been developing genetic circuits that enable the regulation of gene expression in the presence or absence of chemical and environmental inputs [28]. Metabolic engineering makes possible the design of biosynthetic pathways with the purpose of producing products like biofuels. On the other hand, the domain of synthetic biology enables the creation of novel biological functions [29]. Adopting synthetic dynamic control systems can avoid the biosynthesis of unnecessary RNAs/ proteins/metabolites, increase the efficiencies of energy and carbon usage, and allow a host to adjust its metabolic flux to minimize “maintenance loss” [30]. Both metabolic engineering and synthetic biology in tandem aid in the construction of cell factories. For instance, in the construction of a platform cell factory, a synthetic pathway is inserted into the cell factory to enable it to produce the product of interest. However, the quantity of the product of interest produced is upscaled by metabolic engineering which includes pathway optimization. Organisms which are well adapted to industrial conditions are used as platform cell factories. For example, the model organisms like S. cerevisiae and E. coli are considered as cell factories for biofuel production. The continued study of these organisms and the concomitant knowledge generation enable deeper understandings of the organisms and system components and lead to the development of predictive models. Microorganisms play a vital role in biofuel production as they act as biocatalysts in bioethanol fermentation [31]. Different biosynthetic pathways exist for the purpose of biofuels production, and in most of these cases either pyruvic acid or the acetyl-CoA forms important intermediaries [32]. E. coli and S. cerevisiae, are two organisms quite often utilized for the production of biofuels [33,34]. The first-generation biofuels like bioethanol and biodiesel are commonly used renewable liquid fuels for transportation. As regards lignocellulosic waste, the

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breakdown of cellulose derived from agricultural waste demands pretreatment processes and a variety of cellulose degrading enzymes produced by species like Trichoderma spp. The enzymes are endocellulases, exocellulases, β-glucosidases, and cellobiose dehydrogenase [35].

5.9 ENGINEERING MICROBES FOR TOLERANCE TO NEXT-GENERATION BIOFUELS Persistent demand for renewable fuel resources coupled with the rapid strides in metabolic engineering have enabled the identification of ways to upscale the production of biofuel. Nevertheless, the production of biofuel through microbial technology is constrained by the inability of the cells to tolerate the toxic levels of production targets. Often, the biofuels are found to hinder the physiological functioning of the cells and reduce the viability of the cells. This end product inhibition places a limit on the amount of biofuel that can be produced by the cell. Further, inhibitors from the biofuel production process can exhibit cytotoxicity. For instance, residual products like cellulosic hydrolysate produced from the biofuel feedstock can show cytotoxicity. So it is pertinent to develop sustainable strategies so that the biofuel production process is optimized with simultaneous improvement in the tolerance levels of the cells involved in biofuel generation. Recently, there have been several efforts toward engineering strains for biofuel tolerance. Promising methods to engineer strains for biofuel tolerance include engineering biofuel export systems, heat shock proteins, membrane modifications, in situ recovery methods, and media supplements [36]. It is pertinent to synchronize the tolerance engineering efforts with the exploration and engineering of genetically tractable organisms for biofuel production. The microorganisms found in the hydrocarbon-rich environment are valuable resources as they can provide genes of interest to the biofuel production hosts.

5.10 CONCLUSION The widening energy demand supply gap, the volatility in fuel prices, and the growing concerns regarding global climate change have stimulated interest in biofuel production. The materials of biological origin can either be directly burned as solids (biomass) to produce heat energy or converted into liquid biofuels, which have found extensive use in the mobility sector. Biofuels are construed as one of the important sustainable fuel sources and possess huge potential to minimize greenhouse gas emissions. The use of biomass for biofuel production reduces the dependency on fossil fuels and biofuels are considered important from the perspective of climate change mitigation and adaptation. The first-generation biofuels like ethanol, due to their physicochemical properties and the food fuel debate, are considered unsustainable in the long term. Further, alcohols like propyl alcohol and butyl alcohol are fuels of choice as they possess properties like higher energy values/intensity and are comparatively less corrosive. However, the low yield of these biologically-derived hydrocarbons is a major obstacle for commercialization. Great strides in microbiology and biotechnology have built an enviable foundation for understanding cellular processes and metabolism. Of late, metabolic engineering has enabled the improvement of the cellular phenotypes of microorganisms. Metabolic engineering provides a platform to change the metabolic pathways with an aim of augmenting biofuel production. In fact, the metabolic activities of living cells are realized by a regulated, highly coupled network of about 1000 enzyme-catalyzed reactions and selective membrane transport systems. Enzymes are the basic units of metabolism. Several enzymes work in sequence to form a pathway. The enzyme and pathway engineering are the key components of metabolic engineering. The success and efficiency of metabolic engineering is dependent on how we undertake an in-depth study, design, and optimization of the metabolism in an organism. The engineered metabolism can be applied in the production of transportation fuels from easily available, low-cost renewable sources of carbon like biomass. The engineering of microbial biosynthetic pathways enables the production of advanced biofuels like alcohols, esters, and alkanes. Recent developments in the domain of metabolic engineering and synthetic biology have revealed new opportunities for the manipulation of microbes for biofuel production. The successful and sustainable use of microorganisms for biofuel production depends on the organisms’ capabilities to engender biofuels on a scale that is economical, productive, and sustainable at a faster rate and lower cost. This chapter discusses the metabolic engineering for the production of biofuels and emphasizes the need to integrate synthetic biology and systems biology to optimize host organisms and overcome biosynthetic pathway bottlenecks.

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Kriemann, J. Savolainen, S. Schlo¨mer, C. von Stechow, T. Zwickel, J.C. Minx (Eds.), Climate Change 2014: Mitigation of Climate Change. Contribution of Working Group III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge, UK and New York, NY, USA, 2014. [8] F.R. Spellman, The Science of Renewable Energy, second ed., CRC Press, Boca Raton, 2016, pp. 321 401. [9] R. Saxena, D. Adhikari, H. Goyal, Biomass-based energy fuel through biochemical routes: a review, Renewable Sustainable Energy Rev. 13 (1) (2009) 167 178. [10] K. Sekhon, P. Rahman, Synthetic biology: a promising technology for biofuel production, J. Pet. Environ. Biotechnol. 04 (06) (2013) e121. [11] M. Wen, B. Bond-Watts, M. Chang, Production of advanced biofuels in engineered E. coli, Curr. Opin. Chem. Biol. 17 (3) (2013) 472 479. [12] Y. Dhaman, P. Roy, Challenges and generations of biofuels: will algae fuel the world? Ferment. Technol. 01 (05) (2012) 119. [13] P. Peralta-Yahya, J. Keasling, Advanced biofuel production in microbes, Biotechnol. J. 5 (2) (2010) 147 162. [14] P. Peralta-Yahya, F. Zhang, S. del Cardayre, J. Keasling, Microbial engineering for the production of advanced biofuels, Nature 488 (7411) (2012) 320 328. [15] P. Xu, M. Koffas, Metabolic engineering of Escherichia coli for biofuel production, Biofuels 1 (3) (2010) 493 504. [16] J. Bailey, Toward a science of metabolic engineering, Science 252 (5013) (1991) 1668 1675. [17] J. Keasling, Manufacturing molecules through metabolic engineering, Science 330 (6009) (2010) 1355 1358. [18] T. Hanai, S. Atsumi, J. Liao, Engineered synthetic pathway for isopropanol production in Escherichia coli, Appl. Environ. Microbiol. 73 (24) (2007) 7814 7818. [19] T. Jojima, M. Inui, H. Yukawa, Production of isopropanol by metabolically engineered Escherichia coli, Appl. Microbiol. Biotechnol. 77 (6) (2007) 1219 1224. [20] S. Atsumi, A. Cann, M. Connor, C. Shen, K. Smith, M. Brynildsen, et al., Metabolic engineering of Escherichia coli for 1-butanol production, Metab. Eng. 10 (6) (2008) 305 311. [21] M. Inui, M. Suda, S. Kimura, K. Yasuda, H. Suzuki, H. Toda, et al., Expression of clostridium acetobutylicum butanol synthetic genes in Escherichia coli, Appl. Microbiol. Biotechnol. 77 (6) (2008) 1305 1316. [22] D. Nielsen, E. Leonard, S. Yoon, H. Tseng, C. Yuan, K. Prather, Engineering alternative butanol production platforms in heterologous bacteria, Metab. Eng. 11 (4 5) (2009) 262 273. [23] V. Koppolu, V. Vasigala, Role of Escherichia coli in biofuel production, Microbiol. Insights 9 (2016) 29 35. [24] E. Steen, R. Chan, N. Prasad, S. Myers, C. Petzold, A. Redding, et al., Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol, Microb. Cell. Fact. 7 (1) (2008) 36. [25] B. Lange, T. Rujan, W. Martin, R. Croteau, Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes, Proc. Natl. Acad. Sci. U. S. A. 97 (24) (2000) 13172 13177. [26] J. Nielsen, J. Keasling, Synergies between synthetic biology and metabolic engineering, Nat. Biotechnol. 29 (8) (2011) 693 695. [27] J. Nielsen, M. Fussenegger, J. Keasling, S. Lee, J. Liao, K. Prather, et al., Engineering synergy in biotechnology, Nat. Chem. Biol. 10 (5) (2014) 319 322. [28] A.S. Khalil, J.J. Collins, Synthetic biology: applications come of age, in: E. Choffnes, D. Relman, L. Pray (Eds.), The Science and Applications of Synthetic and Systems Biology: Workshop Summary, National Academic Press, Washington, D.C, 2010. [29] V. Colin, A. Rodrı´guez, H. Cristo´bal, The role of synthetic biology in the design of microbial cell factories for biofuel production, J. Biomed. Biotechnol. 2011 (2011) 1 9. [30] F. Zhang, J. Carothers, J. Keasling, Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids, Nat. Biotechnol. 30 (4) (2012) 354 359. [31] A. Agarwal, Biofuels (alcohols and biodiesel) applications as fuels for internal combustion engines, Prog. Energy Combust. Sci. 33 (3) (2007) 233 271. [32] C. Rabinovitch-Deere, J. Oliver, G. Rodriguez, S. Atsumi, Synthetic biology and metabolic engineering approaches to produce biofuels, Chem. Rev. 113 (7) (2013) 4611 4632. [33] J. Clomburg, R. Gonzalez, Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology, Appl. Microbiol. Biotechnol. 86 (2) (2010) 419 434. [34] J. Fortman, S. Chhabra, A. Mukhopadhyay, H. Chou, T. Lee, E. Steen, et al., Biofuel alternatives to ethanol: pumping the microbial well, Trends Biotechnol. 26 (7) (2008) 375 381. [35] Q. Xu, A. Singh, M. Himmel, Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose, Curr. Opin. Biotechnol. 20 (3) (2009) 364 371. [36] M. Dunlop, Engineering microbes for tolerance to next-generation biofuels, Biotechnol. Biofuels. 4 (1) (2011) 32. [37] S. Lee, H. Chou, T. Ham, T. Lee, J. Keasling, Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels, Curr. Opin. Biotechnol. 19 (6) (2008) 556 563.

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6 Bioprospecting Actinobacteria for Bioactive Secondary Metabolites From Untapped Ecoregions of the Northwestern Himalayas Qazi Parvaiz Hassan, Aasif Majeed Bhat and Aabid Manzoor Shah Microbial Biotech Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, India O U T L I N E 6.1 Introduction to Secondary Metabolites

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Acknowledgment

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References

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6.1 INTRODUCTION TO SECONDARY METABOLITES Primary and secondary metabolites are secreted by almost all bacteria. Primary metabolites are directly involved in the normal growth and development of the organism. Secondary metabolites, unlike primary metabolites are not directly involved in the normal growth and development of the organism that produces these substances [1]. Although not exclusive, secondary metabolites are unique to different species and are produced in small amounts as ecological adaptations to different environmental factors. These secondary metabolites are diverse in their structure and function and include substances with different secondary but important functions. The microbes are considered to be the factory manufacturing these secondary metabolites. Microorganisms produce almost all the secondary metabolites such as antibiotics, antitumor agents, enzymes, pigments, toxins, and plant growth regulators. The microbes may or may not possess fungus-like mycelia. The mycelium-bearing microbes possess aerial and substrate hyphae. The substrate hyphae adhere the microbes to the substratum like the soil, tree, rock, or an agar plate. The aerial hyphae, hanging above the substratum, bear two types of mycelia—vegetative mycelium and reproductive mycelium; the latter possess spores and assist in reproduction and the former produce and secrete secondary metabolites during the generation of aerial hyphae. Thus the vegetative mycelia of aerial hyphae are the secondary metabolite production and secretion structures of mycelium-bearing microbes [2]. These secondary metabolite have no primary functions, for example, as an energy source (glucose), cell synthesis (amino acids and fatty acids), or genetic information (nucleic

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acids—DNA and RNA). They are produced in response to certain environmental stimuli as defensive measures to protect these microbes against predators and parasites, in interspecies competition, and in reproductive processes [3]. The microbe that produces the secondary metabolites (antibiotics and mycotoxins) kills other microbes, while sparing itself from the deleterious and toxic side effects of these metabolites. This is accomplished by clustering the genes that code for these metabolites with the genes that confer resistance to that particular compound [1]. This secondary metabolite-resistant genes cluster allows an organism to carry a particular function without itself being affected. Microorganisms produce secondary metabolites during the stationary phase (GO) of the growth cycle. The metabolite production is believed to be triggered by certain optimal conditions such as nutrient exhaustion, decrease in growth rate, and inducer biosynthesis. The microorganisms receive these responses as a signal and then activate or deactivate signal transduction pathways, cause upregulation or downregulation events in chemical differentiation (secondary metabolism), and/or cause morphological differentiation (morphogenesis) [4]. Secondary metabolism is regulated by certain low molecular weight diffusible substances called “autoregulators.” The most well-known autoregulators are γ-butyrolactones (butanolides) of the actinobacteria, oligopeptides of Gram-positive unicellular bacteria, and N-acylhomoserine lactones of Gram-negative bacteria [4]. Microbes are dynamic in the sense that they perceive changing conditions in their surroundings and in response secrete secondary metabolites. Thus the chemical nature of the secondary metabolite is determined by environmental changes. In other words, different environmental conditions facilitate the microorganisms to produce niche specific secondary metabolites. This makes them an exciting pool of novel biodiversity. The isolation of microbial natural products, followed by their screening and their study is a significant scientific approach for the development of prospective therapeutic agents from lesser known and/or new bacterial taxa [5]. It is generally believed by the microbiological researchers that microorganisms are unlimited sources of novel compounds, many of which have therapeutic applications. The microbial populations therefore need to be isolated and screened for the presence of secondary metabolites that could be used as significant pharmaceutical lead molecules. The secondary metabolites produced by a variety of organisms have played important roles in medicine and have revolutionized the pharmaceutical field. In this respect the major contribution has been provided by actinobacteria, especially by the genus Streptomyces.

6.2 INTRODUCTION TO ACTINOBACTERIA “Actinobacteria,” also called actinomycetes, derive their name from two Greek words “atkis” (a ray) and “mykes.” Actinobacteria are a heterogeneous group of high GC Gram-positive bacteria. The GC base-pairing composition of their DNA is more than 55 mol.% of total DNA [6
8]. They are free-living, aerobic, filamentous, saprophytic bacteria, cosmopolitan in their distribution in terrestrial and aquatic environments, are also found colonizing plants, and are the major sources for antibiotic production [9]. They possess varying chemical compositions, are diverse in their morphology, and show distinct evolutionary lineage [10]. They are omnipresent soil microbes of filamentous structures, although some possess pleomorphic and even coccoid shapes. Their taxonomy is as follows: Kingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae, with eight diverse families: Actinomycetaceae, Streptomyceteceae, Actinoplanaceae, Mycobacteriaceae, Micromonosporaaceae, Frankiaceae, Dermatophilaceae, and Nocardiaceae, comprising 63 genera. The morphology of actinobacteria shows mycelia or linear or branched hyphae that give them a resemblance to fungi, but unlike fungi and like bacteria the presence of muramic acid residues in their cell wall structure classifies these microbes as Gram-positive bacteria. However, the fungal morphology distinguishes this group of microbes from other bacteria. Also the molecular basis of nucleic acid sequencing and pairing studies has differentiated these actinobacteria from other bacteria. When the actinobacteria are in their vegetative stage, the reproductive hyphae are absent and all hyphae are vegetative. With maturity, reproductive hyphae bearing asexual spores arise from vegetative hyphae. Although like fungi they possess true hyphae and bear spores [11], they are not fungi in the true sense and form a separate evolutionary lineage of organisms [12]. First discovered by Ferdin and Cohn [13], actinobacteria belong to eubacteria and are one of the largest taxonomic groups within it [14]. Several genera of actinobacteria are being studied but owing to their enormous bioactive potential Streptomycetes are given greater attention and hold a prominent position in drug discovery. Their life cycle is complex, neither exclusively unicellular nor multicellular [2].

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6.3 DISTRIBUTION OF ACTINOBACTERIA Actinobacteria are a widely distributed group of microorganisms that exist in almost all types of habitats, grow in different types of soils, and are filamentous in structure [15]. Their ability to produce different bioactive metabolites, and be responsive to different ecozone adaptations, allows them to survive and flourish in different ecosystems. These prokaryotes constitute a significant component of the microbial population in most soils [16]. They belong to the order Actinomycetales, are principally terrestrial microbes, inhabiting soils of different compositions, are found attached to almost all natural soil substrata, and are the most prominent producers of known antibiotics [17]. These are cosmopolitan soil microbes and inhabit soils of diverse nature and composition. They flourish on the substrata of acidic soils, alkaline soils, fertile soils, alluvial soils, laterite soils, mountain soils, glaciated soils, and fallow land. [18]. One gram of fresh soil sample when spread on a differential growth medium constitutes about 107 out of 109 colony-forming units of actinobacteria [19]. The nature and type of actinobacteria growth depends on the type of soil. Although predominately terrestrial soil microbes, they are also found in freshwater and marine water soils. Among the aquatic habitats the marine environments are also being extensively studied and attempts made in this direction for the isolation and exploration of novel actinobacteria have met with some success [20]. Lentic and lotic bottom and river mud screening have also shown the presence of actinobacteria [21]. They are found in both cultivated and uncultivated soils [22]. Actinobacteria are also widespread in the lacustrine environment and studies also have suggested the cosmopolitan distribution of these bacteria in the oceans [23,24]. Those actinobacteria which grow in extreme latitudes at the North and South Poles in Arctic and Antarctic soils have been found to show excellent antibacterial activity against Gram-positive and Gram-negative bacteria [25]. Most of the actinobacteria live as free-living microbes, a few like Actinomyces israelii are pathogenic species. They grow as infectious agents in humans, other animals, plants [26], and marine sponges [27].

6.4 CHOICE OF ACTINOBACTERIA AS SOURCE OF BIOACTIVE SECONDARY METABOLITES Actinobacteria produce secondary metabolites which are diverse in chemical composition and may account for novel structures. These microbes demand special attention because nearly 61% of the known bioactive microbial metabolites have been isolated from them, especially from the genus Streptomycetes [25]. Actinobacteria produce a broad spectrum of metabolites of bioactive importance Table 6.1. The major producers of biologically important molecules are Streptomyces, Micromonospora, Actinoplanes, Amycolatopsis, and Saccharopolyspora. The genus Streptomycetes alone accounts for about 45%
55% of known antibiotics and is thus a prolific source of bioactive secondary metabolites. Through abiding by natural selection and the principle of the survival of the fittest, actinobacteria during the course of evolution have successfully managed to inhabit different environments and to produce potential bioactive compounds of physiological, biotechnological, therapeutic, and environmental significance. Because actinobacteria are cosmopolitan in their distribution, they inhabit both conventional and nonconventional environments. Conventional environments can be accessed easily and actinobacteria from these regions can readily be tapped so that they can be explored experimentally for the determination of their bioactive potential. This however is not the case with those actinobacteria like thermopiles, halophiles, and other extremophiles that inhabit nonconventional environments. These environments are nonconventional in the sense that the extreme conditions prevent easy access to them and consequently actinobacteria from these regions are largely untapped and unexplored. Thus the genera from these unfamiliar and nascent habitats are yet to be explored and deserve a special mention. These microbes warrant a promising potential to be employed for natural product screening methods to isolate novel species or novel compounds from previously known species. Efforts made in this direction have been successful and are supported by evidence from recent reports on the isolation and characterization of novel actinobacteria from poorly researched habitats [28,29]. Moreover, the same species of actinobacteria allowed to grow in different environments will produce environment-specific metabolites necessary for its survival. Therefore it can be presumed that the screening of untapped regions that possess unexplored actinobacteria will increase the chances of discovering new chemical compounds to be used as a resource for drug discovery and for other biotechnological significance.

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TABLE 6.1 Clinically Significant Antitumor and Antimicrobial Agents Produced by Actinomycetes Actinobacteria

Bioactive compounds

Streptomyces peucetius

Doxorubicin

Streptomyces chrysomallus

Actinomycin D

Streptomyces verticillus

Bleomycin

Streptomyces hygroscopicus

Rapamycin

S. peucetius

Daunorubicin

Streptomyces longisporoflavus

Staurosporine

Streptomyces lavendulae

Mitomycin

Streptomyces carzinostaticus

Neocarzinostatin

Streptomyces halstedii

Vicenistatin

Streptomyces sahachiroi

Azinomycin B

Streptomyces galilaeus

Aclacinomycin

Streptomyces griseus ssp.

Chromomycin

Streptomyces sp.

Nigericin

Streptomyces bikiniensis

Chalcomycin

Salinispora tropica

Salinosporamide A

Streptomyces cinnamonensis

Monensin

Streptomyces olivaceus

Elloramycin

S. carzinostaticus

Neocarzilin

Streptomyces griseoruber

Hedamycin

Streptomyces orientalis

Vancomycin

Streptomyces nodosus

Amphotricin B

Streptomyces mediterranei

Rifampin

Streptomyces kanamyceticus

Kanamycin

Streptomyces viridifaciens

Tetracycline

Streptomyces fradiae

Neomycin

S. griseus

Streptomycin

Streptomyces noursei

Nystatin

Streptomyces venezuelae

Chloramphenicol

Streptomyces erythraeus

Erythromycin

Streptomyces alboniger

Puromycin

S. fradiae

Fosfomycin

Streptomyces garyphalus

Cycloserine

Streptomyces clavuligerus

Cephalosporin

6.5 ACTINOBACTERIA FROM UNUSUAL ENVIRONMENTS The decades of the 1940s and 1950s are considered the “golden era” in antibiotic discovery, when almost all of the important groups of antibiotics, mainly isolated from Streptomyces species, were discovered [17]. Approximately half of the secondary metabolites discovered in this period were found to have bioactive potential. Most of the metabolites function as antibiotics, others as immunosuppressive agents, and a few as antitumor

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agents and enzymes. Large proportions of these secondary metabolites of pharmaceutical significance were isolated from actinobacteria. Almost 80% of the world’s antibiotics are known to come from actinobacteria, mostly from the genera Streptomyces and Micromonospora [30]. Unfortunately only 1%
3% of Streptomyces’ antibiotics have been discovered, the remaining 97%
99% are still undiscovered owing to the difficulty of their isolation by conventional technologies. Modern technologies are thus required for the isolation, selection, screening, and enrichment of these antibiotics from actinobacteria [31,32]. A major problem met during the isolation and screening of actinobacteria from conventional environments is the rediscovery of known species with known compounds [33,34]. The focus of microbiologists has thus changed from conventional to nonconventional environments, from inhabited to uninhabited parts of the globe. The aim is twofold: to attempt to isolate novel actinobacteria strains and/or to detect previously undetected bioactive secondary metabolites. Organisms that grow in unusual habitats have unique physiological and biochemical characteristics. These organisms produce niche specific secondary metabolites. The properties of niche specialization enable them to thrive in extreme conditions of salinity, pressure, temperature, and pH. It is expected that the bioactive compounds produced through this process are unique in structure and function and may have commercial applications.

6.6 NORTHWESTERN HIMALAYAS AS SOURCES OF BIOACTIVE ACTINOBACTERIA The development of drug resistance by pathogenic microbes is a major concern that needs to be addressed. The excessive and inappropriate use of drugs, among other factors, is responsible for the drug resistance developed by these pathogens. The pathogens, due to their genetic variations, have developed drug resistance to overcome the lethal drug doses. A multidrug-resistant strain that is ineffective against a wide range of drugs adds fuel to the fire. The metabolites which were earlier used as drugs against a wide range of pathogens are now ineffective. Thus the need arises to bioprospect alternative sources of natural products and one successful attempt in this regard is to exploit the microbial biodiversity of previously untapped regions, which are expected to be inhabited by unexplored and novel genera of actinobacteria. In this regard the Northwestern Himalayas, a natural region endowed with rich indigenous biodiversity, offers promising potential. The Himalayas are huge mountain ranges, surrounding the whole of northern India from Karakoram to Purvanchal. They are horizontally divided from east to west into the Eastern Himalayas, the Central Himalayas, and the Western Himalayas. The Kashmir Valley, being the northwestern part of India, is surrounded by the Northwestern Himalayas Fig. 6.1. The Kashmir Himalayan region is an integral but geologically younger part of the main Himalayan range. The region, sometimes referred to as the “Switzerland of Asia,” lies between 32 200 to 34 540 north and 73 550 to 75 350 east, comprising an area of 15,948 km2. The mountains of the Himalayas include the Pir Panjal Range in the south and southwest and the Greater Himalayan Range in the north and east. These lofty mountain ranges enclose the deep elliptical bowl-shaped valley of Kashmir. The Himalayan mountain region in the subcontinent stretches from east to west over a distance of about 2500 km and is one of the world’s richest regions of biodiversity with unique microflora, particularly bacteria, fungi, and actinobacteria, of immense biotechnological potential. The need of the hour is to explore these extreme regions with a view to tapping their microbial biodiversity. The region of the Northwestern Himalayas of Jammu and Kashmir is a high-altitude mountain region with different soil textures. These high altitudes have characteristic features like lush green meadows, valleys, alpine glaciers, and a series of different elevations of varying temperature ranges that harbor different plethora of microorganisms, which are sensitive to different ranges of temperatures and soil salinity. The glacier-capped deep gorges are covered with frozen snow (Fig. 6.2) throughout the year. These alpine glaciers are rich sources of extremophilic microorganisms that successfully survive in frozen soils. Included in these microorganisms are psychrophillic/psychrotolerant bacteria and actinobacteria. A thorough literature survey suggests that the actinobacteria present in various niches of the Northwestern Himalayas have remain largely unexplored to date for their antimicrobial and anticancer potential. The research work of the Biotechnology division at CSIR-IIIM Srinagar deserves a special mention. Fieldwork soil sampling from different untapped high-altitude regions of the Northwestern Himalayas, such as Thajiwas glacier, Sinthan top, Khilanmarg, Gulmarg, and Naranag, followed by the isolation of pure cultures and their spectroscopic analysis has shown the presence of a repository of actinobacteria of enormous bioactive potential. Streptomyces tauricus isolated from Thajiwas glacier was for the first time reported to show the presence of an anticancer drug actinomycin D [35]. Furthermore, three new compounds were isolated from a Lentzea violacea strain isolated from the Northwestern Himalayas [36] (Fig. 6.3). These compounds upon chemical analysis were

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FIGURE 6.1 A part of the Northwestern Himalayas in Kashmir, India.

FIGURE 6.2 Gorges covered with frozen snow. NEW AND FUTURE DEVELOPMENTS IN MICROBIAL BIOTECHNOLOGY AND BIOENGINEERING

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FIGURE 6.3 A research team sampling in the Northwestern Himalayas.

found to be a new eudesmane sesquiterpenoid (Compound 1), a new homologue of virginiae butanolide E (Compound 2), and butyl isobutyl phthalate (Compound 3), with Compounds 1 and 2 showing moderate activity against Gram-negative bacteria. Compound 1 was also found to be active against human cancer cell lines. The secondary metabolites of a potent, rare strain, L. violacea, exhibited promising antituberculosis activity [37]. Another actinobacterial strain from the Kashmir Himalayas belonging to the genus Streptomyces has shown the presence of a potent cytotoxic compound that is effective against human colon (HCT-116) cancer cells. Upon characterization the compound was confirmed as alborixin from the species Streptomyces scabrisporus [38]. Soil actinobacteria of the Northwestern Himalayas (with particular reference to the Kashmir Himalayas) have also been evaluated for their antimicrobial activity. In an experiment at IIIM Srinagar, a total of 121 morphologically different actinobacterial strains were isolated and screened for antimicrobial activity against various human pathogens. Many strains showed moderate to high activity, but Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus and Mycobacterium tuberculosis. Based on spectral data analysis, four compounds, namely actinomycin C1, actinomycin C2, actinomycin C3, and actiphenol, were isolated from S. pratensis. Actinomycin C1, C2, and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The study revealed that S. pratensis from the Northwestern Himalayas is a promising strain and could be of great potential for industrial applications [39]. Two compounds reported for the first time from Streptomyces puniceus were characterized as the macrotetrolide Compound 1 (Dinactin) and Compound 2 (1-(2,4-dihydroxy-6-methylphenyl)-ethanone). Dinactin was shown to possess strong antimicrobial activity against all tested bacterial pathogens including M. tuberculosis. It also showed marked antitumor potential against various human cancerous cell lines with few cytotoxicity effects in normal cells [40].

6.7 CONCLUSION Actinobacteria are one of the most promising classes of bacteria that produce novel secondary metabolites with a wide range of applications. Numerous metabolites from actinobacteria have shown to possess antibacterial, antifungal, anticancer, antitumor, cytotoxic, and antiinflammatory properties. These bacteria flourish in soils of different compositions and textures. Their diversity ranges from terrestrial to aquatic (marine and freshwater) ecozones, in which they secrete habitat- and niche-specific secondary metabolites. Vegetative mycelia thrown out from the substrate mycelia of these actinobacteria produce secondary metabolites of immense

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microbiotechnological importance. Due to the rediscovery of already known actinobacteria from conventional environments, there is a renewed interest in the exploration of actinobacteria from nonconventional and untapped ecozones of the Northwestern Himalayas for the production of bioactive metabolites. The high-altitude extreme ecosystems of the Northwestern Himalayas allow the growth of novel actinobacteria and the production of nascent temperature-sensitive secondary metabolites. These metabolites that originate from unusual habitats add to the molecular biodiversity of the Northwestern Himalayas. The pool of secondary metabolites shows promising potential to be used to combat multidrug-resistant pathogens. Actinobacterial flora from high-altitude untapped regions of the Northwestern Himalayas have not been fully explored to date. There are great chances to find novel antibiotics from the actinobacterial flora of these ecoregions which can be used more effectively against human diseases.

Acknowledgment We are grateful to Elsevier group for enabling us to write on a topic of hot and recent trend in microbiological research and for advising required changes throughout the manuscript. We are deeply indebted to our sample collection team members Dr. Phalisteen Sultan, Mr. Mohd Ayub Bhat, Mr. Riyaz Ahmad Bhat, Mohd Musa Malik, and Mr. Showkat Ahmad Wani who left no stone unturned to walk along the difficult terrains of Himalayas to search out the best possible samples, the research on which adds to this manuscript.

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C H A P T E R

7 Microbial Metabolites: Peptides of Diverse Structure and Function Des Raj Kashyap Department of Microbiology and Immunology, Indiana University School of Medicine-NW, Gary, IN, United States O U T L I N E 7.1 Introduction

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7.3 Classification of Microbial AMPs 7.3.1 Class I: Posttranslationally Modified Bacteriocins 7.3.2 Class II: Unmodified Bacteriocins

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References

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Further Reading

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7.1 INTRODUCTION Microbial metabolites continue to play pivotal roles in the discovery of new therapeutic candidates. As a whole, microbes produce primary and secondary metabolites. Primary metabolites have an essential function in the organism itself, whereas secondary metabolites may have important functions for their producers, or they could simply be a waste product. A majority of the secondary metabolites produced by microorganisms may not participate directly in their growth and development; however, they play an important role in the organism’s ecological interactions with other organisms. Many of these products have therapeutic and agricultural applications and have been crucial to the health and well-being of humans. In many cases, such metabolites are being used for the treatment of human diseases such as cancer, bacterial infections, inflammation, and others [1 3]. The accidental discovery of penicillin by Alexander Fleming in 1928 in tandem with the advent of sulfanilamide marked the beginning of the antibiotic era. However, contrary to a report by the US surgeon William H. Stewart in 1976 “..we had essentially defeated infectious diseases and could close the book on them (infectious diseases)..,” the golden era barely lasted half a century [4]. Today, because of frequent and increasing resistance of bacteria to antibiotics, the emergence of persister (antibiotic-tolerant) bacteria, the failure to develop new or improve existing antibiotics, and detrimental changes to the beneficial microbiome caused by frequent use of antibiotics, bacterial infections are a major cause of morbidity and mortality. The World Health Organization (WHO) has expressed great concern about the current status of human health. According to WHO estimates, one billion individuals will be infected with Mycobacterium tuberculosis between 2000 and 2021, and about 35 million of those affected will die as a result of an antibiotic-resistant form of tuberculosis [5,6]. Regardless of several steps taken to avoid the development of microbial resistance, it is imperative that new antimicrobial agents and new strategies continue to be developed. Future therapies should apply target-specific antibiotics to single-out

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pathogens while leaving commensal microbes unaffected. Among the possibilities, antimicrobial peptides (AMPs) produced by bacteria offer a viable contributor. Because of their narrow killing spectrum, AMPs offer an advantage as they mostly kill bacteria that are closely related to the producers [4,7]. Since the field of AMPs is rather broad and covering them all is beyond the scope of this chapter, I will focus on the AMPs of microbial origin.

7.2 ANTIMICROBIAL PEPTIDES AMPs are evolutionarily conserved short protein molecules found in all forms of life, from multicellular organisms to bacterial cells (Table 7.1). The UNMC Department of Pathology and Microbiology at the Nebraska Medical Center in Omaha, NE, maintains a comprehensive database of AMPs (http://aps.unmc.edu/AP/main.php). The database contains 2830 AMPs from six kingdoms (294 bacteriocins and peptide antibiotics from bacteria, four from archaea, eight from protists, 13 from fungi, 341 from plants, and 2116 from animals). In higher organisms, AMPs contribute to innate immunity and are a crucial part of the first line of defense against harmful microorganisms. Conversely, in bacteria, these peptides provide a territorial advantage to the producers [4]. Their extraordinary range of antimicrobial activity and the ability to modulate host innate responses has spurred research and clinical interest in these AMPs, as potential therapeutic option against all pathogens [18]. Bacterial AMPs are not new to us. An antimicrobial substance, called gramicidin, isolated from Bacillus brevis in 1939, is believed to have started the era of bacterial AMPs. Gramicidin was successfully used to treat wounds on guinea pig skin, confirming its therapeutic potential [19]. Later the peptide was shown to exhibit activity against a wide range of Gram-positive bacteria [20,21]. This was the first AMP of bacterial origin to be commercially manufactured as an antibiotic [22]. However, the advent of penicillin and streptomycin in 1943 led to a rapid loss of interest in the therapeutic potential of the AMPs. In the early 1960s, with a realization that the “Golden Age of Antibiotics” had ended due to a rise in the multidrug-resistant microbial pathogens, a renewed interest in host defense molecules was awakened [23,24]. Some sources consider this point in time to be the true origin of AMP research [25]. But as the emergence of bacterial resistance to antibiotics grew, so did the efforts to either discover new drugs or chemically modify existing drugs. Unfortunately, these discoveries and modifications could not guarantee the prevention of bacterial resistance [16]. Today bacterial resistance and the emergence of persister (antibiotic-tolerant) bacteria remain the key elements causing morbidity and mortality. Therefore the TABLE 7.1 A Comprehensive List of Antimicrobial Peptides (AMPs) From Different Sources Source of AMPs

AMPs

Antimicrobial activity

Reference

Mammals

α-Defensins, β-defensins, LL-37, indolicidin, lactoferrin, histatin, protegrin

Bacteria, fungi

[8 10]

Amphibians Buforins, japonicin-1 and 2, brevinin, temporin, magainin, tigerin-1, bombinins, pseudin-2, dermaseptin, distinctin

Bacteria, fungi, protozoa

[9,11,12]

Plants

Bacteria, fungi, insects, nematodes

[13]

Crustaceans Callinectin, astacidin 2, armadillidin, homarin, scygonadin, penaeidin, crustin, hyastatin, arasin, stylicin, hemocyanin-derived peptides

Bacteria, fungi

[14]

Fungi

Plectasin, echinocandins, aculeacins, mulundocandins, FK463, aureobasidin, leucinostatins, helioferins

Bacteria

[9,15]

Bacteria

Iturin A, fengycin, surfactin A, entianin, bacitracin, bacthuricin F4, thuricin Bn1, amylolysin, Bacteria, fungi plantazolicin, subtilosin, bacillomycin D, haloduracin, pumilacidin, sonorensin, lactosporin

[16]

Insects

Defensins (sapecins), drosocin, pyrrhocoricin, hymenoptaecin, coleoptericin, drosomycin, melittin, bombolitins, diptericin, cecropin A, bicarinalin, ponericin G2, gambicin, attacins, sarotoxin IA, ceratotoxin, stomoxyn, spinigerenin, thanatin, heliomicin, Alo3, defensin A, smD1, gallerimycin, termicin, royalisin, metchnikowin, apidaecin IA, abaecin, formaecin, lebocin,

[9,12,17]

Knottin-peptides, puroindolines, snakins, heveins, peptide PvD1, plant defensins, lunatusin, vulgarinin, hispidulin, Lc-def, cicerin, arietin, shepherins, cyclotides, thionins (types I V), lipid transfer proteins

Bacteria, virus, fungi, protozoa

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obvious need to discover and develop new approaches to antibacterial therapy with new inhibitory mechanisms stimulated a new interest in AMP research [26]. These novel peptides offer a great alternative as they can either be used alone, as a replacement for the existing antibiotics, or be used in combination with other drugs to adequately combat antibiotic-resistant pathogens [4].

7.3 CLASSIFICATION OF MICROBIAL AMPS Microbial AMPs are a heterogeneous group of peptides having diverse structures and functions. Commonly referred to as bacteriocins, microbial AMPs share some common features with their eukaryotic counterparts. These features include their small size (20 50 aa), as well as cationic, amphiphilic, and hydrophobic properties. Yet there are also important differences between the two. For example, microbial AMPs, although often very potent, have a very narrow target spectrum. On the contrary, eukaryotic AMPs have a broader spectrum, targeting a larger diversity of bacteria [27]. The antimicrobial activity spectrum of these peptides largely hinges on their mechanism of action, and the relative distribution of their target among different bacterial species. According to their biosynthetic pathways, AMPs can be classified into either ribosomal or nonribosomal peptides [28]. Ribosomally synthesized AMPs, also known as bacteriocins [29], are the most studied subgroup of AMPs [26]. Most bacteriocins are products of Gram-positive bacteria, as reported in the BACTIBASE dataset (http://bactibase.pfba-lab-tun.org/statistics.php). According to this source, 156 of the 177 bacteriocins sequenced are the product of Gram-positive organisms and 18 of Gram-negative organisms. Spread across 31 genera, the lactic acid bacteria (LAB) make up the most prominent group of these bacteria, responsible for 113 bacteriocins. Few bacteriocins have been reported from the domain Archaea [30]. The current approach taken to classify bacteriocins is based on the model used for the classification of bacteriocins from the LAB [26]. Consequently the peptides that undergo posttranslational alteration are classified as Class I peptides, whereas the peptides that undergo modest modifications are classified as Class II peptides. These modifications include the formation of disulfide bridges and the circularization or the addition of N-formylmethionine [31]. According to this approach, larger proteins must be excluded from the bacteriocin category. In Gram-negative bacteria, most bacteriocins have been characterized from Escherichia coli and other enterobacteria. Subject to their size, the ribosomally synthesized antimicrobials are known as microcins (small peptides, ,10 kDa) or colicins (larger proteins, .20 kDa) [32,33]. The general consensus is that the designation “bacteriocin” should be retained for peptide antimicrobials [26], and thus we will not discuss ribosomally synthesized antimicrobial proteins in this chapter. With only 16 identified to date, microcins form a very restricted class of bacteriocins [34]. This can be attributed to their hydrophobic nature. Microcins are encoded by several genes on either a plasmid or chromosome. These genes are usually arranged in an operon and encode the precursor and proteins required for synthesis and activities such as secretion, self-immunity, and in some cases posttranslational modification of the microcins [35]. Based on their molecular masses, structure, gene synteny, the presence or absence of disulfide bonds, and posttranslational modifications, microsins are grouped into two classes [36] (see Fig. 7.1). Microcins that are plasmid-encoded and have a molecular mass less than 5 kDa are classified as Class I microcins. Examples of Class I microcins include B17, C (or C7 C51), and J25 produced by E. coli. Microcins belonging to this class undergo extensive backbone posttranslational modifications. On the other hand, microcins with a molecular mass greater than 5 10 kDa are classified as Class II microcins. Microcins belonging to this class can further be subdivided into two classes, Class IIa, and Class IIb. Microcins belonging to Class IIa, are plasmid-encoded peptides and do not undergo posttranslational modifications. These peptides are formed as an assembly of three peptides having two, one, or no disulfide bond(s). Example of Class IIa microcins are L, V, and N microcins. On the contrary, microcins belonging to Class IIb, are chromosome-encoded linear microcins. Examples of microcins belonging to Class IIb include microcins E492, M, and H47. Microcins belonging to this subclass may or may not carry a C-terminal modification. Microcins have a unique structure: a C-terminal core region and an N-terminal leader peptide [34]. During their export, the N-terminal leader peptide gets cleaved by the export machinery. As an example, CvaA/CvaB/TolC cleaves a 15-amino acid, double-glycine leader peptide from a Colicine V precursor during export [34]. In the past, there have been several attempts to classify bacteriocins of Gram-positive bacteria. These attempts involved classifying the bacteriocins based on determinants including the spectrum of activity, heat resistance, trypsin sensitivity, and cross-reactivity between different bacteriocins and host combinations [37,38]. These

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Gram-negative bacteriocins Microcins (Peptides,