Peptide Antibiotics Biosynthesis and Functions: Enzymatic Formation of Bioactive Peptidesand Related Compounds 9783110837834, 9783110084849

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Peptide Antibiotics Biosynthesis and Functions: Enzymatic Formation of Bioactive Peptidesand Related Compounds
 9783110837834, 9783110084849

Table of contents :
I. Introduction
A Survey of Enzymatic Peptide Formation
On Biosynthesis of the Cyclic Antibiotics Gramicidin S and Tyrocidine, and of Linear Gramicidin
II. Pathways and Genetics
Chemistry and Biosynthesis of the Poly (y-D-Glutamyl) Capsule in Bacillus licheniformis
Recent Developments with Cell free Extracts on the Enzymic Biosynthesis of Penicillins and Cephalosporins
Accumulation of

Citation preview

Peptide Antibiotics Biosynthesis and Functions

Peptide Antibiotics Biosynthesis and Functions Enzymatic Formation of Bioactive Peptides and Related Compounds

Editors Horst Kleinkauf

Hans von Döhren

w DE


Walter de Gruyter • Berlin • New York 1982

Editors Horst Kleinkauf, Professor, Dr.rer.nat. Hans von Döhren, Dr.rer.nat. Institut für Biochemie und Molekularbiologie Technische Universität Berlin Franklinstraße 29 D-1000 Berlin 10


der Deutschen


Peptide antibiotics - biosynthesis and functions : enzymatic formation of bioactive peptides and related compounds / ed. Horst Kleinkauf ; Hans von Döhren. - Berlin ; New York : de Gruyter, 1982. ISBN 3-11-008484-8 N E : Kleinkauf, Horst [Hrsg.]

Library of Congress Cataloging in Publication Data

Peptide antibiotics—biosynthesis and functions. Bibliography: p. Includes index. 1. Antibiotics--Physiological effect. 2. Peptide synthesis. 3. Microbiological synthesis. I. Kleinkauf, Horst, 1930. II. Döhren, Hans von, 1948QP801.A63P46 615'329 82-1391 ISBN 3-11008484-8 AACR2

Copyright © 1982 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Typesetting: W. Tutte, Druckerei GmbH, Salzweg. - Printing: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany


In late 1979 when we started organizing the first symposium on the enzymatic biosynthesis of peptides to be held in Berlin in 1980, we encountered a surprising response that enabled us to extend the programme. Since a wide coverage of the field was achieved, we decided to publish the contributions presented at this meeting in extended form, together with other articles written upon special request; at the present time, no comparable review or monograph is available on the subject. Work on the biosynthesis of peptides has been carried out for more than 30 years, and dates back to the time when enzymes were thought to be connected with protein biosynthesis catalyzing amino acid polymerization and fragment condensation reactions. As the determination of protein structure progressed, the need for nucleic acid templates, a general polymerization mechanism and ribosomal machinery became evident. In the sixties, some attention continued to be focused on the processes independent of nucleic acids. Lipmann's, Laland's and Kurahashi's groups evaluated during the course of their work on gramicidin S and tyrocidine in the late sixties two main features: (1) the activation of amino acids with subsequent aminoacylation is similar to the tRNA-charging in the ribosomal system and (2) the similarity of 4'phosphopantetheine-mediated transport of intermediate peptides to the polymerization of acetate units in fatty acid biosynthesis. During the last 10 years several multienzyme systems have been evaluated, some of which contain multifunctional polypeptides. However, the biosynthesis of many peptides has still not been elucidated, due to the instability of enzymes involved. These include penicillins, cephalosporins, actinomycins and valinomycin. Certain advances have come from genetic studies and protoplast work. On the other hand, structures such as the complex dodeca-peptide bacitracin and the modified nonadeca-peptide alamethicin have been obtained by enzyme preparations. From the limited number of peptide structures available, one is tempted to propose a limit of 30 to 40 specific residues for an enzymatic system, while identical units such as the y-glutamyl-capsule of Bacillus licheniformis may be repeated several thousand times. Peptides containing non-protein constituents may also be derived from precursors of ribosomal origin, as has been proposed for nisin and subtilin. No definite conclusions have yet been reached regarding the functions of peptides, nor of the regulation of their biosynthesis as mostly secondary metabolites. Several investigations proposing transcriptional regulation and membrane functional modification have been presented. It is the aim of this monograph to summarize the current advances in the field of enzymatic peptide biosynthesis and functions. We want to thank all authors for their contributions and their cooperation. The symposium from which this book has been derived was made possible through financial support from the Deutsche Forschungsgemeinschaft, the Senator für Wirtschaft und Verkehr (Berlin), and the Technische Universität Berlin. We also wish to thank the Bayer AG, Beckman Instruments GmbH, Braun Melsungen AG, Hoechst AG, LKB Instruments G m b H and Schering AG for their generosity. For their help in the preparation of the book we


are indebted to E. Denaro, H. Kühnauer, J. McGuirk and C. Stadler. Finally, we acknowledge the cooperation and patience of Mrs. E. Glowka and R. Weber of Walter de Gruyter Publishers. Horst Kleinkauf Hans von Döhren


It is my great honor to write the Foreword in this book which contains a thorough review of recent advances in studies of the biosynthesis of peptide antibiotics. It is said that microorganisms in nature compete with each other and produce antibiotics to suppress the growth of competitors, although this has not been fully studied experimentally. In contrast, it is certain that antibiotics are not involved in the functions of growth and multiplication in microbial cells. Antibiotics are secondary metabolites of microorganisms and divided into various groups, each one containing a common structural part. Studies on antibiotics suggest that the genes involved in the biosynthesis of the common structural parts were generated and distributed among different strains belonging to different species or genera. It is not true that all genes and their products are necessary for the growth and multiplication of the cells containing or producing them. It is certain that not only those genes which are necessary for growth but also those which are seemingly useless have been generated. The production of secondary metabolites may be dependent upon such genes which are useless in their producing strains. It seems that these genes have been transferred to cells of other strains, because the ability to produce a given group of antibiotics is widely distributed among strains of different species and genera. As described in this book, peptide antibiotics are synthesized by multifunctional enzymes. Protease inhibitor peptides which I have found are also synthesized in a similar manner. Leupeptin acid (acetyl-L-leucyl-L-leucyl-L-arginine), which has no biological activity, is synthesized in a reaction mixture containing acetylleucine, leucine, arginine, ATP, and leupeptin acid synthetase. The reaction sequence of this multifunctional enzyme is as follows: acetylleucine -> acetylleucylleucine —• leupeptin acid. Leupeptin acid is reduced to leupeptin, acetylleucylleucylargininal, which is rapidly released extracellularly. Leupeptin inhibits trypsin, papain, cathepsin B, etc., but does not function in its producing cell. The principle of the involvement of multifunctional enzymes of enzyme complexes in the biosynthesis of peptide antibiotics holds true also in the case of other secondary metabolite peptides. The genes for multifunctional enzymes can be cloned, and the deoxynucleotide sequences in them, determined. The results may indicate evolutionary relationships of these genes with those required for the growth and multiplication of microbial cells. How the genes for the biosynthesis of secondary metabolites were generated, evolved, and transferred to cells of other strains may be solved by investigation of peptide antibiotics. Such a study is important for a detailed understanding of the microbial world. However, such a study has been overlooked by investigators concerned with life science. The principle which has been found in the biosynthesis of peptide antibiotics may also hold true for the biosynthesis of other antibiotics. I am especially interested in the mode of biosynthesis of the structural part common to each group of antibiotics. It seems plausible that the biosynthesis of secondary metabolites other than peptides may depend upon multifunctional enzymes. I have been engaged in a study of aminoglycoside antibiotics and their derivatives. This



study has indicated that amino groups of aminoglycosides play a predominant role in antibacterial action, whereas hydroxyl groups do not. These findings are consistent with the fact that there are known antibacterial peptide antibiotics which behave similary to aminoglycosides in this respect. Their mechanism of action involves the inhibition of protein synthesis at the ribosomal level. Analysis of mechanism(s) of action of peptide antibiotics will also provide us with useful information for the study of other antibiotics. Investigation of secondary metabolites is, of course, necessary to understand the structure and function of nature, especially those of the microbial world. In this area, the study of the biosynthesis and function of peptide antibiotics has made a great contribution. In closing, I should like to emphasize once again that the principles found by study of peptide antibiotics may hold true for secondary metabolites in general. Professor Hamao Umezawa

December 15, 1981 Microbial Chemistry Research Foundation Institute of Microbial Chemistry Tokyo/Japan


I. Introduction


H. Kleinkauf, H. v. Döhren A Survey of Enzymatic Peptide Formation F. Lipmann On Biosynthesis of the Cyclic Antibiotics Gramicidin S and Tyrocidine, and of Linear Gramicidin


II. Pathways and Genetics



F.A. Troy Chemistry and Biosynthesis of the Poly (y-D-Glutamyl) Capsule in Bacillus licheniformis 49 D.J. Hook, R.P. Elander, R.B. Morin Recent Developments with Cell Free Extracts on the Enzymic Biosynthesis of Penicillins and Cephalosporins 85 H. Shirafuji, M. Yoneda Accumulation of ¿-(L-a-Aminoadipyl)-L-Cysteinyl-D-Valine Derivatives by Mutants of Cephalosporium acremonium 97 U. Keller, M. Ebert, H. Kleinkauf Genetic Studies on the Biosynthesis of Actinomycin 101 S. Sengupta, S.K. Bose Role of Peptides from Mycobacillin-Synthesizing System in the Biosynthesis of Mycobacillin 109 III. Fermentations and Peptide Production E.J. Vandamme, D. Leyman, D. De Buyser, P. De Visscher, J. Spriet, G . C . Vansteenkiste, O. Nimi, A. Poirier, A.L. Demain Environmental Influences on the Dynamics of the Gramicidin S Fermentation H.J. Aust, H. v. Döhren Production of Gramicidin S-Synthetase from Bacillus brevis ATCC 9999 by Batch Fermentation C.-W. Chiu, T. Bernhard, H.W. Dellweg Studies on the Gramicidin S-Synthetase Production in Bacillus brevis ATCC 9999 in C- and P-Limited Chemostat Cultures H.I. Haavik, 0 . Fröyshov On the Role of L-Leucine in the Control of Bacitracin Formation by Bacillus licheniformis U. Keller, R. Zocher, G. Kraepelin Current Research in Ergot Peptide Synthesis by Claviceps purpurea H.v. Döhren Applications of Multienzyme Systems in the Production of Peptide Antibiotics





155 161 169



IV. Enzyme Systems Gramicidin S-Synthetase S.G. Laland, K. Aarstad, T.L. Zimmer The Fidelity of Gramicidin S-Synthetase with Particular Reference to the Amino Acids Cyclohexylalanine and Phenylalanine Y. Saito Some Characteristics of Gramicidin S-Synthetase Obtained from Mutants of Bacillus brevis which could not form D-Phenylalanyl-L-ProlylDiketo-Piperazine R. Kittelberger, M. Altmann, H. v. Döhren Kinetics of Amino Acid Activation in Gramicidin S Synthesis J. Vater, N. Mallow, S. Gerhardt, H. Kleinkauf The Temperature Dependence of the Partial Processes Involved in the Biosynthesis of Gramicidin S D. Bothe, H. v. Döhren, H. Zschiedrich, A. El-Samaraie, M. Krause, H. Kleinkauf Further Characterization of Multienzyme Fragments of Gramicidin SSynthetase obtained from Gramicidin S Nonproducer Mutants M. Altmann, H. v. Döhren, A. El-Samaraie, M. Pore, R. Kittelberger, H. Kleinkauf Limited Proteolysis: Studies on the Multienzyme GS 2 of Gramicidin SSynthetase K. Aarstad, 0 . Frciyshov Tryptic Cleavage of the Heavy Enzyme of Gramicidin S-Synthetase C. Schröter, W. Rönspeck, M. Altmann, H. v. Döhren, H. Kleinkauf Use of Affinity Chromatography in Purification of Gramicidin S-Synthetases C. Christiansen, B. Nordvi, T.L. Zimmer, S.G. Laland A Survey on the Use of Affinity Chromatography for Studying the Mechanism of Gramicidin S Formation



195 209



243 253 259


Other Synthetases R. Zocher, U. Keller, H. Kleinkauf Enniatin-Synthetase: Studies on the Activation of Residues Involved in Enniatin Synthesis K. Kurahashi, S. Komura, K. Akashi, C. Nishio Biosynthesis of Antibiotic Peptides Polymyxin E and Gramicidin A H. Ishihara, I. Ogawa, K. Shimura Component I Protein of Bacitracin Synthetase: A Multifunctional Protein . . . L. Vitkovic, P. Pfaender Bacitracin Synthetase Subenzymes and Tail Peptide Formation in Bacitracin . 0 . Frciyshov, A. Mathiesen Bacitracin Synthetase. Tryptic Cleavage of Enzymes B and C Z. Kurylo-Borowska, J. Heaney-Kieras Edeine Synthetase: Evidence for Bidirectional Synthesis of Edeines

269 275 289 297 307 315



K. Suzukake, M. Hori, H. Hayashi, H. Umezawa Biosynthesis of Leupeptin 325 K. Bauer, P. Jungblut, H. Kleinkauf Biosynthesis of Carnosine and Related Peptides 337 J.E. Seely, F . D . Marshall Inhibition of Carnosine Synthetase by Spermidine 347 B. Shane Corynebacterium Species Folylpoly-y-Glutamate Synthetase 353 Peptide Synthetases - Problems and Properties. Summary of a Round-Table Discussion 369 V. Possible Functions of Peptide Antibiotics in their Producer Organisms M.A. Marahiel, H. v. Döhren A Survey of Possible Functions of Peptide Antibiotics in the Producer Organisms H. Ristow, J. Russo, E. Stochaj, H. Paulus Tyrocidine Induced Sporulation of Bacillus brevis in a Medium Lacking a Nitrogen Source M.A. Marahiel, W. Danders, G. Kraepelin, H. Kleinkauf Studies on the Role of Gramicidin S in the Life Cycle of its Producer Bacillus brevis ATCC 9999 J.F. Preston,B.E.C. Johnson, M. Little,T. Romeo, H.J. Stark, J.E. Mullersman Investigations on the Function of Amatoxins in Amanita Species: A Case for Amatoxins as Potential Regulators of Transcription L. K. Ramachandran, B.R. Srinivasa, G. Radhakrishna Structural Requirements for the Biological Activity of Polymyxin B J. Salnikow, G. Haeselbarth Gramicidin S - Fragments Generated by Mild Acid Hydrolysis of the Antibiotic N.S. Kaur, G . N . Chandrasekaran, N. Vasantha, K. Jayaraman Relevance of the Association of Polymyxin Biosynthetic Complex with Membrane DNA During Growth and Sporulation of Bacillus polymyxa ... Author Index Subject Index

373 375



399 427 445

453 467 469

I. Introduction In the survey of enzymatic peptide formation we first discuss the current state of research on synthetases. A brief compilation of major contributions is followed by a comment on enzyme systems, their possible classification and organization. Multifunctional protein structures are discussed in relation to the produced peptides, as well as origins of structural diversities of secondary metabolites. Finally the activation of peptide constituents and possible mechanisms of their sequential addition are discussed. In the second contribution Lipmann gives an account of the major contributions of his laboratory at Rockefeller University. We consider this summary an excellent introduction to the so-called thiotemplate mechanism, since here the stepwise elucidation of the reaction sequence is documented.

A Survey of Enzymatic Peptide Formation Horst Kleinkauf, Hans von Döhren Institut für Biochemie und Molekulare Biologie Technische Universität Berlin Franklinstraße 29, D-1000 Berlin 10, F.R. of Germany

1. Introduction Several biosynthetic routes to peptides not utilizing mRNA as a primary template have been evaluated during the past 25 years. A survey is given in table I. The field has not been reviewed comprehensively, and in this volume most aspects will be covered. The major problem originating from the nonlinear structure of enzymes serving as templates is the organization of the sequential addition of amino acids to peptides. As is indicated in figure 1 the information leading to a peptide sequence is by no means independent of nucleic acids. Once the protein template is constructed it contains the information and cannot be changed, at least in vivo. Thus Crick's central dogma (1) that „once information has passed into protein, it cannot get out again" ist not violated, despite several speculative comments (2, 3). The enzymology of peptide synthetases is fairly complex, so that most enzymes have been described as multienzyme complexes, multienzyme systems (4), or synthetases containing subenzymes (5). There are thus general difficulties introducing multienzymes into the enzyme classification scheme. The only enzyme numbered so far is the multienzyme 1 of gramicidin S synthetase (GS1) or phenylalanine racemase (6) (E.C. Epimerization of Phe however is only one function, while a two-step activation of Phe, and the transfer of DPhe to the multienzyme 2 (GS 2), are being neglected. Secondly the product, DPhe, is not released from the enzyme, but remains enzyme-bound. This problem could be solved by multiple notations of individual functions, or by introduction of multienzyme complexes and interacting multienzyme systems. However some rules for function priorities are needed. Such rules could be based on the structure of the products formed, but multiple notations cannot be avoided if several products originate from partially identical multienzymes.

aalRNA DNA—4mRNÄ}^

P e " ,ide

DNA—»- mRNA —•( enzymes -y-*- peptide 38 peptide acid aatRNA Fig. 1

Flow of information and origin of templates in ribosomal and enzymatic peptide formation.

Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • New York


H. Kleinkauf, H. v. Dôhren

Table I

Survey of major advances in enzyme and process description in peptide biosynthesis

I. Single step processes and enzymes1 glutathione carnosine 4'phosphopantothenyl-Cys peptidoglycan S. aureus 5-peptide interpeptide bridges without tRNA with tRNA tRNA Gly-structure transpeptidation aminoacyl transferases y-poly-Glu synthetase poly-(y-DGlu)capsule

Block et al. 1949-55 (31) Kalyankar & Meister 1959 (32) Bauer & Jungblut 1980 (33) Brown 1959 (34) Strominger et al. 1962-64 (35-37) Mizuno et al. 1973 (38) Strominger et al. 1970-72 (39-42) Strominger et al. 1968-70 (43-49) Kamiryo & Matsuhashi 1969 (50) Thorndike & Park 1969 (51) Roberts 1974 (52) Strominger et al. 1965-68 (53-55) Wise & Park 1965 (56) Soffer et al. 1966-71 (57, 58) Griffin & Brown 1964 (59) Shane 1980 (60, 61) Troy 1973 (62, 63)

II. Multistep processes and multienzymes2 gramicidin S

tyrocidine enterochelin bacitracin edeine linear gramicidin mycobacillin alamethicin enniatin polymyxin leupeptin

Kurahashi et al. 1967-71 (64, 65) Lipmann et al. 1968-71 (66-68) Laland et al. 1968-70 (69-71) Kleinkauf et al. 1976-80 (72-73) Kurahashi et al. 1968-71 (74-76) Lipmann et al. 1970-73 (77-79) Bryce & Brot 1972 (80) Ishihara & Shimura 1974 (81) Frriyshov et al. 1974-75 (82, 83) Kurylo-Borowska 1974 (84) Lipmann et al. 1972, 77 (85, 86) Kurahashi & Akashi 1977-80 (87, 88) Sengupta & Bose 1974 (89) Mohr & Kleinkauf 1978 (90) Zocher & Kleinkauf 1978 (91) Komura & Kurahashi 1979-80 (92-95) Suzukaka et al. 1979-80 (96-98)

1 In this section also enzymes have been listed performing repeated indentical additions. We must admit however, that there are some arguments to include these into Section II, and vice versa. 2 Generally no intermediates are accepted by these systems, contrary to Section I peptides. Leupeptin and mycobacillin may be exceptions, but multienzymes are involved in their formation.

A Survey of Enzymatic Peptide Formation



HOsA/COiH Paa I—»Ala, ATP P a a /3Ala


I HO-P /V-CC!zH i --frSf OH OH ,,



4'P- Pan I—Cys.ATP





4'P - PanCys




4' P - p a n t e t h e i n e

I CoA Fig. 2


Steps leading from pantoic acid to coenzyme A.

Another difficulty stems from the different organizations of enzymes and multienzymes, that may be disorganized in vitro generating dissociated or even artefactual complexes. This may be illustrated with the biosynthesis of CoA shown in figure 2. While individual enzymes have been isolated from rat liver and E. coli (7-10), a complex catalyzing all functions has been found in yeast (11) and applied for the enzymatic synthesis of the compound (12). Stabilization of such complex structures is at present a general difficulty, only solved if individual enzymes form stable aggregates or are covalently linked. Stabilizations have not been achieved in systems forming penicillins, cephalosporins, actinomycins, or valinomycin. On the other hand, compounds from similar microorganisms like enniatins, alamethicin, or leupeptin have been successfully produced in vitro from isolated enzymes. Procedures of enzyme isolation from active protoplasts, organelles, vacuoles or membranes are largely a matter of trial and error today. So enzyme systems catalyzing sequences of many reactions are so far not well characterized, considering protein structure and specific activities in vitro and in vivo (13, 14). In an attempt to classify antibiotics, B6rdy stated that sufficient information on biosynthesis of peptide antibiotics is not available for a systematic approach from this side (15). As we have pointed out, any classification scheme not based on the chemical structure of the compounds involved is only of limited value (16). We have proposed from analysis of a recent compilation of bioactive peptides (17) that peptide structures can be linearized pointing to a linear precursor generally involved, and that cyclic structures derived from linear peptides can be divided into only 4 common types (acarboxyl-a-amino links, ring closure withft,y, or ¿-carboxyl functions, lactones, and depsipeptides with two or more ester linkages). These features could provide the basis of a structural classification scheme and help to detect some similarities in the enzymology of peptide formation (Table II).


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(I) (II) (III)


Reaction 4 Since the stereochemical configuration of the product of the first reaction, I, catalyzed by the membrane-associated L-glutamic acid activating enzyme is L, it is clear that stereochemical inversion must occur subsequent to activation. The proposed Reactions 2 and 3 are less well understood since only indirect evidence supports their existence. Since a step in the polymerization reaction after activation is inhibited by sulfhydryl group blocking agents and that chemical treatments known to liberate thioesterified polypeptide chains release membrane-bound polyglutamic acid (reaction product III), it is reasonable to conclude that a thioester is functionally involved. What is less certain is the existence of the monoglutamyl thioesterified product, II. The fact that A M P is inhibitory for polymer formation supports the existence of intermediate II and also the reversibility of the second reaction. Alternatively, it is possible that the activated glutamyl adenylate is transferred directly to a growing oligo- or polyglutamyl chain (III) and that the stereochemical inversion occurs concomitantly with polymerization. The apparent high molecular weight of the labeled y-D-glutamyl polymers released by treatment of the membranes with hydroxylamine or at pH 10 further supports the existence of III above. Nothing is known concerning the nature of the proposed thioprotein in II or III. That only partial release of radioactivity from the membrane occurs with hydroxylamine is supportive of the nature of IV. Also compatible with this view is the observation that in vitro, polymer growth occurs by the addition of glutamyl residues to an endogenous acceptor which contains substantial amounts of D-glutamic acid. The proposal that a thioester may serve as an acceptor of activated glutamyl moieties and thus function to anchor growing polyglutamyl chains in the multienzyme complex is analogous to the known role which enzyme-bound 4'-phosphopanthetheine plays in peptide antibiotic synthesis (33, 35). It is envisioned that such an intermediate may function, as seen in Reaction 2 and 3 above, to facilitate the transfer of activated monomers to the growing polymeric chain. This would be in accord with the observation that these polymers are extended by the addition of new glutamyl residues to the amino terminus end of growing nascent chains. This mechanism of elongation is in contrast to polypeptide antibiotic synthesis where the activated oligopeptide is transferred to an activated monomer (35). Another difference between peptide antibiotic



and (y-D-glutamyl) capsular polymer synthesis is that the latter is dependent upon the structural integrity of the cell membrane cell wall complex (1). Lysozyme and detergent sensitivity of the polyglutamyl polymerization reaction(s) is interpreted to mean that a particular conformation of the membrane-bound enzymes and endogenous acceptors is required. Thus, definitive characterization of the linkages involved and the interactions between the components of the membrane-bound polyglutamyl synthetase complex awaits further study.

4. Future directions and experimental strategies Many questions regarding the mechanism of synthesis and assembly of the y-Dglutamyl capsular polymers remain to be answered. They include: 1) the nature of the regulation of the synthesis of the multienzyme complex; 2) the molecular architecture and transbilayer orientation of the enzymes and acceptors; 3) the mechanism whereby the capsular polymers are translocated across (through) the membrane; 4) the exact role of membrane lipids in the assembly process; 5) the precise mechanism by which chain elongation is initiated and the mechanism by which it is terminated; and 6) the chemical identity of the thioester postulated to serve as an acceptor of activated glutamyl moieties and thus function to anchor growing polyglutamyl chains in the multienzyme complex. Indeed it seems likely that a number of the reactions and therefore future problems involved in assembly of these polymers may be similar to those involved in synthesis of the peptide antibiotics. It also seems likely that application of recent advances in cell-free translation methods, recombinant DNA technology, immunochemistry and high resolution nuclear magnetic resonance and fluorescent spectroscopy techniques may play a key role in future studies directed at studying these questions. Since PGA and peptide antibiotics both appear late in the growth cycle, it should be possible to isolate cellular messenger RNA molecules enriched in information for synthesis of these components. The products of in vitro translation of this isolated RNA by one or two-dimensional gel analyses may reveal important information about the number and size of molecular components comprising a complex. The specificity of these analyses would be enhanced if an antibody was available to any of the components of the complex. Immunoprecipation of the translatable products would enhance information about the relationship of the translatable products to the enzyme complex. While isolation of mutants conditionally defective in synthesis of antibiotics, and perhaps PGA, will continue to provide meaningful information, cloning and amplification of the genomes responsible for these products by recombinant DNA technology should provide a major advance in the field. In principle, a DNA fragment containing information for PGA (or peptide antibiotic) can be covalently joined to a plasmid and the recombinant DNA molecule introduced into an appropriate host cell (e. g. E. coli) by viral infection or transformation. Cells containing the recombinant DNA would be selected for by their ability to synthesize a capsule (colony morphology) or produce the antibiotic. These cloning techniques can also be used to study the regulation of gene expression which may be a powerful method to probe the likely complexity of the genomes involved in the construction of these polyenzyme complexes.

Chemistry and Biosynthesis of the Poly(y-D-Glutamyl) Capsule in Bacillus licheniformis


The physical interactions of membrane components involved in the PGSC is amenable to study by 3 1 P-NMR and fluorescent spectroscopy techniques. 3 1 P-NMR can be used to study the motional characteristics of phospholipid headgroups and other phosphoryl components such as 4'-phosphopanthetheine. Information from the phosphorous resonances of 4'-phosphopanthetheine in the polyenzyme complexes in peptide antibiotic synthesis might also be productive to investigate - particularly under conditions where synthesis is initiated in reconstituted systems. While not limited to the phosphorous nuclei, information regarding the molecular motions, conformation and dynamics of these multienzyme complexes can also be obtained from 1 3 C and 2 Henriched enzyme preparations. Additional information regarding physical changes in the PGSC associated with polymer synthesis can be obtained by fluorescent spectroscopy employing diphenylhexatriene (DPH) and cw-parinaric acid (9,11,13,15-m, trans, trans, cisoctadecatetraenoic acid, c«-PnA) and iraws-parinaric acid (9,11, 13, 15 - all transoctadecatetraenoic acid, trans-PnA) as fluidity probes of the lipid environment. Cisand trans-PnA are both naturally occurring conjugated fatty acids and are probes of both membrane fluidity and lipid-protein interactions (36-38). 7rans-PnA partitions strongly in the solid phospholipid phase while the cw-derivative partitions with a preference for the fluid phases (39). A quantitative method to assess the distribution of these probes between coexisting phases has been published (39) and we shall utilize this method to determine the mole fraction of PnA in the solid phase and fluid phase of the PGSC as a function of temperature. Information about lateral phase separations and partitioning of the polyglutamyl synthetase complex between ordered and fluid domains will eventually be important in understanding the organization and assembly of the complex. By analogy, it would not be surprising if similar molecular motions would be important in the multienzyme complexes involved in peptide antibiotic synthesis - particularly if the 4'-phosphopanthetheine arm is shuttling activated amino acid moieties around as proposed. Finally, future studies directed at understanding the effect of lipids on the function and assembly of the PGSC may require knowledge about the interactions of specific annular or boundary lipids with the activating enzyme, racemase, polymerase or other components of the complex. To this end, extension of the technique of photolabeling of membrane proteins with arylazidophospholipids will be applied (40). This technique features the capability to reveal which domains of the hydrophobic sector of proteins interact with the lipid. Information about their approximate position within the membrane can also be obtained and this approach may also be revealing in how these polymers find their way across or through the bilayer - an intriguing problem which also remains to be convincingly documented for even the much smaller peptide antibiotics. Thus, the real significance of this symposium lies in its stimulus to the future discovery of such mechanisms and answers to the questions posed above.

References 1. Troy, F.A.: J. Biol. Chem. 248, 305 (1973). 2. Troy, F.A.: J. Biol. Chem. 248, 316 (1973).



3. Gardner, J., Troy, F.A.: J. Biol. Chem. 254, 6262 (1979). 4. Troy, F.A.: Ann. Rev. Microbiol. 33, 519 (1979). 5. Housewright, R.D.: The Bacteria, Vol. III (I.C. Gunsalus, R.Y. Stanier, eds.) p. 389, Academic Press, New York, 1962. 6. Thorne, C.B., Leonard, C.G.: J. Biol. Chem. 233, 1109 (1958). 7. Thorne, C.B., Gomez, C.G., Housewright, R.D.: J. Bacterid. 69, 357 (1955). 8. Thorne, C.B., Molnar, D.M.: J. Bacteriol. 70, 420 (1955). 9. Williams, W.J., Thorne, C.B.: J. Biol. Chem. 210, 203 (1954). 10. Williams, W.J., Thorne, C.B.: J. Biol. Chem. 211, 631 (1954). 11. Williams, W.J., Thorne, C.B.: Amino Acid Metabolism, Johns Hopkins Press, Baltimore, pg.107, 1955. 12. Williams, W . J , Litwin, J., Thorne, C.B.: J. Biol. Chem. 212, 427 (1955). 13. Leonard, C.B, Housewright, R.D.: Biochim. Biophys. Acta 73, 530 (1963). 14. Troy, F . A , Heath, E.C.: Fed. Proc. 27, 345 (1968). 15. Troy, F . A , Frerman, F . E , Heath, E.C.: J. Biol. Chem. 246, 118 (1971). 16. Troy, F . A , Frerman, F . E , Heath, E.C.: Methods in Enzymology, Vol XXVIII, V. Ginsburg, e d . Academic Press, New York, pp.602, 1972. 17. Inouye, M. (Ed.): Bacterial Outer Membranes: Biogenesis and Function, John Wiley & Sons, New York, 1972. 18. Sutherland, I. W. (Ed.) : Surface Carbohydrates of the Prokaryotic Cell, Academic Press, New York, 1977. 19. Osborn, M.J.: Structure and Function of Biological Membranes, L.I. Rothfield, ed. Academic Press, New York, pp.343, 1971. 20. Smyth, D . G , Blumenfeld, O.O., Königsberg, W.: Biochem. J. 91, 589 (1964). 21. Roskoski, R , Jr., Gevers, W , Kleinkauf, H , Lipmann, F.: Biochemistry 9, 4839 (1970). 22. Roskoski, R , Jr., Kleinkauf, H , Gevers, W , Lipmann, F. : Biochemistry 9, 4846 (1970). 23. Manning, J . M , Moore, S.: J. Biol. Chem. 243, 5591 (1968). 24. Halpern, B , Westley, J.W.: Biochem. Biophys. Res. Commun. 19, 361 (1965). 25. Halpern, B , Westley, J.W.: Tetrahedron Lett. 21, 2283 (1966). 26. Pardee, A.: J. Biol. Chem. 190, 757 (1951). 27. Waley, S.G.: J. Chem. Soc, 517 (1955). 28. Schachman, H.K.: Methods Enzymol. 4, 32 (1957). 29. Schumaker, V . N , Schachman, H.K.: Biochim. Biophys. Acta 23, 628 (1957). 30. Chervenka, C.H. : A Manuel of Methods for the Analytical Ultracentrifuge, Spinco Division of Beckman Instruments, Inc., Palo Alto, Calif, p. 56, 1969. 31. Koväcs, J , Bruckner, V.: J. Chem. Soc, 4255 (1952). 32. Kent, L . H , Record, B . R , Wallis, R.G.: Phil. Trans. Roy. Soc. London 250, 389 (1957). 33. Kurahashi, K.: Ann. Rev. Biochem. 43, 445 (1974). 34. Lehninger, A. L. : Biochemistry, 2nd Edition, Worth Publishers, Inc., New York, pp.933, 1975. 35. Kleinkauf, H.: Chemie in unserer Zeit 14, 105 (1980). 36. Sklar, L . A , Hudson, B.S, Simoni, R.D. : Proc. Natl. Acad. Sei. 72, 1649 (1975). 37. Sklar, L . A , Hudson, B.S, Simoni, R.D.: Biochemistry 16, 813 (1977). 38. Sklar, L . A , Hudson, B.S, Simoni, R.D.: Biochemistry 16, 5100 (1977).

Chemistry and Biosynthesis of the Poly(y-D-Glutamyl) Capsule in Bacillus



39. Sklar, L.A., Miljanich, G.P., Dratz, E.A.: Biochemistry 18, 1707 (1979). 40. Bisson, R., Montecucco, C., Gutneniger, H., Azzi, A.: J. Biol. Chem. 254, 9962 (1979).

Recent Developments with Cell free Extracts on the Enzymic Biosynthesis of Penicillins and Cephalosporins Derek J. Hook, Richard P. Elander and Robert B. Morin* Bristol-Myers Company, Industrial Division and Bristol Laboratories* Syracuse, New York 13201, U.S.A.

1. Introduction Recent studies on the biosynthesis of penicillin and cephalosporins have focused on the use of cell-free extracts to further our understanding of the involved oxidative cyclizations of peptide intermediates. This has solved the major previously encountered problem, namely the impermeability of the intact mycelia of the penicillin and cephalosporin-producing organisms to precursors more complex than simple amino acids. Initial progress in this direction had been made in the preparation of protoplasts from Penicillium chrysogenum and Cephalosporium acremonium and the study of their biochemical properties (18). These protoplasts were capable of respiration, maintaining intracellular amino acid pools and synthesizing antibiotic. Later, Fawcett et al (20) and Bost and Demain (11) lysed protoplasts to prepare cellfree extracts capable of carrying out various activities related to the /^-lactam antibiotics. This technique of preparing cell-free extracts by lysis of protoplasts of P. chrysogenum and C. acremonium has led to considerable progress in understanding pencillin and cephalosporin biosynthesis and holds promise for future detailed studies. The use of the same technique has led also to a better understanding of the related pathways in Strep tomyces. Following the discovery of a tripeptide ( tripeptide in these experiments. Additionally, Meesschaert et al (27) indicated that chromatographic behavior of the unknown from cell-free incubation and a synthetic sample were the same. Experiments to establish whether or not thiazepine and thiazepine sulfoxides are intermediates indicate that no formation of bioically active material occurred from the L-aamino-adipyl thiazepine (4) under conditions in which ACV tripeptide was converted to biologically active material (42). R-NH


R = i - ( L - a - aminoadipyl)






R = (L) - CO-(CH2)3-CH(NH2)-COOH (3)

R= t -(L-a-aminoadipyl) (4)


D.J.Hook, R.P.Elander, R.B.Morin

3. Ring expansion of penicillins to cephalosporins Kohsaka and Demain (46) reported the stimulation of cephalosporin biosynthesis in cell-free extracts of C. acremonium by the addition of penicillin N to the extracts. They also indicated that the formation of cephalosporin was unaffected by inhibitors of protein synthesis. This report suggesting the intermediacy of penicillin N in cephalosporin C synthesis stimulated many laboratories to try to repeat these results but without much success. At the 1978 Madison meeting, Demain and co-workers presented work that showed (43) that penicillin N was converted to desacetoxycephalosporin C (5e) by cell-free extracts of C. acremonium CW-19 and several mutant strains. Several of these mutants were shown to be deficient in their ability to convert penicillin N to a cephalosporin in cell-free extracts. The partially purified cephalosporin produced by these cell-free extracts was identified as desacetoxycephalosporin C by TLC in three solvent systems; paper chromatography, and paper electrophoresis, all followed by bioautography. On the basis of this data, Demain and co-workers concluded that a linear pathway of formation of cephalosporins from penicillins was more likely than a branched pathway. Penicillin N, a potential key intermediate, has been difficult to obtain in good purity because of its general instability, but recently, Baldwin et al (9) has described a synthesis of penicillin N by a simple method which can be utilized to introduce a label into both the a-aminoadipyl side chain and the penicillin nucleus. This has facilitated studies of the ring expansion reaction as described above. The identity of the product of the ring expansion reaction was established by doublelabelled isotope feeding experiments by Baldwin's group (10). These experiments used extracts of C. acremonium CW-19 mutant M0198. 3 H and 14 C were incorporated from singly and doubly labelled penicillin N into desacetoxycephalosporin C. In these studies, use of a HPLC separation system indicated the product of the reaction to be desacetoxycephalosporin C. It was further shown that the desacetoxycephalosporin C isolated from the cell-free reaction by isotope dilution and by formation of derivatives had the same 3 H/ 1 4 C ratio as the starting penicillin N. Meanwhile, a group at Bristol was using Demain's cell-free system and encouraging preliminary results were obtained indicating ring expansion activity. Stimulated by the work of Turner et al (39) on the oxygenation of desacetoxycephalosporin C to desactylcephalosporin C (5 b), a biosynthetic transformation requiring ascorbate, 2-oxoglutarate and ferrous cations as cofactors, the possibility that cofactors were involved in the conversion of penicillin N to desacetoxycephalosporin C was investigated. These same cofactors were tried in the cell-free system that Demain had developed. Ascorbate and ferrous ions did indeed stimulate the conversion of penicillin N to a cephalosporin (24) and could be regarded as cofactors for the reaction. Following the report of the Bristol group at the Madison meeting, other workers were able to confirm Demain's findings on the conversion of penicillin N to desacetoxycephalosporin C. Discussion at the meeting indicated also that a number of factors were important in obtaining active cell-free extracts and these were later summarized by Sawada et al (34). The several factors involved: (i) lack of the cofactors; (ii) chemical liability and impurity of penicillin N; (iii) instability of the supersensitive assay organisms used to test for the reaction products, and (iv) lack of predictability of the time of the peak of

Enzymic Biosynthesis of Penicillins and Cephalosporins



R.NH^j-j^S R3 CO2H



(a) D -^-aminoadipyl



(b) D - « - a m i n o a d i p y l



( c ) D - « -aminoadipyl



(d> D - « - a m i n o a d i p y l



( e ) D -•< -aminoadipyl




enzyme activity of the producing organism. Recently, the development of techniques for the chemical synthesis of specifically radio-labelled and unlabelled, unstable potential intermediates that might be involved in the biosynthetic pathway has facilitated the study of these biosynthetic systems. Sawada et al (34) confirmed that ascorbate and FeS0 4 stimulated the conversion of penicillin N to desacetoxycephalosporin C and the optimum concentrations found were 0.04 mM FeS0 4 and 0.67 mM ascorbic acid. If ATP was used (0.83 mM) an energy generating system was found not to be necessary. 2-oxo-glutarate showed no effect on the reaction. The optimization of the cofactor requirements, control of microbial growth and time of harvest, led to a system for preparation of cell-free extracts with ring expansion activity that could be reproducibly obtained. Further studies by Saw da (35) indicated that Triton X-100 treatment or sonication of the protoplast lysate enhanced enzyme activity. They also found that active extracts could be obtained by merely sonicating intact mycelia. Studiesaby Baldwin et al (10) also showed that the product of the ring expansion reaction in the presence of ascorbate and ferrous ions was also desacetoxycephalosporin C. In these experiments the efficiency of conversion of penicillin N to desacetoxy-cephalosporin C ranged from 9 to 16%.

4. Functionalization of the 3-methyl group of cephalosporins Recent work on the functionalization of the 3-methyl group of desacetoxycephalosporin C was initiated by Turner et al (39) who showed that conversion of the 3-methyl group to the 3-hydroxymethyl group was catalyzed by cell-free extracts of both C. acremonium and Streptomyces clavuligerus. Stevens et al (47) suggested that the enzyme responsible was an oxygenase on the basis of incorporation of oxygen directly from molecular oxygen by the use of 1 8 0 2 . Turner et al (39) showed that partially purified enzymes for C. acremonium and S. clavuligerus required 2-oxo-glutarate, Fe 2+ -cations and a number of reducing agents (DTT and ascorbic acid) for activity,


D.J.Hook, R.P.Elander, R.B.Morin

thus exhibiting the properties of 2-oxoglutarate-linked dioxygenases. Both enzymes were highly specific for cephalosporins with the natural D-a-aminoadipyl side chain, since a number of 1-fi side chain derivatives of the 3-methylcephems were tested, and only with those having the D-a-aminoadipyl side chain was the 3-methyl group oxidized at a detectable rate. Interestingly, these studies showed a difference between the Cephalosporium and Streptomyces enzymes, since the enzyme from C. acremonium, but not that from S. clavuligerus, was activated 10 fold when it was preincubated with a reaction mixture lacking 2-oxoglutarate and desacetoxycephalosporin C, and Fe 2 + cations seemed to play a role in the activation. Fujisawa et al (22) showed that the acetylation of desacetylcephalorin C leads to cephalosphorin C (5a) in C. acremonium. Preliminary experiments had shown that cell-free extracts of Streptomyces clavuligerus synthesis cephamycins from carbamoylphosphate (13). A more detailed investigation (12) has indicated that the enzyme responsible is an ATP-dependent carbamoyl phosphate o-carbamoyltransferase. The enzyme responsible was purified and found to be fully active only in the presence of Mn 2 + and Mg 2 + cations. It was stimulated by ATP, although the role of ATP is not known at present. In contrast to the dioxygenase responsible for the formation desacetylcephalosporin C from desacetoxycephalosporin C the O-carbamoyltransferase is tolerant of a wide range of 7-/? side chains on the cephem nucleus.

5. Introduction of the 7-methoxy group into cephalosporins Studies have shown that methionine provides the methyl group of the 7-a-methoxy cephalosporins (41) and the oxygen in the 7-a-methoxy group is derived from molecular oxygen (29). Recently, O-Sullivan et al (31) have demonstrated that cell-free extracts of S. clavuligerus convert cephalosporin C and O-carbamoyl desacetylcephalosporin C into 7-amethoxy derivatives and that desacetyl-cephalosporin C was not a substrate and desacetoxycephalosporin C was only a poor substrate. Thus, it appears that the final stage in synthesis of the cephamycins (5 c, d) is the introduction of the 7-a-methoxy group.

6. Biosynthesis of the semi-synthetic penicillins The structures of the two most common solvent extractable penicillins, penicillin G and penicillin V are shown in 2 a and 2 b. Most of the early work on the formation of the semi-synthetic penicillins focused on the properties of the transacylase enzyme from P. chrysogenum (32, 33). Studies on purified preparations of the enzyme (23, 37, 38, 40) indicate that four activities are observed for this enzyme, the ratios which remain constant throughout the various prufication stages. This suggests that the multiple activities (penicillin acyltransferase, 6-APA acyl transferase, penicillin acylase and phenylacetyl hydrolase) were performed by either the same enzyme or multi-enzyme complex. Loder (26) showed that cell-free extracts of P. chrysogenum were capable of converting (l- 14 C)-phenylacetyl CoA to solvent extractable penicillin in the presence of 6-APA

Enzymic Biosynthesis of Penicillins and Cephalosporins


(2c) or isopenicillin N but not penicillin N. These results were confirmed later by Fawcett et al (19) who reported the incorporation of 3 H into solvent extractable penicillin from isopenicillin N and 6-APA labelled in the 2-/3 methyl group, either with crude extracts or with partially purified extracts in the presence of phenylacetyl CoA. Additionally, no incorporation was detected on incubation of these extracts with penicillin N. Meesschaert et al (27) also reported that their cell-free preparations were capable of exchange reactions between 6-APA and isopenicillin N thus supporting earlier reports by Abraham (2) of an enzyme capable of converting isopenicillin N into 6-APA and aaminoadipic acid. Further studies are needed to clarify the exact role of isopenicillin N and 6-APA in the biosynthesis of penicillins and also to determine whether the enzyme responsible for the biosynthesis of solvent-extractable penicillins has multiple activities. 13 C N M R has proved useful by the identification of the cyclic lactam of a-aminoadipic acid (2-oxo-piperdine-6-carboxylic acid) in production fermentation of penicillin G and V, as well as unprecursed fermentations (14). The finding that this lactam is present to the extent of 30 mole % of the amount of penicillin V as well as the finding that the lactam as isolated being essentially recemic has potential implications concerning our understanding of the terminal transacylase step of penicillins. Friederich and Demain (21) have indicated that a-aminoadipic acid can be recycled up to 10 times per mole of penicillin produced in resting cells of P. chrysogenum and reports by Abraham (2), and Meesschaert et al (27) have indicated an exchange between aaminoadipic acid and isopenicillin N in cell-free systems. The findings of the aaminoadipic acid lactam and these other reports indicate that further studies are needed to more fully understand the metabolism of a-aminoadipic acid in penicillin biosynthesis.

7. Future areas of investigation In the next few years there will be considerable progress in the purification of the individual enzymes involved in penicillin and cephalosporin biosynthesis and an understanding of their specificity and cofactor requirements. The introduction of high field high resolution 1 3 C N M R instrumentation, should see the application of in vivo 13 C N M R techniques to the study of penicillin and cephalosporin biosynthesis. Still unanswered questions relate to the tetrapeptide complex found in Penicillium, Cephalosporium, and Paecilomyces (7,17, 25) and their relation to the ACV tripeptide and penicillin biosynthesis. One hypothesis is that these peptides are shunt metabolites and not related to the mainstream of /Mactam biosynthesis. Recently, Neuss et al (28) reported on the discovery of a number of new peptides (aminoadipyl-seryl-valine, aminoadipyl-alanyl-valine, aminoadipyl-alanyl-isodehydrovaline) related to the ACV tripeptide in P. chrysogenum fermentation broths also ask questions relating to the underlying biochemical mechanisms of the oxidation cyclizations involved in penicillin and cephalosporin biosynthesis. Furthermore, future studies must focus on the regulation of penicillin biosynthesis and the production of penicillin and cephalosporin in relation to the growth of the producing organisms.


D.J. Hook, R.P. Elander, R.B. Morin

Recent findings and theories regarding penicillin and cephalosporin biosynthesis also have implications relating to speculations on the mechanisms of biosynthesis of other /¡-lactams of the nocardicin group where it should prove fruitful to search for peptides of the type phenylglycyl-seryl-phenylglycl or phenyglycyl-alanyl-phenylglycyl or similar peptides. In strains of Streptomyces producing the thienamycin, clavulanic and olivanic acid series of compounds, peptides might also be found related to the structure of these compounds through transformations of the type suggested by Baldwin's free radical hypothesis of cyclization. The next several years should result in other new interesting facets in addition to those involved in the biosynthesis of penicillins, cephalosporins and other /Mactam antibiotics.

References 1. Aberhart, D.J., Tetrahedron 33, 1545-59 (1977). 2. Abraham, E.P., J. Antibiotics Suppl. 30, S l - 2 6 (1977). 3. Adrieris, P., Meesschaert, B., Eysser, H. and Vanderhaeghe, H., FEMS Microbiol. Lett. 4, 15-18 (1978 a). 4. Adriens, P., Vandehaeghe, H., FEMS Microbiol. Lett. 4, 19-21 (1978 b). 5. Arnstein, H.R.V., Crawhall, J.C., Biochem. J. 67, 180-7 (1957). 6. Arnstein, H.R.V., Clubb, M.E., Biochem. J. 68, 528-35 (1958). 7. Arnstein, H.R.V., Artman, M., Morris, D., Toms, E.J., Biochem. J. 76, 353-7 (1960). 8. Baldwin, J.E., Wan, T.S., J. Chem. Soc. Chem. Comm. 249-50 (1978). 9. Baldwin, J.E., Herchen, S.R., Singh, P.D., Biochem. J. 186, 881-7 (1980a). 10. Baldwin, J. E., Singh, P. D., Yoshida, M., Sawada, Y., Demain, A. L., Biochem. J. 186, 889-95 (1980b). 11. Bost, P.E., Demain, A.L., Biochem. J. 162, 681-7 (1977). 12. Brewer, S.J., Taylor, P.M., Turner, M.K., Biochem. J. 185, 555-64 (1980). 13. Brewer, S.A., Boyle, T.T., Turner, M.K., Biochem. Soc. Trans, 5,1026-9 (1977). 14. Brundidge, S.P., Gaeta, F.C.A., Hook, D.J., Sapino, C.A., Elander, R.P., Morin, R.B., J. Antibiotics 33, 1348 (1980). 15. Chan, J. A., Maung, F.C., and Sih, C.J., Biochem. J. 15,177-80 (1978). 16. Demain, A. L., Biosynthesis of Antibiotics, Vol. 1., L.J. Snell ed., Academic Press, Inc. N. Y., p. 29-94 (1966). 17. Enriquez, L.A., Pisano, M.A., Antimicrobial Agents Chemother. 16, 392-7 (1979). 18. Fawcett, P. A., Loder, P.B., Duncan, M.J., Beesley, T.J., Abraham, E.P., J. Gen. Microbiol. 79, 293-309 (1973). 19. Fawcett, P. A., Usher, J. J., Abraham, E.P., Biochem. J. 151, 741-6 (1975). 20. Fawcett, P.A., Usher, J.J., Huddleston, J.A., Bleaney, R.C., Nisbet, J.J., Abraham, E.P., Biochem. J. 157, 651-60 (1976). 21. Friedrich, C.C., Demain, A.L., Arch. Microbiol. 119, 43-7 (1978). 22. Fujisawa, Y., Shirafuji, H., Kida, M., Nwa, K., Yoneda, M., Kanzaki, T., Nature (New Biol.) 246, 154-5 (1973). 23. Gatenbeck, S., Brunsberg, U., Acta. Chem. Scand. 22, 1059-61 (1968).

Enzymic Biosynthesis of Penicillins and Cephalosporins


24. Hook, D.J., Chang, L.T., Elander, R.P., Morin, R.B., Biochem. Biophys. Res. Commun. 87, 258-265 (1979). 25. Loder, P. B., Abraham, E.P., Biochem. J. 123, 177-81 (1971). 26. Loder, P.B., Post. High. I. Med. Dosw. 26, 493-500 (1972). 27. Meesschaert, B., Adriens, P., Eysser, H., J. Antibiotics 33, 722-30 (1980). 28. Neuss, N., Miller, R.D., Affolder, C.A., Nakatsukasa, W., Mabe, J., Huckstep, L. L., DeLa Higuera, N., Hunt, A. H., Occolowitz, J. C., Gillam, J. H., Helv. Chim. Acta 63, 1119-29 (1980). 29. O'Sullivan, J., Aplin, R.T., Stevens, C.M., Abraham, E.P., Biochem. J. 179, 4752 (1979a). 30. O'Sullivan, J., Bleaney, R.C., Huddleston, J. A., Abraham, E.P., Biochem. 184, 421-6 (1979 b). 31. O'Sullivan, J., Abraham, E.P., Biochem. J. 186, 613-6 (1980). 32. Paterson, W.H., Wideburg, N.E., Proc. 4th Intern. Congr. Biochem. Vienna 15, 136 (1960). 33. Pruess, D.L., Johnson, M.J., J. Bacteriol. 94, 1500-8 (1967). 34. Sawada, Y., Hunt, N.A., Demain, A.L., J. Antibiotics 32, 1303-10 (1979). 35. Sawada, Y., Solomon, N.A., Demain, A.L., Biotechnol. Lett. 2, 43-8 (1980). 36. Sjoberg, B., Thelin, H., Nathorst-Westfelt, L., Van Tamelen, E. E., Wagner, E. R., Tetrahedron Lett. 281-6 (1965). 37. Spencer, B., Biochem. Biophys. Res. Comm. 31, 170-5 (1968). 38. Spender, B., Maung, C., Biochem. J. 118, 29P-30P (1970). 39. Turner, M.K., Farthing, J.E., Brewer, S.J., Biochem. J. 173, 839-50 (1978). 40. Vanderhaeghe, H., Classer, M., Vlietinck, A., Parmentier, G., Appl. Microbiol. 16, 1557 (1968). 41. Whitney, J.G., Brannon, D.R., Mabe, J. A., Wickes, K.J., Antimicrob. Agents, Chemother. 1, 247-54 (1972). 42. Wolfe, S., Hook, D.J., Morin, R.B., Gaeta, F.C.A., Bowers, R„ Jokinen, M.G., 1980, unpublished results. 43. Yoshida, M., Konomi, T., Kohsaka, M., Baldwin, J.E., Herchen, S., Singh, P., Hunt, N.A., Demain, A.L., Proc. National Acad. Sci., U.S. 75, 6253-7 (1978). 44. Young, D.W., Morecombe, D.J., Sen, P.K., Eur. J. Biochem. 75, 133-47 (1977). 45. Konomi, T., Herchen, S., Baldwin, J. E., Yoshida, M., Hunt, N. A., Demain, A.L., Biochem. J. 184, 427 (1979). 46. Kohsaka, M., Demain, A.L.: Biochem. Biophys. Res. Comm. 70, 465 (1976). 47. Stevens, C. M., Abraham, E. P., Haung, F. C., Sih, C. J.: Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 625 (1975).

Accumulation of 0,15 h~ 1 and < 0,45 '). It remains possible that oxygen only inactivates some of the 18 to 19 catalytic functions of the GS synthetase complex, still displaying the amino acid activation reactions but not anymore initiation, elongation and/or cyclization. Another hypothesis would be that under high aeration conditions, GS constituent amino acids are not formed or are preferentially used for cell formation. Whether the slower growth during the active growth phase, obtained under low aer-


E.J. Vandamme et al.

ation conditions, also favours antibiotic synthesis is not clear. It has indeed been shown in chemostat experiments that GS synthetase formation is a function of growth rate and that a high growth rate (n > 0,50 h~ is incompatible with GS synthetase formation (6, 28). Chemostat experiments run at fixed low dilution rates (n < 0,50 h~ r ), under varying aeration rates and D.O.T. levels should now be undertaken to help to answer these questions. It is quite possible that maintaining a low D.O.T. establishes a gradual decrease in growth rate and forces the cells into "transient growth" kinetics, thus allowing for extra GS synthetase and GS formation (20). Seddon and Fynn (32) have found that a decrease in growth rate coincided with an increase in tyrothricin production by B. brevis ATCC10068, as a result of a lack in the supply of oxygen necessary for oxidative metabolism. GS formation and growth rate of B. brevis ATCC 9999 are thus both clearly determined by the D.O.T. level, while growth extent is less dependent. 2.3. Effect of pH on GS fermentation dynamics 2.3.1. Complex YP-medium studies The early cessation of growth in complex YP-medium fermentations (Figure 1, 2, 4 and 5) is probably due to high pH (> 8,5), rather than to low aeration rate or nutrient exhaustion. A 5 liter-fermentation was conducted at constant pH 7,3 (by automatic controlled addition of 2 N HC1) with all other parameters similar to the oxygen limited fermentation (Figure 2). This pH value of 7,3 was arbitrarily chosen as a mean value close to the optimum pH for growth (pH = 7,0) and the pH value of 7,6, normally used for in vitro activity measurements of GS synthetases 2 and 1 (45).

Fig. 6 Gramicidin S fermentation pattern in a 7,5 1 fermentor in complex YP-medium; aeration rate 5 1 of air/min; 300 rpm; pH controlled at 7,3 (Symbols as in Figure 1; maximal O.D. = 18,3 g DCW/1).

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


No literature data are available on the effect of environmental pH on GS fermentations. Usually, media have an initial pH value of 7,0 to 7,4 and the pH is allowed to change freely during the course of the fermentation. Controlling environmental pH at 7,3 during fermentations by B. brevis ATCC 9999 increased specific GS production in complex YP-medium from 0,170 mg/mg DCW to 0,220 mg/mg DCW (4100 mg GS/1) (compare Figure 6 with Figure 2). Growth extent was also considerably increased (18,3 g DCW/1) due to a more favourable pH throughout the entire growth cycle, though growth was slow. During constant pH fermentations in YP-medium, GS production occurs early and throughout the entire growth cycle and not solely at the end of it, as is normally found in fermentations without pH control. Other researchers have also found that, depending on cultural conditions which affect pH patterns, secondary metabolites can be produced during active growth (14-16). The frequently observed syntheses of oligopeptide antibiotics after active growth might thus be a pH controlled phenomenon. 2.3.2. FlKt-medium


A large drop in pH was apparently the cause of the relatively higher specific antibiotic levels in the previous 5-liter-F 3/6 fermentation (Figure 3) despite a much lowered aeration rate. Keeping the pH constant at 7,3 under lowered aeration conditions, resulted in a doubling of biomass (5,5 g DCW/1) and in a 4-fold increase in GS production (1200 mg GS/1 or 0,220 mg/mg DCW) (Figure 7). A distinct diauxic lag was observed. The antibiotic was produced throughout the entire second growth phase, a



Fig. 7 Fermentation pattern of gramicidin S in a 7,51 fermentor in chemically defined F3/6-medium: aeration rate 21 of air/min; 250 rpm; pH controlled at 7,3 (Symbols as in Figure 1 ; maximal O.D. = 5,5 g DCW/1).


E. J. Vandamme et al.

behaviour similar to that observed in controlled pH GS fermentations in complex medium. Haavik (14,15) demonstrated the beneficial effect of controlled pH on bacitracin production by B. licheniformis ATCC 10716 in chemically-defined medium. Neutralization of the growth medium with CaC0 3 or increasing the phosphate level prevented rapid pH changes and led to an increase in bacitracin titer; controlling pH at 8,0 resulted in a 50% increase in antibiotic production. Metabolic changes in the medium during the fermentation cycle as well as catabolic products (i. e. acetic acid and pyruvic acid) greatly affect the internal cell-pH, which can determine the level of antibiotic produced. It is quite possible that the accumulation of such organic acids, during oxygen-limited growth of B. brevis ATCC 9999 in F 3/6 medium, lowers external pH and subsequently internal pH, which causes inhibition of GS synthesis. Keeping the internal pH constant, by imposing a controlled pH to the cell environment, would save ATP and could allow for a continuous and prolonged GS synthesis. It is clear from our work that both aeration rate and pH effect the level of D.O.T. during growth of B. brevis ATCC 9999 in complex and defined medium and that a low D.O.T. correlates directly with a decrease in growth rate and an increase in GS formation. Our data further suggest that careful control of D. O. T., allowing a high degree of oxygen transfer during very early growth and a low degree later, combined with pH control, would lead to an improved GS fermentation process. 2.4. Nutrient utilization patterns during GS fermentations 2.4.1. Complex medium studies The complex medium that was found best for GS production by B. brevis ATCC 9999 was YP-medium, containing 5% yeast extract and 5% peptone. GS fermentation dynamics in this medium have been described earlier (39-41). Lowering the high (5%) concentration of yeast extract and peptone in YP-medium (especially below 3%) resulted in a gradually decreased growth and antibiotic formation, followed by extensive lysis of he culture. Udalova et al. (36, 37) had also observed that a large surplus of carbon and nitrogen sources is favourable for growth and antibiotic production by B. brevis var. G. B. It was earlier found that the only carbon sources which increased the growth extent were D-fructose, glycerol and meso-inositol (39-41). Asatani and Kurahashi (9) demonstrated that this defect in carbohydrate utilisation could be attributed to lack of the transport system(s) for various carbohydrates in B. brevis ATCC 9999. Table I

Effect of glycerol, D-fructose and meso-inositol in YP-medium on growth and GS production.

Carbon source added -(=YP) + 2,5% glycerol + 2,5% D-fructose + 2,5% meso-inositol

DCW (g/1)



12,7 21 18 20

1140 840 550 1200

(mg/mg DCW) 0,090 0,041 0,030 0,090

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


Fig. 8 Utilization pattern of glycerol during gramicidin S fermentation in a 281 fermentar in 3% YPmedium (aeration rate 81 of air/min; 500 rpm; Symbols as in Figure 1).

Of these three carbon sources, only meso-inositol increased growth in YP-medium without inhibition of GS formation (Table I). It is tempting to suggest that the inhibitory effects of glycerol and fructose on GS production are due to catabolite repression of the GS-synthetases. Since the yeast extract component of YP-medium contains both glycerol and meso-inositol, their utilization pattern was followed in function of the fermentation cycle. Both polyols were quantitated by gaschromatography. Fructose is not an inherent component of YP-medium. Meso-inositol utilization displayed no special features. However, GS formation is indeed only initiated upon depletion of glycerol (Figure 8). These data suggest indeed that glycerol, an inherent constituent of the complex YP-medium, might effect catabolite repression of GS synthetase formation, and could explain, why addition of glycerol - but not of »jeso-inositol - , to the complex YP-medium specifically results in a lowering of GS formation as shown in Table I. Because amino acids are nutritionally important for growth of B. brevis (18, 31), we followed amino acid utilization patterns in a fermentation cycle in YP-medium (3%) run at a high aeration level (Figure 5). Amino acids in the spent medium were quantitated with a Technicon Sequential Multi-Sample Amino Acid Analyser. In Table II, changes in amino acid levels are given for early-log, mid-log and late growth phases. These utilization patterns are quite complex, but nevertheless certain amino acids behave in an identical manner. Glutamine or/and glutamic acid, glycine and aspartic acid or/and asparagine are preferentially used in the early log-phase, while histidine and tyrosine (albeit in limited amounts) are metabolised throughout the total active


E.J. Vandamme et al.

Table II Changes in amino acid levels in the supernatant during YP-fermentation (utilization = + ; excretion = —).

Growth-phase (see Fig. 5)



late growth

Time period (min. after inoculation; see Fig. 5)







Glu or/and Gin Gly Asp or/and Asn

+ 1,72 + 1,09 +0,64

+ 0,88 + 0,28 — 0,13

+0,37 -0,28 -0,07

His Tyr

+0,13 +0,2

+0,13 +0,23

0,01 +0,1

Phe Val Ser Leu

+0,17 +0,22 +0,07 + 0,02

+0,22 +0,33 + 0,27 +0,3

+0,58 + 0,57 + 0,43 + 1,3

Ala Met

+0,17 +0,01

+0,94 + 0,2

+0,57 + 0,02

Pro lie Arg

+0,37 +0,19 +0,37

+0,16 +0,16 +0,05

+0,29 +0,56 + 1,61

Lys Thr

-0,25 -0,14

+0,4 +0,5

+0,23 +0,44

DCW (g/1) Amino a c m ^ ^ ^ A conc. (g/1) —

growth cycle. Phenylalanine, valine, serine and leucine are metabolised throughout the whole growth cycle, but their utilization is more pronounced towards the end of growth. Alanine and methionine - on the contrary - are rapidly utilized in the mid-log phase. Remarkably, the uptake of proline, iso-leucine and arginine is highest in the early log-phase and towards the end of growth. Production of certain amino acids (free or as oligopeptides) was also observed: lysine and threonine appear in the supernatant in the early log-phase; aspartic acid or asparagine appears in the middle-log, while glycine is produced at the end of growth. More detailed investigations concerning the utilization of specific amino acids have been performed in the chemically defined F3/6-medium (Section 2.4.2.). 2.4.2. F3/6-medium studies In the chemically defined F 3/6-medium, utilization patterns of D-fructose, of the five growth stimulatory amino acids and of the precursor L-phenylalanine were followed

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


Fig. 9 Utilization patterns of amino acids and fructose and excretion of amino acids during gramicidin S formation in F3/6-medium (aeration 21 of air/min; 250 rpm; p H controlled at 7,3; maximal O . D . = 5,5 g DCW/1).

(Figure 9) at controlled pH 7,3 and a lowered aeration rate (21 of air/min, 250 rpm). DFructose was measured colorimetrically according to the modified cysteine-carbazol method (30). Free amino acids in the spent medium were quantitated with a Technicon Sequential Multi-Sample-Amino Acid Analyser. L-Glutamine was separated and quantitated by thin layer chromatography on silicagel G with as solvent npropanol/34% NH 3 -solution: 70-30. These data might help to elucidate the nutritional, physiological and regulatory functions of these compounds during biomass and/or GS production phases. The results demonstrated that initial growth occurs at the expense of L-arginine and L-glutamine as carbon- and nitrogen sources. L-Proline was also slightly metabolised during this early growth phase. These data correspond with amino acid utilization patterns observed in complex YP-medium. Rapid GS synthesis started at the time L-glutamine and L-arginine were completely metabolized. Subsequently, D-fructose and L-histidine were utilised, respectively as a carbon and nitrogen source. During this period, L-proline was not metabolised. Upon depletion of D-fructose and L-histidine, growth abruptly stopped, indicating that these nutrients are limiting for biomass production. At this stage, L-proline was further metabolised and probably used for maintenance or sporulation processes. Methionine as a sulfur source was gradually broken down throughout the whole fermentation cycle. L-Phenylalanine was metabolised from the moment GS production started, and its disappearance was directly proportional to the amount of antibiotic formed, supporting its precursor-function (5). During growth in F 3/6-medium, B. brevis ATCC 9999 synthesises and excretes some amino acids: L-lysine (max: l,110mg/l), L-alanine (max: 312mg/l), L-valine (max: 278 mg/1) and L-serine (max: 133 mg/1). Especially L-lysine - as also observed in YP-


E.J. Vandamme et al.

medium fermentation - is synthesized early in that part of the growth cycle where Larginine and L-glutamine serve as C- and N-source. Alanine, serine and valine though not excreted in YP-medium fermentation - are here produced during the second growth phase. Cultural conditions favouring synthesis of these amino acids are not yet defined. In fermentations yielding high biomass levels, these amino acids were partially or completely metabolised during the late growth or differentiation stages. Generally, few data are available on amino acid synthesis and excretion in Bacillus species (3, 44) and - as far as the authors are aware - not at all for Bacillus brevis species. In the fully aerated culture, with uncontrolled pH, nutrient utilization rates and extent were comparable to the above controlled pH-fermentation. The oxygen limited, pHuncontrolled fermentation (Figure 3) displayed a much slower and incomplete utilization pattern, caused most probably by the strong acidification of the medium due to a shift to "anaerobic" metabolism. The diauxic growth patterns, observed here in all 5 liter F 3/6 fermentations, reflect primary growth at the expense of the amino acids L-arginine and L-glutamine and secondary growth at the expense of D-fructose as carbon sources. Rapid GS formation starts at the onset or during the second growth phase. Transition of growth phases seems to be determined by nutritional parameters and their regulatory effects rather than by aeration rate, D.O.T. or pH. This might indicate that initiation of GS synthesis is regulated by amino acid-metabolism, while extent of GS formation is controlled by D.O.T. and pH. 2.5. Dynamics of GS-synthetases 1 and 2 formation in B. brevis ATCC 9999 cultures The high yielding chemically defined F3/6 as well as the complex YP-medium have been used for further microbiological and biochemical studies on the dynamics and regulation of GS synthetase 2 and 1 formation as a function of the fermentation cycle. The two soluble enzymes are rapidly produced in a coordinate fashion in the transition period following th early logarithmic growth phase (2, 29, 34, 39-42). Both activities rapidly decline as the cells move towards the stationary phase or shift diauxic growth phases. Several hours after the onset of enzyme synthesis, GS becomes detectable, but its formation continues for a reasonable period even after the complete disappearance of soluble GS synthetase activity. Rapid disappearance of soluble synthetases can be retarted by utilizable carbon sources or ATP (4). Such kinetics of appearance and disappearance have been observed in both complex and defined media, irrespective of their nutrient composition, and environmental (i.e. aeration) conditions (Fig. 10 and 11).

In complex YP-medium, the enzymes are rapidly synthesized during a 3 to 4 hour interval in the 24 hour fermentation cycle i. e. in the transition period following the log phase (Figure 10). They reach a peak as the cells proceed into the stationary phase. Only at peak synthetase levels does GS become detectable, but the antibiotic continues to be formed at a constant rate even though the soluble synthetase levels drop drastically (see also Figure 4). In chemically defined F 3/6-medium, where initial growth occurs at the expense of L-

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


Fig. 10 Dynamics of GS-synthetases formation in complex YP-medium (shake flask fermentation) (250 Klett units correspond to 1 g of DCW/1; Symbols as in Figure 1 and 4).

glutamine and L-arginine (and where fructose and other nutrients are utilised after a short diauxic lag), the soluble enzymes are mainly formed during this transition phase. They are again produced in a coordinate fashion, reaching a peak and disappearing within an interval of about 8 hours, in a fermentation cycle lasting about 40 hours (Figure 11). GS can be detected only during the second growth phase. The onset of GS-synthetase formation seems to be related to the establishment of a low growth rate and is apparently not a result of the continuous variations in the medium constituents during the batch growth cycle. This hypothesis was established in chemostat studies with different nutrient limitations (28). We have obtained evidence that GS-synthetases occur in an insoluble as well as in a soluble form in growing B. brevis cells (38, 41, 42). Treatment of cell pellets, after extraction of soluble GS-synthetases, with detergent (Triton X-100) resulted in a liberation of additional GS-synthetases 2 and 1 activity, even from pellets prepared from late-stage cells with no apparent soluble GS-synthetase activities (Figure 12). These data indicate that GS-synthetase activities exist in both soluble and particle-bound forms, the latter being more resistant to in vivo degradation or inactivation processes, and responsible for late GS production after inactivation of the soluble form. It is also possible that the soluble enzymes become membrane-bound as the cells proceed towards the stationary phase and enter early sporulation events. Similar observations were independently made in the cases of tyrocidine, bacitracin and edeine biosyntheses


E.J. Vandamme et al.

Fig. 11 Dynamics of soluble GS-synthetases formation in F 3/6-medium (shake flask fermentation; Symbols as in Figure 1 and 4).

(9, 24-26), though an opposing trend has recently been found for polymyxin B synthetase (46). This phenomenon, which might be common to all oligopeptide synthetases, might be envisaged as a built-in enzyme immobilization process, to protect enzyme-complexes against increasing proteolytic activities in the cell during early (germination, outgrowth) or late (sporulation) cell differentiation stages. Arginine, although not required for growth of B. brevis, stimulates growth and specific production of GS to a much greater extent than any of the other four stimulatory amino acids in the F 3/6-medium. On the other hand, arginine lowers the specific activity of soluble GS-synthetase and inhibits its activity. We recently gained an understanding of these paradoxical effects. Arginine stimulates growth presumably because endogenous arginine limits protein synthesis. Arginine stimulates specific production of GS by providing ornithine, a constituent of the GS molecule, and by increasing GSsynthetase specific activity of the particulate fraction of the cell. Although growth on arginine lowers soluble GS-synthetase specific activity, its effect on the particulate enzyme complex is so marked that overall GS-synthetase activity is increased. Arginine acts either as an inducer of GS-synthetase or exerts a compartmentation effect favoring the particulate form of the enzyme. This is important for GS production since the particulate activity appears much more stable in vivo than the soluble form (4).

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


Fig. 12 Dynamics of particulate-solubilized GS-synthetase I formation in F3/6-medium; insoluble cell fractions treated with Triton X-100 (shake flask fermentation; Symbols as in Figure 1 and 4).

2.6. Concluding Remarks Environmental parameters greatly influence the dynamics of the GS fermentation process; cell yield, GS production and GS synthetase 1 and 2 formation are affected by external factors such as medium constituents, aeration rate and pH. It is hoped that these studies will contribute to a better understanding of the physiology of the GS producing microorganism and of the regulatory mechanisms involved in the biogenesis process of this secondary metabolite. Furthermore, these studies indicate how complex and interrelated microbial phenomena are and that only close cooperation between biochemists, microbiologists and biotechnologists can lead to further progress and understanding of microbial productivity.

Acknowledgements The authors are indebted to all the members of the MIT-Enzyme Engineering Group and of the Fermentation Microbiology Laboratory, Department of Nutrition & Food Science, Massachusetts Institute of Technology (MIT), Cambridge, USA, for valuable


E.J. Vandamme et al.

help and discussions. Part of this work emanated from a NATO-postdoctoral fellowship held at MIT by E.J. V. A.L.D. gratefully acknowledges the support of the National Science Foundation for grant GI-34284. P.D. V. and D.D.B, are recipients of fellowships, administered by the Belgian "Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL)"; G. V. and J. S. are indebted to the Belgian "Nationaal Fonds voor Wetenschappelijk Onderzoek (NFWO)" for financial support. Prof. A. Wilssens, head of the department, University of Ghent, is thanked for encouragement and support. The technical assistance of Wilfried Pieters is gratefully acknowledged.

References 1. Asatani, M. and Kurahashi, K.: J.Biochem. 81, 813 (1977) 2. Barraclough, R. and Laland, S.: Biochem. Soc. Transactions, London, 3, 534 (1975) 3. Chattopadhyay, S.P. and Banerjee, A.K.: Z. Allg. Mikrobiol. 18, 243 (1978) 4. Demain, A. L.; Agathos, S.; Poirier, A. and Nimi, O.: Abstracts Ann. Meet. Soc. Industrial Microbiol. Flagstaff, Arizona, USA, p. 29 (1980) 5. Demain, A. L. and Matteo, C. C.: Antimicrob. Agents Chemother. 9, 1000 (1976) 6. Demain, A.L.; Piret, J.M.; Friebel, T.E.; Vandamme, E.J. and Matteo, C.C.: In Microbiology 1976 (Ed. D. Schlessinger) p. 437, Amer. Soc. Microbiol., Washington D.C. (1976) 7. Friebel, T.E. and Demain, A.L.: J.Bact. 130, 1010 (1977) 8. Friebel, T.E. and Demain, A.L.: Fems Microbiol. Lett. 1, 215 (1977) 9. Froyshov, O.: Febs Lett. 81, 315 (1977) 10. Froyshov, O., Zimmer, T.L. and Laland, S.G.: In International Review of Biochemistry: Amino Acid and Protein Biosynthesis II, 18, (Ed. H. R. V. Arnstein) p. 49, University Park Press, Baltimore (1978) 11. Fynn, G.H. and Davison, J.A.: J. Gen. Microbiol. 94, 68 (1976) 12. Glazer, V.M.; Silaeva, S.A. and Shestova, S.L.: Biokhimiya 31, 1135 (1966) 13. Gus'Kova, T. M. and Udalova, T. P.: Priklady Biokhimiya Mikrobiologiya 9, 718 (1973) 14. Haavik, H.I.: J. Gen. Microbiol. 81, 383 (1974) 15. Haavik, H.I.: J. Gen. Microbiol. 84, 226 (1974) 16. Haavik, H.I. and Froyshov, O.: Nature (London) 254, 79 (1975) 17. Hamilton, B.K., Montgomery, J.P., Wang, D.I.C.: in Enzyme Engineering 2, (Pye, E.K., Wingard, L.B., eds). p. 153, Plenum Press, New York (1973) 18. Knight, B.C.J.G., Proom, H.: J. Gen. Microbiol. 4, 508 (1950) 19. Koischwitz, H., Kleinkauf, H.: Biochim. Biophys. Acta 429, 1052 (1976) 20. Koplove, H.M., Cooney, C. L.: in Advances Biochem. Eng., Immobilised Enzymes II, 12 (Eds. T. K. Ghose, A. Fiechter, N. Blakebrough), p. 1, Springer Verlag, Berlin 1979 21. Korshunov, V.V., Egorov, N.S.: Mikrobiologiya 31, 515 (1962) 22. Kupletskaya, M.V.: Mikrobiologiya 34, 905 (1965) 23. Kupletskaya, M.V., Maksimov, V.N., Kasatkina, T.B.: Priklady Biokhimiya Mikrobiologiya 5, 541 (1969)

Environmental Influences on the Dynamics of the Gramicidin S Fermentation


24. Kurylo-Borowska, Z.: Biochim. Biophys. Acta 399, 31 (1975) 25. Lee, S.G.: in Lipmann Symposium: Energy, Biosynthesis and Regulation in Molecular Biology (D. Richter, ed.) p. 368 Walter de Gruyter, New York 1974) 26. Lee, S.G., Littau, V., Lipmann, F.: J. Cell. Biol. 66, 233 (1975) 27. Lipmann, F.: Acc. Chem. Res. 6, 361 (1973) 28. Matteo, C.C., Cooney, C.L., Demain, A.L.: Microbiol. 96, 415 (1976) 29. Matteo, C. C., Glade, M., Tanaka, A., Piret, J., Demain, A.L. : Biotechnol. Bioeng. 17, 129 (1975) 30. Nakamura, M. : Agric. Biol. Chem. 32, 701 (1968) 31. Proom, H., Knight, B.C.J.G.: J. Gen. Microbiol. 13, 474 (1955) 32. Seddon, B„ Fynn, G.H.: J. Gen. Microbiol. 74, 305 (1973) 33. Silaeva, S.A., Glazer, V.M., Shestakov, S.V., Prokofiev, M.A.: Biokhimiya 30, 947 (1965) 34. Tornino, S., Yamada, M., Itoh, H., Kurahashi, K. : Biochem. 6, 2552 (1967) 35. Udalova, T.P., Fedorova, R.I.: Mikrobiologiya 34, 631 (1965) 36. Udalova, T.P., Guskova, T.M., Paramonov, N.Y., Silaev, A.B.: Antibiotiki 16, 304 (1971) 37. Udalova, T.P., Guskova, T.M., Silaev, A.B.: Mikrobiologiya 41, 280 (1972) 38. Vandamme, E.J.: D. Sc. Thesis, University of Ghent, Belgium (1976) 39. Vandamme, E.J., Demain, A.L.: Antimicrob. Agents Chemother. 10, 265 (1976) 40. Vandamme, E. J., Demain, A. L. : in Developments in Industrial Microbiology 17 (L. A. Underkofler, ed.), p. 51 Amer. Inst. Biol. Sciences, Washington D. C. (1976) 41. Vandamme, E. J., Demain, A. L. : Abstracts 6th Intern. Fermentation Symposium (H. Dellweg, ed.) p. 224 Berlin (West) (1976) 42. Vandamme, E.J., Demain, A.L.: IVth Fems Symposium Vienna (1977) 43. Vater, J., Kleinkauf, H.: Biochim. Biophys. Acta 429, 1062 (1976) 44. Yamada, K., Kinoshita, S., Tsunoda, T., Aida, K. : The Microbial Production of Amino Acids. Kodansha Ltd., Tokyo (1972) 45. Zimmer, T. L., Laland, S.G.: in Methods in Enzymology; Antibiotics 43, (Ed. J.H. Hash), p. 567, Academic Press, New York (1975) 46. Vasantha, N., Balakrishnan, R., Kaur, S., Jayaraman, K.: Arch. Biochem. Biophys. 200, 40 (1980)

Production of Gramicidin S-Synthetase from Bacillus brevis ATCC 9999 by Batch Fermentation Hans Jürgen Aust and Hans von Döhren Institut für Biochemie und Molekulare Biologie Technische Universität Berlin Franklinstrasse 29, D-1000 Berlin 10, F.R. of Germany

1. Introduction Although there have been extensive investigations in the fermentative production of secondary metabolites, little attention has been given to the enzyme systems producing them. As soon as the multienzymic nature of the peptide synthesizing system gramicidin S-synthetase was established (for reviews see Lipmann (1), Kurahashi (2), and Kleinkauf & Koischwitz (3)), its usefulness for preparative scale peptide antibiotic production has been recognized (4, 7). In a program exploring gramicidin S formation from amino acids and ATP by the soluble multienzyme system gramicidin Ssynthetase as a model for cell-free biosynthesis, fermentation procedures have been developed for enzyme production (Tzeng et al. (5), Demain et al. (6)). Our investigations on the fermentative aspect for the production of gramicidin S-synthetase have brought forth similar results. We were also able to make some improvements on the medium composition and the handling of the batch fermentation.

2. Selection and stock culture maintenance (Fig. 1) Spores of B. brevis ATCC obtained from the Institute of the American Type Culture Collections were allowed to grow on potato agar slant after 12 h incubation in 1% Yeast Peptone medium (YP) (37 °C). After the outgrowth, cells were transferred onto a 1% Yeast Peptone agar plate. After 24 h incubation at 37 °C, 50 single colonies were chosen and allowed to grow in shake flasks overnight. The gramicidin S-synthetase activity was then assayed. The culture with the highest activity was selected and allowed to sporulate in Hanson medium (14). The spores obtained were then divided in 2 ml portions, 2% glycerol added, and frozen in dry ice/acetone. They served then as inoculate for the subsequent fermentations. Thus the influence of the inoculate could more or less be eliminated; otherwise it is difficult to compare the results obtained from different fermentations.

3. Comparison of different media Different media for the fermentation were tested. A comparison is given in table I. It is clear that fermentations with 10% Yeast Peptone medium yield the highest activity, and the equipment cost is low. On the other hand, the cell yield per gram of substrate is lower in 10% YP medium; and the subsequent purification of the enzyme is more Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • New York


H.J.Aust, H. v.Dôhren















50 O V E R N I G H T




— * GS








2 ML SPORE S U S P E N S I O N + 2 % G L Y C E R I N E



Fig. 1



Selection scheme of B. brevis

difficult because of the higher content of other proteins, and higher proteinase activities. The stability of the enzyme is lower, and the disruption of the cells more difficult, the reason behind this is not yet clear. In view of this and of the higher price of YP medium - although a strict analysis of the production cost was not made, we tried to improve the 1 % glutamate medium, because it is easier to elucidate the effect of each substance. We found that the phosphate concentration played a significant role in regard to the production of gramicidin Ssynthetase. At lower concentration of phosphate, a significantly higher activity is achieved (Fig. 2, 3). This confirmed the general assumption that phosphate plays a significant role in the regulation of the biosynthesis of secondary metabolites (8); but up till now no investigation has been carried out on the phosphate regulation of the biosynthesis of the gramicidin S-synthetase. Only Vandamme (9), a few years ago, has shown the effect of phosphate on the production of gramicidin S. Subsequent investig-

Production of Gramicidin S-Synthetase by Batch Fermentation

Table I


Comparison of enzyme yields and stabilities at different fermentation conditions



tl/2 h

DCW g/1

generations spec, cell time min. activity

vol. activity


YP 10% YP 1% Glu 1 Glu 2 Glu 3

0.3 0.3 0.3 0.5 0.3

10.93 1.71 1.37 2.34 1.56

30 30 45 50 45

11 4.0 6.4 2.4 2.4

7.213 0.410 0.526 0.336 0.224


YP 10% YP 1% Glu 1 Glu 2 Glu 3

0.5 0.3 0.2 0.5 0.3

4.2 1.8 1.75 2.75 1.75

50 30 60 30 45

23.52 3.32 7.76 8.77 3.01

5.927 0.358 0.814 1.447 0,316


YP 10% Glu 2

0.8 0.5

11.35 1.8

40 30

12.00 4.4

8.172 0.475

Aeration rates were maximum and temperature was 37 °C by all fermentations. YP 10% = 5% yeast extract and 5% Bacto peptone YP 1% = 0.5% yeast extract and 0.5% Bacto peptone Glu 1 = 1 % glutamate, five gramicidin S amino acids and 1.36% phosphate Glu 2 = 0 . 1 % glutamate, 0.2% phosphate and spec, trace elements Glu 3 = 1 % glutamate and 1.36% phosphate 1 1/2 = half-life of enzyme at 37° spec, cell activity = nM GS x min" 1 x g cells Vol. activity = nM GS x h" 1 x litre"1

ations on this are not known. In our opinion, it is important to look deeper into the phosphate regulation mechanism in order to achieve a still higher enzyme production. We have also tried to elucidate the effect of manganese on the production of the enzyme complex. Preliminary results showed that a slightly higher activity might be achieved with an absence of manganese in the culture medium. It is interesting to note that although the enzyme production increased in the absence of manganese in the medium, gramicidin S production could not be detected. We suspect that in the absence of manganese the pool concentration of certain amino acids might be dropped to a minimum so that no gramicidin S could be synthesized although the enzyme complex is active. Further investigations are needed to give a quantitative evaluation of the effect of the manganese concentration on the enzyme production, and to give a satisfactory explanation of this phenomenon.


H.J.Aust, H.V.Döhren



Fig. 2


The influence on specific cell activity of B. brevis at various phosphate concentrations. NM GS



Fig. 3 The influence of various phosphate concentrations in minimal-medium on the specific enzyme activity of gramicidin-S-synthetase.

Production of Gramicidin S-Synthetase by Batch Fermentation


4. Influence of fermentation parameters on the enzyme production 4.1. Impeller system Our experience showed that for our purpose the intensor system (Fig. 4) equipped with a propeller and a draught tube guiding the circulation of the culture broth is superior to the vaned disc impeller. With this system, we could achieve a relatively good mixing and a high oxygen transfer rate with a comparatively small sheer force; whereas with a vaned disc impeller with baffle, a higher shear could not be avoided which might be the cause of the poorer production of gramicidin S-synthetase by fermentations with this system. The foaming in the fermenter with intensor system is less of a problem than in the fermenter with vaned disc impeller.

4.2. Agitation A higher agitation rate at the beginning of the fermentation resulted often in a poor cell growth; this may be due to the sudden change of environment from the shake flask to the fermenter. Therefore, we started the fermentation with a lower agitation rate while maintaining a sufficient dissolved oxygen tension such that the cell growth would not be otherwise inhibited. The agitation rate was then stepwise increased to meet the increased demand of oxygen owing to the increase in cell concentration. The oxygen supply in fermentations with 1 % medium could be guaranteed by a maximal agitation rate and aeration rate even at the end of fermentation. This was not the case by AIR



Two systems of agitation.


H.J.Aust, H.v. Dôhren

fermentations with 10% YP medium, so that the cell growth might be said to be 0 2 limited. An economical usage of the substrate here is not possible. An oxygen enrichment of the culture was then tried. 4.3. Oxygen supply Owing to the difficulty of oxygen supply near the end of the fermentation with 10% YP medium, an enrichment with oxygen to keep the DOT a level of 10%, 20%, and 30% oxygen partial pressure respectively, has been carried out. The results are shown in table II. The highest enzyme activity and cell yield could be obtained by maintaining the DOT at 20% by means of 0 2 -enrichment. No significant improvement on the enzyme activity could be made by 0 2 -enrichment by fermentations with 1 % minimal medium. 4.4. Temperature Fermentations at 37, 46, and 50 °C were also performed hoping that the gramicidin Ssynthetase isolated from cells grown at a higher temperature might be more stable Table II

Oxygen-enriched aeration

(501 fermenter

YP 10% - medium)

%p02 t 1 / 2 (h) DCW g/1

10% 0.3 4.9

20% 0.3 6.82

30% 0.3 6.25

Max 0.5 4.2

n M GS „ min x g cells Generations time min. Temperature °C nM GS Vol. act. , " M , G S h x litre





30 37

45 37

45 37

50 37





t 1 / 2 = half-life of enzyme at 37°C Table III

Effect of temperature on enzyme production

Temperature ti/2 (h) DCW g/1 nM GS min x g cells Generations time min. Aeration Vol. act. , n M , G S h x litre t 1 / 2 = half-life of enzyme at 37°C

37°C 0.3 10.93 11 30 Max 7.213

(101 fermenter

YP 10% - medium)

46 °C 0.2 4.62

50 °C 0.15 2.453



30 Max 0.221

24 Max 0.00736

Production of Gramicidin S-Synthetase by Batch Fermentation


towards heat inactivation (table III). This assumption was unfortunately not confirmed by the results (11). We could only obtain very poor enzyme activity and even a slight decrease in stability expressed in t 1 / 2 . With the increase in temperature the cell yield was also poorer. 4.5. Cell harvest Owing to the fact that the maximum in gramicidin S-synthetase activity appears only for a very short period during the fermentation, special attention should be given to the harvest point, such that the appearance of the maximum coincides with the harvest. The greatest difficulty in achieving this lies in the lack of a quick assay method for the gramicidin S-synthetase, therefore other parameters must be looked for to pin-point the harvest. The drastic drop in the respiratory rate near the end of the fermentation was shown to be a very good and reliable signal for the harvesting of the cells (Fig. 5). Although the physiological relationship between this drop in the respiratory rate and the increase in the gramicidin S-synthetase activity is not clear, we suspected that there must be some connection between them, because the drastic drop generally reflects a drastic change in the metabolism. We might even suppose that this is the division point of the primary and the secondary metabolism. This phenomenon was also observed by others (12), but until now no further investigations are known to deal with this phenomenon. The redox potential and the 0 2 and C 0 2 analysis of the exit gas might also be drawn to pin-point the harvest. As we do not possess the necessary equipment for that,


Agitation UPMxtO2-

a pH



Enzyme Activity 30

nM G5

triirv g

Fig. 5A 50 L batch fermentation at 37°C in 1% minimal médium (1% glutamate, 1% phosphate) and 20% p 0 2 .


H.J. Aust, H. v. Dohren

h Fig. 5B Parameters currently used in estimation of gramicidin S-synthetase production phase: Dissolved oxygen tension (DOT), mean generation time (MGT), and pH. The drop in respiratory activity corresponds to highest enzyme activity. This opens up the possibility of harvesting the culture at the moment of its highest enzyme activity which is one of the most difficult problems to solve up till now.

these possibilities remain to be tested. A rapid cooling down of the culture broth to 4°C was found to be necessary particulary by large scale fermentation so that the loss of enzyme activity could be kept to a minimum. 4.6. Scale U p The scale-up of fermentation from bench-type fermenter to pilot fermenter is often difficult with some microorganisms. As pointed out by Oldshue (9) the various properties of the fermenter change differently with scale-up. Our experience from fermentations in 10, 20, 50, 200, 500 and 40001 fermenters showed that the B. brevis ATCC 9999 was relatively tolerant towards these changes, if a sufficient level of DOT above 20% and a not too high agitation rate were maintained. The inoculation relationship of 1:10 was found to be appropriate. A 5001 fermentation has been illustrated in fig. 6,

Production of Gramicidin S-Synthetase by Batch Fermentation

» — *

Fig. 6A



Cell growth and the specific cell activity of Bacillus brevis A T C C 9999 during the fermentation.

Fig. 6B Concentration of gramicidin S-synthetase shown by 5% SDS gels during growth in the 500 litre fermentation.


H.J.Aust, H.V.Döhren

m H-*

2 19,14

Fig. 6C





Scanning of SDS gels permit estimation of the enzyme concentration during the fermentation.

E 650

Fig. 7 3).




501 batch fermentation at 37 °C of Bacillus brevis ATCC 9999 in glutamate-minimal-medium (Glu

showing also the quantitation of the multienzyme GS 2 in crude extracts by Polyacrylamide gel electrophoresis. Growth curves are quite reproduceable (Fig. 8), but may vary at different fermentation volumes (Fig. 7).

Production of Gramicidin S-Synthetase by Batch Fermentation

Fig. 8


Reproducable 10 litre fermentation of B. brevis A T C C 9999 in 10% YP-medium at 37 °C.

Acknowlegement We wish to thank W. Wania, and H. Schüler from Gesellschaft für Biotechnologische Forschung Stöckheim Braunschweig for their cooperation and support in the fermentation, A. El-Samaraie for PAGE-analysis, and Th. Bernhard for bioassay of gramicidin S; E. Denaro, D. Schmidt and C.W. Chiu for their help in preparation of the manuscript. This work was supported by grants from the Bundesministerium für Forschung und Technologie (PTB 8013) and the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 9.

References 1. Lipmann, F.: Adv. microb. Physiol. 21, 227 (1980) 2. Kurahashi, K.: Ann. Rev. Biochem. 34, 445 (1974) 3. Kleinkauf, H., Koischwitz, H.: in: Hahn, F.E. (ed) Progress in molecular and subcellular biology, vol. VI, p. 59, Springer, Berlin-Heidelberg-New York (1978) 4. Wang, D.I.C., Fleischaker, R., Stramondo, J.: in: Biotechnol. Appl. Proteins Enzymes (Z. Bohak, N. Sharon, eds.) p. 183 Academic Press, New York (1977)


H.J. Aust, H. v. Döhren

5. Tzeng, C. H., Trasher, K. D., Montgomery, J. P., Hamilton, B. K., Wang, D. I. C. : Biotechnol. Bioeng. 17, 143 (1975) 6. Demain, A. L., Piret, J., Friebel, T.E., Vandamme, E.J., Matteo, C. C.: in Microbiology, 1976 (Washington D.C.) p. 437, (1976) 7. Matteo, C.C., Glade, M., Tanaka, A., Piret, J., Demain, A.L. : Biotechnol. Bioeng. 17, 129 (1975) 8. Martin, J. F., and Demain A.L.: Microbiol. Rev. 44, 230 (1980) 9. Vandamme, E.J.: Proefschrift, Rijksuniversität, Gent (1976) 10. Oldshue, S.Y.: Biotechnol. Bioeng 8, 3 (1966) 11. Zehnpfennig, Angelika: Dipl. Arbeit 1979 and unpublished results 12. Kraepelin, G. and Vandamme, E.J.: personal communications 13. Zimmer, T.L. and Laland S.G.: Methods in Enzymology 43, 567 (1975) 14. Hanson, R.S., Blicharska, J., Szulmajster, J., Biochem. Biophys. Res. Commun. 17, 1 (1964)

Studies on the Gramicidin S-Synthetase Production in Bacillus brevis ATCC 9999 in C- and P-Limited Chemostat Cultures Chung-Wai Chiu, Thomas Bernhard and Hans Werner Dellweg Institut für Biochemie und Molekulare Biologie Technische Universität Berlin Franklinstrasse 29, D-1000 Berlin 10, F . R . of Germany Abbreviations'. GS: gramicidin S; D : dilution rate; Sco: carbon substrate concentration in feeding medium; Spo: phosphate substrate concentration in feeding medium; V: working volume of fermenter.

1. Introduction So far little has been known of the regulation of the biosynthesis of the gramicidin Ssynthetase system in B. brevis. The knowledge of the regulation mechanism is interesting not only for the understanding of the physiology, but also for the improvement of the production of the two multienzymes complex. Some investigations in this direction were made by the Matteo and Demain group (1). They showed that in phosphate, nitrogen, or sulphur limited chemostat cultures, a high production of gramicidin Ssynthetase could be attained. How does the microorganism regulate its production? Does it need some low molecular weight mediators? These are certainly questions that need time and effort to answer. Preliminary investigations were made in our laboratory aiming to give first of all a clearer picture of the influence of the major nutrient components on the biosynthesis of the gramicidin S-synthetase before taking further steps to investigate the regulation at its molecular level. Experiments were carried out in a chemostat using a basal defined medium developed by one of us (T. B.) with fumarate as the sole carbon source. Pulse and shift experiments were also made to elucidate the transient response of the cells as this might give a deeper insight into the complicated biological processes (2-3).

2. C-limited chemostat culture 2.1. Effect of dilution rate In a C-limited chemostat culture with 0.2% fumarate and 20 mM phosphate in the infeeding medium, good sporulation was observed at dilution rates lower than 0.2 h~ but no gramicidin S-synthetase activity could be detected throughout the whole range of dilution rates. On the contrary, in another C-limited chemostat culture containing 1.3% fumarate and 2 mM phosphate in the infeeding medium, gramicidin S-synthetase activity could be detected at dilution rates higher than 0.2 h ~ 1 with a maximum at D = 0.27 IT 1 (Fig. 1). Owing to the lack of an effluent gas analyzer, a complete balance of Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • New York


C.-W.Chiu, T.Bernhard, H.Dellweg





dilution rate in h Fig. 1 Effect of dilution rate on gramicidin S-synthetase activity in B. brevis ATCC 9999 grown in a Climited chemostat.

material is not possible and we cannot say whether the system is under carbon limitation or under energy limitation - a point which might be important in the evaluation of the results. 2.2. Transient response A pulse of fumarate (27.5 g) added to a fumarate limited chemostat culture (1.3% fumarate) growing at D = 0.17 h " 1 caused an increase in gramicidin S-synthetase activity (Fig. 2) within 2 hours, but after that, the activity decreased again rapidly.The decrease was faster than an exponential washout so that an enzyme inactivation must have taken place. The cause of this inactivation is not known. It is interesting to note fumarate pulse (27,5 g)

~r in

i u time in hours Fig. 2 Transient response caused by a fumarate pulse given to a C-limited chemostat culture of B. brevis ATCC 9999.

Studies on the Gramicidin S-Synthetase Production in C- and P-Limited Chemostat Cultures


that the phosphate concentration in the culture broth ran through a minimum corresponding to the maximum of the gramicidin S-synthetase activity. Judging from these results, we suspect that the increase in activity might be the result of an induction or derepression leading to de novo synthesis of the multienzymes. Of course, it would be too simple and too early to declare that fumarate is an effector of the regulation. A similar response was also obtained by pulsing fumarate to a C-limited chemostat culture growing at a higher dilution rate of D = 0.22 h~ On the other hand, pulsing phosphate to a C-limited culture caused no substantial change in the enzyme activity.

3. P-limited chemostat culture 3.1. Effect of dilution rate A maximum in gramicidin S-synthetase activity was observed at D = 0.22 h - 1 (Fig. 3), and the activity level was much higher than that obtained in a C-limited chemostat culture. This activity is comparable to that normally observed in batch fermentations with YP-medium. The result suggests an economical alternative for the production of gramicidin S-synthetase. Contrary to the C-limited chemostat culture, no sporulation could be detected under P-limitation.

Fig. 3 Effect of dilution rate on gramicidin S-synthetase activity in B. brevis ATCC 9999 grown in a Plimited chemostat

3.2. Transient response An immediate decrease in gramicidin S-synthetase activity was observed after pulsing phosphate to a P-limited chemostat culture growing at D = 0.22 h " 1 . The decrease in activity followed approximately the exponential washout. This suggests a repression of the biosynthesis of gramicidin S-synthetase. The repression lasted approximately 3


C.-W. Chiu, T. Bernhard, H. Dellweg

hours (Fig. 4).Thereafter, the activity increased again and reached gradually its original level. A similar response was also observed by pulsing phosphate to a P-limited chemostat culture growing at D = 0.27 h ~ 1 . In both cases the recovery from the repression started after the phosphate concentration in the culture broth had decreased to a level lower than 5 m M (Fig. 4a, b). phosphate pulse



time in hours Fig. 4 Transient response caused by a phosphate pulse given to a P-limited chemostat culture at D = 0.27, 0.22 and 0.17 h" 1 .

Studies on the Gramicidin S-Synthetase Production in C- and P-Limited Chemostat Cultures


A different response was obtained however, by pulsing phosphate into a P-limited chemostat culture growing at D = 0.17 h~ The gramicidin S-synthetase activity did not return to its original level after the washout of the phosphate added; instead, sporulation (10%) was observed 10 hours after the pulse, and the gramicidin Ssynthetase then was hardly detectable (Fig. 4). 3.3. Shift experiment Further more downshift of phosphate concentration in the infeeding medium from 1 mM to 0.5 m M was made. The steady state specific gramicidin S-synthetase activity was slightly lower than that before the shift, showing that the enzyme production can hardly be improved by further lowering the phosphate concentration. Thus, the optimal phosphate concentration may be about 1 mM. The important results were summarized as follows: Table I

The important results were summarized as follows:

Kind of limitation


GS-synthetase activity

0.2% fumarate 20 m M phosphate

yes at D 0.2 h " 1

not detectable


1.3% fumarate 2 m M phosphate

yes at D 0.2 h " 1

active at D 0.2 h " 1


1.3% fumarate 1 m M phosphate



Pulse C





no effect



It should be noted that in continuous and in batch cultures with fumarate minimal salts medium, gramicidin S could only be detected in the culture broth and not within the cells, a phenomenon contrary to that observed in batch cultures with YP-medium, where mainly intracellular gramicidin S could be detected. The results show that a P-limitation favours the gramicidin S-synthetase production, and a high phosphate concentration causes repression of the biosynthesis of the gramicidin S-synthetase. This confirms the results obtained by J. Aust and H. v. Dôhren (4). The phosphate pulse experiments show that repression of the biosynthesis takes place only when phosphate is taken up by the cells. It has to be discussed how far repression is related with phosphorylated compounds like the nucleotide triphosphates, or the highly phosphorylated nucleotides. It should not be too surprising if that is the case, as a stringent factor and a stringent factor-like system were already detected in B. brevis ATCC 8185 (5). Of course, the effector needs not necessarily to be a phosphorylated compound. Further investigations are needed before one can make more precise predictions. Furthermore, the results show that sporulation occurs only when the gramicidin Ssynthetase activity is practically undetectable (Fig. 4c and Tab. 1).This further supports the viewpoint that gramicidin S is not a prerequisite of sporulation (6).


C.-W.Chiu, T.Bernhard, H.Dellweg

Acknowledgement We thank H. Kleinkauf and H. v. Döhren for their help in initiating these studies. This work was supported by grants of the Bundesministerium für Forschung und Technologie (PTB 8013 and 8310) and the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 9.

References 1. Matteo, C.C., Cooney, C.L., Demain, A.L.: Journal of Gen. Microb. 96, 415 (1976). 2. Koplove, H.M., Cooney, C.L.: Adv. in Biochem. Eng. 12, 1 (1979). 3. Harrison, D.E.F., Topiwala, H.H.: Adv. in Biochem. Eng. 3, 167 (1974). 4. Aust, J., Döhren, H.v.: this volume, p. 137 5. Sy, J.: in "Regulation of macromolecular synthesis by low molecular weight mediators" p. 95, Eds.: Koch, G., Richter, D., Acad. Press (1979). 6. Kambe, M., Imae, Y., Kurahashi, K.: J. Biochem. 75, 481 (1974).

On the Role of L-Leucine in the Control of Bacitracin Formation by Bacillus licheniformis Hans Ivar Haavik and Oystein Freiyshov, Department of Research and Development, A/S Apothekernes Laboratorium for Specialpraeparater, Skdyen, Oslo 2, Norway

1. Introduction The dodecapeptide antibiotic bacitracin consists of ten different amino acids (1). It is produced by strains of Bacillus licheniformis by the thiotemplate mechanism (2). The control mechanisms for its formation are under investigation. Addition of bacitracin to the bacitracin synthetase inhibited the enzyme activity in the presence of several different metal ions and it was suggested that product-metal-ion-complexes exert feedback inhibition on the enzyme activity (3). Furthermore, it has been shown that the bacitracin production by the high yielding mutant Bacillus licheniformis strain AL was powerfully stimulated by the addition of the amino acid leucine to a synthetic medium (4). In the present paper in vivo and in vitro experiments which suggest that leucine plays an inducer role in the regulation of the bacitracin synthetase are described.

2. Methods 2.1. Strains and Media The bacitracin producing strains Bacillus licheniformis ATCC10716 and AL were kept as spore suspensions at 4 °C throughout the investigation. The synthetic media had the following composition (g/1 distilled water): L-glutamic acid, 20.0; L-alanine, 0.2; citric acid, 1.0; N a H 2 P 0 4 • 2 H 2 0 , 2.0; KC1, 0.5; Na 2 S0 4 , 0.5; MgCl 2 • 6 H 2 0 , 0.2; CaCl 2 • 2 H 2 0 , 0.01; FeS0 4 • 7 H 2 0 , 0.01; MnS0 4 • H 2 0 , 0.01. The pH was adjusted to 7.0 with 10N NaOH before autoclaving at 121 °C for 20 minutes (M2-medium). The M20 medium had the same composition as the M2 medium except that the N a H 2 P 0 4 • 2 H z O content was 20.0 g/1 and the start pH was 6. The MO 20 medium had the same composition as the M20 medium except that Lglutamic acid was substituted with L-ornithine. L-leucine (lg/1) was added before autoclaving when it was a component of the medium.

2.2. Growth Bacterial growth was measured as the absorbance at 650 nm (E 650 ) in a Spectronic 20 spectrophotometer. Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • N e w York


H. I. Haavik, ©. Frriyshov

2.3. Assay of Bacitracin Bacitracin was determined by an agar diffusion method: Ten ml of seed agar (Difco Antibiotic Medium no 1) inoculated with a predetermined amount of Micrococcus luteus was added directly to the 9 cm sterile Petri dishes. Six stainless steel cylindres were placed on the surface of the seeded plates by a mechanical dropping devise. Three of the cylinders were filled with our working standard of concentration 1 i. u. bacitracin/ml. The other three cylinders were filled with the unknown sample to be determined. For each sample there were used three plates. After incubation at 37 °C for 24 hours the zones of inhibition were measured with a Fisher-Lilley zone reader. The potency of the unknown sample was determined by the use of a standard curve.

2.4. Production of auxotrophs Leucine auxotrophs were made by spreading spores on Bacto Nutrient agar and irradiate with UV for 15 seconds. After incubation at 37 °C for about 16 hours colonies were formed on the plates. The colonies were replicated on M 2 medium solidified with Bacto agar and with and without leucine (1 g/1).

2.5. Bacitracin synthetase Frozen cells were thawed and lyzed with the aid of lysozyme. A43-49% saturated ( N H 4 ) 2 S 0 4 fraction was prepared as in (5).

2.6. Cell free synthesis of Bacitracin The reaction mixture is described in (5). The incubations were carried out at 37°C for 30 minutes.

3. Results Fig. 1 shows that leucine markedly increases the amount of bacitracin synthetase and bacitracin formed by Bacillus licheniformis strain AL when grown in the synthetic M 20 medium. When leucine was added to the original bacitracin producing strain B. licheniformis ATCC 10716 these effects were not observed (results not shown). The addition of leucine to the cell free preparation of the bacitracin synthetase had no stimulatory effect upon bacitracin formation. This is shown for strain AL in Table I. When more than 1.0 m M leucine was added to the cell free reaction mixture less 14 Cbacitracin seems to be produced. This may be due to leucine substitution at the isoleucine site (6). In medium MO 20 where glutamic acid is substituted with ornithine both B. licheniformis strain AL and strain ATCC 10716 showed increased bacitracin production in the presence of leucine (Table II and III). We made five auxotrophs of B. licheniformis ATCC 10716 which required leucine for growth. Three of the mutants produced more bacitracin than the parent strain when grown in the M 2 medium in the presence of leucine (Table IV).

On the Role of L-Leucine in the Control of Bacitracin Formation



10 Period

11 of

12 growth











Fig. 1 Bacitracin production and bacitracin synthetase formation in medium M20 with and without Lleucine (1 g/1). Spores of B. licheniformis strain AL were suspended in 200 ml of the medium, incubated on a rotatory shaker (16 hours, 37°C) and used to inoculate 8 1 medium in a New Brunswick fermentor. The cells were grown at 37°C with stirring (500 rev/min.). At invervals 20 ml aliquots were withdrawn. The absorbance ( o ) of the culture was measured, the bacitracin content (A) was determined and the bacitracin synthesizing activity ( • ) was analyzed as described in Methods. For the medium M20 with 1 g/1 leucine open signs were used. For the medium M20 without leucine closed signs were used. The total amount of 14 C-L-isoleucine in each incubation mixture was 120000 cpm.

Table I Effect of leucine upon the cell free synthesis of bacitracin by the bacitracin synthetase from B. licheniformis strain AL

Added leucine (mM) 0.001 0.01 0.1 1.0

Synthesis of bacitracin (c.p.m.) 5100 4820 3920 4120

Total amount of radioactivity in each incubation mixture was 70900 c.p.m.


H. I. Haavik, 0 . Fróyshov

4. Discussion The addition of leucine to the M20 medium resulted in the formation of relative large amounts of bacitracin synthetase and stimulated the bacitracin production about 15 fold (Fig. 1). This stimulatory effect of leucine upon bacitracin production was also seen in the M 2 medium (4). Leucine had no stimulatory effect upon the bacitracin synthetase when added in vitro. Thus leucine does not seem to influence the specific activity of the synthetase. Our results may indicate that leucine plays an inducer role in the regulation of the bacitracin synthetase.

Table II Effect of leucine upon bacitracin production by Bacillus licheniformis ATCC 10716 in the M 0 2 0 medium

Added leucine (g/1)

Growth after 22 h (E 6 5 0 )

none 1 2 Table III medium

Bacitracin production 22 h (i.u./ml)

10.0 9.8 9.8

12.8 22.8 20.3

Effect of leucine upon bacitracin production by Bacillus licheniformis strain A L in the M 0 2 0

Added leucine (g/1) none 1 2

Growth after 22 h (E 6 5 0 )

Bacitracin production 22 h (i.u./ml)

3.8 9.6 9.2

0.1 11.0 12.2

Table IV Growth and bacitracin production by five auxotrophic mutants of B. licheniformis 10716 in the M2 medium with 1 g/1 leucine


ATCC 10716 Mutant LI Mutant L2 Mutant L3 Mutant L4 Mutant L5

Growth after 22 h (Eeso) 9.0 5.4 9.4 7.0 8.6 8.8

Bacitracin production 22 h (i.u./ml) (%) 14.4 7.0 20.0 14.1 24.3 22.4

100 49 139 98 169 156


On the Role of L-Leucine in the Control of Bacitracin Formation


Leucine may be a part of a more comprehensive amino acid control mechanism for bacitracin formation (7). Amino acids may also have inducer roles in the formation of other antibiotics (8). The original bacitracin producing strain B. licheniformis ATCC 10716 did not show increased bacitracin production in the presence of leucine when grown in the M2 or M20 medium. It is possible that this strain produces enough internal leucine to fully express the inducer role of leucine when grown in these media. Strain ATCC 10716 may be a better producer of internal leucine than strain AL (4). When grown in the MO 20 medium leucine stimulated bacitracin production by both strains. This indicates that leucine has a general inducer role in the formation of bacitracin by strains of B. licheniformis. Strain AL showed poor growth in the MO 20 medium without the addition of leucine. It is possible that the strains make less internal leucine when grown in this medium than in the M2 and M20 medium. Our results indicate that leucine has a somewhat different inducer role in the high yielding mutant strain AL than in strain ATCC 10716. This supports the hypothesis that high yielding mutants may have various defects or modifications in their control mechanisms for antibiotic formation (9,10). Since leucine auxotrophs may produce different amounts of bacitracin in the presence of the same amount of leucine, it is likely that some other factors may influence the inducer role of leucine. We thank Mr. T. Heiyland, Director of Research and Development, for his support, and Mrs. Tove Nordgarden and Mrs. Anita Mathiesen for excellent technical assistance.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Galardy, R.E., Printz, M.P. and Craig, L.C.: Biochemistry 10, 2429 (1971) Frriyshov, 0 . and Laland, S.G.: Eur. J. Biochem. 46, 235 (1974) Frctyshov, 0., Mathiesen, A. and Haavik, H. I.: J. gen. Microbiol. 117,163 (1980) Haavik, H. I. and Vessia, B.: Acta path, microbiol. scand. Sect. B, 86, 67 (1978) Fr^yshov, 0 . and Mathiesen, A.: This volume Frriyshov, 0 . : FEBS 12th Meeting abstr. commun. 2922 (1978) Haavik, H.I.: Folia Microbiol. 24, 234 (1979) Drew, S.W. and Demain, A.L.: Ann. Rev. Microbiol. 31, 343 (1977) Demain, A.L.: Stadler Genet. Symp. University of Missouri, Columbia 8, 41 (1976) 10. Hostalek, Z.: in Regulation of secondary product and plant hormone metabolism (M. Luckner, K. Schreiber, eds.) p. I l l , Pergamon Press Oxford 1979

Current Research in Ergot Peptide Synthesis by Claviceps purpurea Ullrich Keller, Rainer Zocher and Gunda Kraepelin Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstraße 29, D-1000 Berlin 10, F . R . of Germany Abbreviation: N T G : N-Methyl-N'-Nitro-N-Nitrosoguanidine

1. Introduction Ergot peptides are produced by members of the genus Claviceps (1). The chemical structure of these compounds attracted the interest of many investigators and prompted them to elucidate the mechanism of biosynthesis (2). Ergot peptides contain D-lysergic acid in the N-terminal position of a peptide chain as shown in the case of ergotamine (Figure 1). Replacement of residues in this chain by other amino acids of more or less similar structure results in different peptide alkaloids. The cyclol bridge between the carboxyl-group of proline and a-hydroxyalanine is a peculiar property of this class of substances and seems to be introduced after the formation of the Dlysergyl-peptide (3). Studies of ergot peptide formation in a cell-free system were not yet successful. However, ergo line ring synthesis was achieved in vitro (4). In our laboratory, attempts were made to establish a protoplast system actively synthesizing ergot peptides as a prerequisite for the preparation of a cell-free system. Parts of this work have already been published elsewhere (5). In this contribution we describe some further aspects of the physiology of ergot peptide synthesis in mycelia and protoplasts of C. purpurea with special regard to the role of nitrogen-metabolism in starved and fresh mycelia and amino acid pools in cellular compartments.

OC O C — N


HN Fig. 1

Structural formula of ergotamine.

Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • N e w York


U. Keller, R. Zocher, G. Kraepelin

2. Materials and Methods 2.1. Strains Clavicepspurpurea ATCC 20102 and mutants of this strain with increased capacity for ergotamine formation were used during this work. 2.2. Culture conditions Strains of Claviceps purpurea were kept on medium T 2 at 0°-2°C (6). Transfer was made at one month intervals. Frozen stocks were kept at — 32°C under liquid paraffin. Conditions for cultivation in liquid medium were the same as described previously (5). 2.3. Mutagenic procedures Mutagenesis was performed by transfer of mycelium grown for six days in an inoculum medium (7) into medium T25 (8) containing various concentrations of NTG (1030 ng/ml). Those cultures, where after a three days lag further growth was observed, were harvested and treated with ^-glucuronidase in medium PM (5) in order to get protoplasts. Protoplasts were regenerated as described previously (5) and resultant single colonies were isolated and tested for alkaloid production in submerged culture. 2.4. Isolation of vacuoles Vacuoles of C. purpurea were prepared as described previously (9). Protoplasts were treat with dilute buffer in the presence of 0.025% Triton X-100 and after release of vacuoles these were stabilized by the addition of 12% Ficoll 400. Vacuoles were purified by centrifugation in a Ficoll step gradient of 10% and 8%. 2.5. Measurement of ergot peptide synthesis Ergot peptide synthesis was followed by measuring the incorporation of 14 C-Phe into ergotamine. 109 protoplasts or 70-80 mg mycelium were usually incubated for three hours in a final volume of 1 ml (medium PM) in the presence of 1 |iCi radiolabel. Other chemicals were present as described in the text. Isolation and determination of radioactive ergot peptide was done as described previously, with the exception that carrier ergotamine was used only in the case of the wild type strain (5). Ergot peptide concentration in culture media was estimated by the van Urk reaction with ergonovine maleate as the standard (10). 2.6. Methods of analysis Amino acid pools of whole protoplasts and isolated vacuoles were determined as described previously (5). The distribution of alkaloid in the cultures between cells and medium was measured by total extraction of homogenized cultures and separate determination of alkaloid in the cell-free culture fluid after standard procedures (8). It was

Current Research in Ergot Peptide Synthesis by Claviceps purpurea


found that at least 90% of total alkaloid was always present in the medium. D-lysergic acid was chromatographed on thin-layer plates of silicagel (Merck) by development in ethanol-water (80:20) (11). In this solvent system it runs with an R f -value of 0.48, whereas in the solvent system chloroform-methanol-ammonia (80:20:0.2) which is also used for the separation of alkaloids, it has an R f -value of about 0.3.

2.7. Chemicals All chemicals used were from commercial sources with the exception of D-lysergic acid. This was prepared according to Stoll et al. (12). [U- 14 C]-L-phenylalanine (19 TBq m o l e - x) and [5- 3 H]-L-tryptophan (926 TBq m o l e - *) was purchased from the Radiochemical Centre, Amersham.

3. Results and Discussion Claviceps purpurea ATCC 20102 is an efficient source for protoplasts actively synthesizing ergot peptides. In our recent studies with protoplasts of this strain we observed ergot peptide synthesis to be enhanced by the addition of D-lysergic acid. It was also noted, that both mycelium and protoplasts were much more active with respect to synthetic activity and uptake of the radioactive precursor after a 12 hours incubation period in the protoplast medium (lacking N-source). Cells tested immediately after harvest from the growth cultures were much less active. It was concluded from the above findings that the D-lysergic acid concentration within the cells is a rate limiting factor in ergot peptide synthesis and that nitrogenmetabolism is of importance for the synthetic process.



4 8 12 D - L y s e r g i c acid (mM)

Fig. 2 Dependence of ergotamine synthesis on D-lysergic acid concentration in incubations with mycelia of three strains of C. purpurea. h — a strain 10-29, o — o strain 20-21, x — x parent wild type


U.Keller, R.Zocher, G.Kraepelin

Table I

List of strains of C. purpurea

Ergotamine production after 7 days of growth. (mg/1)


Wild type (parent ATCC 20102) 20-21 10-29

20-30 100-150 600-800


[WT] •fKf'


PU |> * • J K, * • ,-jf», a

The original strain obtained from ATCC (wild type) produces 20-30 mg/1 ergot alkaloid in the growth medium after 7 days of cultivation. Although this is a low titre, the incorporation of radioactive precursor into ergot alkaloid proceeded efficiently especially when cells incubated for 12 hours in medium PM were used. Mutants producing higher levels of ergot peptides were obtained from C. purpurea ATCC 20102 by treatment with NTG. Three isolates tested showed increased levels of ergot peptide concentration in liquid culture (Table I). When mycelia or protoplasts of these strains, and also of the wild type, were subjected to N-starvation in PM for 12 hours and then tested for ergot peptide synthesis, the highest incorporation rate of 14 C-Phe was found in the mutant 10-29 and the lowest in the wild type (Figure 2). Increasing concentrations of externally added D-lysergic acid stimulated ergot peptide synthesis remarkably in the mutant 20-21 and in the wild type, whereas in strain 10-29 further increase was weak, indicating that sufficient quantities of D-lysergic acid for peptide alkaloid synthesis were present in the cells. In order to prove this interpre-

Current Research in Ergot Peptide Synthesis by Claviceps purpurea


tation, we incubated 5 ml (75 mg/ml wet weight) of starved cells of 10-29 with 3 Htryptophan for 5 hours and subsequently extracted the alkaloid synthesized. In the solvent systems besides radioactive ergot peptide we found a radioactive band which showed a slight fluorescence. Cochromatography with authentic D-lysergic acid did not show any difference. In strain 20-21 and in the wild type, we never detected free Dlysergic acid. Obviously, the increased level of ergotamine in 10-29 is due to a mutation affecting the ergoline-ring synthesis. The fact that cells preincubated for 12 hours in PM were more active with respect to uptake of 14 C-Phe and incorporation into alkaloid than freshly harvested mycelium seems to be a consequence of N-starvation, in particular of some changes in the amino acid metabolism. The influence of various nitrogen-sources on ergotamine synthesis was therefore tested in a series of experiments. Synthesis was measured by incubation of mycelial suspensions in PM (ca. 80 mg/ml) in the presence of 1 jxCi 14 C-Phe for a period of 3 hours. When the mycelium was incubated for 12 hours in PM and in the presence of a nitrogen-source like NH4 , asparagine, methionine, tryptophan or glutamic acid, and then tested for its synthetic activity, all three strains 20-21, 10-29 and the wild type showed a significant reduction in the formation of radioactive ergot peptide when compared with controls incubated without a nitrogen-source (Table II). A possible explanation for this reduction is that addition of an N-source activated vegetative growth since the medium PM essentially differs from growth medium by lacking any N-source. Therefore, fresh mycelia of the two mutants 20-21 and 10-29 were incubated immediately after harvest for three hours with 1 (j.Ci 14 C-Phe in the presence of two different nitrogen sources at various concentrations (Figure 3). As can be seen also in these experiments, ergot peptide formation is drastically reduced by adding rather low concentrations of Asn (Cys or Glu not shown). NH4 in this case, had only a limited effect. Wild type freshly harvested could not be measured due to its low incorporation rate. Table II Formation of 14 C-ergotamine in mycelia of three different strains of C. purpurea preincubated in the presence of various nitrogen-sources.

N-source present during a 12 h preincubation of mycelia in PM

wild type ( + 5 mM D-lysergic acid)



ergotamine (cpm) tryptophan methionine NHi asparagine glutamate cysteine

(10 mM) (10 mM) (50 mM) (20 mM) (20 mM) (20 mM)

6500 2100 1900 620 1840 1700 -

11000 7100 7400 3000 -

52000 39500 35000 34600 28000 30000 24500


U. Keller, R. Zocher, G. Kraepelin


10 20 30 ¿0 50


10 20 30 ¿0 50

Concentration (mM)

Fig. 3 Influence of externally added amino acids (asparagine) and NH 4 C1 on in freshly harvested mycelia of strains 10-29 ( • ) and 20-21 ( ED ).


C-ergotamine formation

The same experiment was done with mycelia preincubated for 12 hours in medium PM (Figure 4). Also with these starved mycelia both nitrogen-sources reduced the incorporation of 14 C-Phe into alkaloid. Only in the high producer NH4 had a slightly stimulating effect. The uptake of 1 4 C-Phe was never observed to be affected by the presence of an amino acid or NH4 in all three strains. It is therefore assumed that external addition of a nitrogen-source activates primary metabolism at the expense of ergot peptide synthesis. The measured incorporation rates cannot be explained on the basis of a mere dilution of the radioactive precursor. These results are an additional support of our recent findings that ergot peptide synthesis is directly fed and eventually regulated by the intracellular amino acid pool. The slight stimulatory effect of NH4 in strain 10-29 is not yet clear. However, we cannot exclude that NH4 plays an additional role in the regulation of N-metabolism. If one compares the behaviour of the three strains wild type, 20-21 and 10-29, it turns out that starved wild type resembles fresh 20-21 and starved 20-21 resembles fresh 1029. This is deduced from the lysergic acid effect and also from the increased sus-




E 60n 50a) 0 400

0 30u CD CL


20 10 0

0 10 20 30 40 50 10 20 30 ¿0 50 Concentration (mM)

Fig. 4 Influence of externally added amino acid (asparagine) and NH 4 C1 on I 4 C-ergotamine formation in mycelia preincubated 12 h in medium PM. 10-29 ( • ); 20-21 ( E3 ); parent wild type ( • ).

Current Research in Ergot Peptide Synthesis by Claviceps purpurea


ceptibility to N-sources. Besides this finding, it may be noted that the wild type forms a mycelium of the sphacelial type while that of strain 10-29 has strongly scleratial characteristics. The increased level of ergotamine production of the latter strain agrees with the observation of Spalla's group and also that of Pazoutava et al. correlating ergot peptide synthesis with a sclerotial phenotype of cultures (13, 14). Also the strong vacuolization and lipid content of cells actively synthesizing ergot peptides indicates a high metabolic turnover of endogeneous N-reserves, which reacts very sensitively upon addition of external N-sources. Therefore, further studies of vacuoles from C. purpurea should provide more information about ergot peptide synthesis. As described previously, we have succeeded in developing a procedure to isolate vacuoles from protoplasts of the wild type strain of C. purpurea (9). This procedure involves lysis of protoplasts with dilute buffer in the presence of Triton X-100 with subsequent stabilisation by Ficoll and centrifugation in Ficoll step gradients. Usually vacuole preparations were contaminated with varying portions of lipid bodies. Despite this difficulty, determinations of the relative composition of the amino acid pool in the vacuole fraction were possible, and are shown in Table III. These preliminary data clearly indicate that the vacuolar amino acid pool differs from that of the whole protoplast. Determination of absolute concentration of single amino acids in the vacuoles was not possible because of the lack of a vacuolar marker. Therefore attempts are currently being made to meet this difficulty. Table III PM.*)

Relative amino acid pool of vacuoles and whole protoplasts of wild type after 12 h incubation in

amino acid alanine glycine proline glutamate aspartate lysine arginine histidine

protoplasts (moles/mole Ala) 1 0.104 0.175 0.422 0.12 0.64 0.103 0.046

vacuoles (moles/mole Ala) 1 0.20 0.286 0.667 0.095 0.721 0.325 0.121

*) Protoplasts were prepared during a 12 h period in medium PM by action of /(-glucuronidase. From these protoplasts, vacuoles were prepared and subjected to analysis.

References 1. Groger, D.: Planta medica 28, 37 (1975). 2. Floss, H . G . : Tetrahedron 32, 873 (1976). 3. Belzecki, C.M., Quigley, R.F., Floss, H.G., Crespi-Perellino, N., Guicciardi, A.: J. Org. Chem. 45, 2215 (i980). 4. Heinstein, P.F., Lee, S.-L., Floss, H . G . : BBRC 44, 1244 (1977).


U. Keller, R. Zocher, G. Kraepelin

5. Keller, U., Zocher, R., Kleinkauf, H.: J. Gen. Microbiol. 118, 485 (1980). 6. Amici, A.M., Minghetti, A., Scotti, T., Spalla, C., Tognoli, L.: Appi. Microbiol. 15, 597 (1967). 7. Amici, A. M., Minghetti, A., Scotti, T., Spalla, C., Tognoli, L. : Experientia 22, 415. 8. Amici, A.M., Minghetti, A., Scotti, T., Spalla, C., Tognoli, L.: Appi. Microbiol. 18, 464 (1969). 9. Keller, U., Zocher, R., Kleinkauf, H.: In "Abstracts of the VIth International Fermentation Symposium", London (Canada) 1980, p. 25. 10. Hofmann, A.: Die Mutterkornalkaloide, Verlag Enke, Stuttgart 1964, p. 116. 11. Rucman, R.: J. Chromât. 121, 353 (1976). 12. Stoll, A., Hofmann, A., Becker, B.: Helv. Chim. Acta 26, 1602 (1943). 13. Spalla, C., Marnati, M.P.: In "Antibiotics and other Secondary Metabolites", Hùtter, R., Leisinger, T., Nûesch, J., Wehrli, W., Ed., Academic Press, London, 1978, p. 218. 14. Pazôutovâ, S., Rehâcèk, Z., Vorisëk, J.: Can. J. Microbiol. 26, 363 (1980).

Applications of Multienzyme Systems in the Production of Peptide Antibiotics H. v. Döhren Institut für Biochemie und Molekulare Biologie Technische Universität Berlin Franklinstrasse 29, D-1000 Berlin 10, F . R . of Germany

I. Chemistry of biosynthesis 1.1. C a r b o x y l activation (1) The large number of bioactive polypeptides and polyketides are derived by polymerization of organic acids. Most of the known enzymatic reactions are pratically irreversible (2). Several general observations concerning the biochemical activation of carboxylic groups can be made. Simple carboxylic acids are generally found as CoAderivatives (Fig. 1), and are accepted by synthetases in this activated state (e.g. fatty acid synthetase, 6-methyl-salicylic acid synthetase, acylation reactions). On the other hand, we find amino acids and oc-hydroxy acids not as CoA-derivatives but in an activated state bound to a macromolecule like t R N A or an enzyme. Polymerization proceeds either on enzyme surfaces or mRNA-directed on the ribosome. Although these remarkable differences can be seen, the primary activation reaction of carboxyl is the cleavage of the c/.-ß or ß-y bond of nucleoside triphosphates. By this reaction, enzyme-attached anhydrides of phosphoric or adenylic acid are formed (Fig. 2). These

uh2 JL



0© Fig. 1

Structures of CoA and 4'-phosphopanteteine

0) Fig. 2

Structures of aminoacyladenylate (1) and aminoacylphosphate (2)

Peptide Antibiotics © 1982 Walter de Gruyter & Co., Berlin • New York



are then generally transferred by enzymic catalysis to the thiol or hydroxyl acceptor groups. An especially effective intramolecular transport system has been found in certain multienzymes containing 4'phosphopantetheine. This enzyme-bound cofactor is involved in the transport of intermediate substrates (carboxyl compounds and peptides).

1.2. Nonribosomal and ribosomal mechanisms Each step in polymerization reactions consists of the addition of one unit to the substrate to be elongated. Nearly all known peptide elongation reactions proceed by "head growth" (3), which means that the activated head (peptide carboxyl) will react with the acceptor amino acid (Fig. 3). The peptide or starter acid is thus called donor, R l X^ H H T' I I 0 - C I- * — II N - CI •- c

Donor Fig. 3



Definitions of d o n o r and acceptor compounds in peptide bond formation (11)

while the added substrate is called acceptor. In enzymatic peptide formation, the activated amino acids are acceptors, while the peptide to be elongated and transported is the donor compound. In ribosomal polypeptide formation, aminoacyl-tRNAs are acceptor compounds, while peptidyl-tRNA is the donor. In amino acid acylation reaction, the acyl-CoA compound is donor, while an aminoacyl-tRNA may also be donor in cell wall interpeptide bridge formation, with a peptide functioning as acceptor. Although the exact mechanisms of catalysis of polyketide and polypeptide formation are not known, many similarities are expected. This has been illustrated by the tentative mechanism proposed by Kleinkauf and von Dohren (4).

1.3. Chemical applications Both mixed anhydrides of phosphoric acids and reactive thiol esters have been applied in organic synthesis of peptides. If we again consider the reaction sequence of amino acid activation in multienzyme systems, we differentiate 3 steps: aminoacyl adenylate foration, thiolaminoacylation and peptide bond formation (Scheme 1).

RCO^H Scheme 1

,pf/OK RCO2P IpOR'

2. RJ-SH




Application of Multienzyme Systems in the Production of Peptide Antibiotics


The first two reactions have been realized in laboratory conditions in solution in the preparation of thiol esters using diethyl phosphorocyanidate (I) or diphenyl phosphorazidate (II). The realization of step 3 O NC-P-OEt Scheme II







has been achieved by coupling of alkylthiol esters with amino acid derivates in the presence of pivalic acid or 2-hydroxypyridin (5). Thiol esters serve in this case as both reactive and protective agents. The reaction proceeds without racemisation. Other principles of enzymatic biosynthesis of peptides have so far not been applied. It has not been checked if in cyclic peptide synthesis a biomimetic route of cyclization is advantageous. Gramicidin S e.g. is formed enzymatically from the fragments D-PhePro-Val-Orn-Leu-X by head-to-tail dimerization. Synthetic routes proceed from different or protected pentapeptides (6). The principle of noncovalent protection can be found in enzyme catalyzed reactions. Reactive groups like the ¿ - N H 2 group of ornithine are protected by enzyme structure during elongation reactions against side reactions. Peptide biosynthesis on multienzymes represents in principle a reverse solid phase synthesis, where the growing peptide chain is transported to the amino acid attached to the polymer. Then the peptide formed is again polymer-attached, and being in reactive form may be transferred. The exclusive reaction of peptide with the activated amino acid remains to be clarified in order to make use of this mechanistic principle.

2. Peptide synthetases 2.1. Definitions [7] To avoid confusion, some general definitions concerning multienzymes will be given in Fig. 4. We start by numbering individual catalytic functions of the enzymes involved; covalent connections between enzymes or supports are indicated by a solid line. Noncovalent interactions (stable complexes) are indicated by dotted lines. Any complicated enzyme structure with more than one catalytic function could be termed multifunctional enzyme. We further differentiate between stable complexes of subunits, and multifunctional enzymes, as well as multifunctional subunits which are composed of single polypeptide chains. For this last covalent connection, the term "complex" should not be used. In addition, we propose the term multienzyme system for interacting multienzymes.

2.2. The thiotemplate mechanism [8,9] The aminoacylation of enzyme thiol groups with subsequent peptide transport and elongation by 4'phosphopantetheine has been termed the "thiotemplate" mechanism.


H. v. Dòhren

© enzyme

© enzyme








Fig. 4 Terminology of multienzymes. All the structures presented have been termed multienzyme complexes. Here a different terminology is proposed. Each catalytic function is numbered and called enzyme. If several different enzymes are co-immobilized this should be termed enzyme system. Several functions linked physically or chemically can be called multifunctional proteins. We differentiate single subunit complex of associated enzymes as enzyme complex, covalently linked enzymes with no subunits (multienzymes) which may consist of a single polypeptide chain, and complexes of multienzymes, which may also contain enzymes. Interacting enzymes with no stable complex formation are systems of multienzymes or enzymes.

This term indicates that the enzymic template resembles an arrangement of active thiol groups. Already in 1954, Lipmann had proposed the existence of polyenzymes representing linear arrangements of activated amino acids. The enzymes responsible for biosynthesis of the peptide antibiotics gramicidin S, tyrocidine and linear gramicidin, indeed show similarities with this model. They are multienzymes or polyenzymes, which carry activated amino acids in a specific linear sequence, although the protein structure has a three-dimensional set up. For functioning of this polyenzyme model, a peptide carrier protein is required, as is illustrated in Fig. 5. A model put forward by Laland et al. illustrates very well the reaction sequence (Fig. 6). However, this model does not correlate enzyme and substrate dimensions, and does not explain the selection of peptide elongation steps. Since the size of gramicidin S synthetase has been determined with the electron microscope, a refined model has been proposed [10] (Fig. 7). In this model the sequence control is thought to originate from recognition of the intermediate peptide before elongation. Each activating enzyme could be considered a specific peptide synthetase.

Application of Multienzyme Systems in the Production of Peptide Antibiotics


Fig. 5 Extension of the polyenzyme model by introduction of a transport function. Top, transthiolation reaction, bottom, transpeptidation 3 (33)

2.3. Other mechanisms [9, 11] Different mechanisms have been elucidated for the biosynthesis of compounds like glutathion, pantetheine (CoA), and peptidoglycan. No covalent attachment of intermediates is observed, and free intermediates can be incorporated into the final product. Generally, peptide carboxyls are activated, quite often with y-carboxyls of terminal glutamic acid. These systems often permit single step additions to peptides.

3. Examples of applications 3.1. In vivo systems Generally, the majority of active peptides are still isolated from stationary microbial cells. Limited manipulations of some in vivo systems are summarized in Table I. Some new peptides may be produced by incorporation of specified precursors (directed biosynthesis). Transport of precursors and reaction rates can be influenced by cell immobilisation {Bacillus licheniformis), protoplast formation (Streptomyces antibioticus), or the use of organelles (mitochondria of rat liver).


H. v. Dôhren

Fig. 6

Schematic representation of gramicidin S formation by gramicidin S synthetase (8)

Application of Multienzyme Systems in the Production of Peptide Antibiotics


A M P / A T P b i n d i n g site ^

adenylate formation J

Pro b i n d i n g • Pro-thiolester

4'-phosphopantetheine dipeptide binding site

Orn-' thiolester

I »

tripeptide b i n d i n g site

Fig. 7 Hypothetical scheme of transpeptidation and transthiolation of activated peptides on gramicidin S-synthetase. Dimensions as deduced by electron microscopy have been tentatively correlated with dimensions of substrates and cofactor. Intermediates are thought to be bound on each amino acid activating functional unit before peptidation occurs.

We would like to point out some recent observations on peptide production. In a study of oxygen-enriched aeration in batch fermentation, Flickinger and Perman [12] increased bacitracin production rate and yield several fold, while growth rate and carbohydrate uptake was decreased. The authors suggested that the conditions simulated a carbon limitation. By immobilizing producing cells of B. licheniformis with polyacrylamide, Suzuki et al. [13,14] obtained a system operating in 0.5% peptone solution with a half-life of 10 days at 30 °C. The productivity was superior to the conventional continuous fermentation process at comparable dilution rates. In gramicidin S production, Vandamme [15] induced product formation in growing cells and increased the yield under controlled p H conditions.



Table I

Directed biosynthesis of peptides. Preparation of peptide analogs by in vivo substitution (9)


replaced amino acid







Trichoderma viride

Kirby and Robins 1976

penicillin N



Acremonium chrysogenum (Lys auxotroph)

Troonen et al. 1976

etamycin A




griseoviridus Chopra et al. 1979

neoviridogrisein I Allo-hydroxyD-Pro



griseoviridus Okumura 1979



azetedine-2caboxylic acid sarcosine pipecolic acid





p-Cl-Phe p-F-Phe

Claviceps purpurea strains Beacco et al. 1978


Val, Ile, Leu

nor Val


Val, Ile, Leu

nor Leu Abu 5,5,5,-F-Leu /?-OH-Leu

Formica and Apple 1976

If appears that generally important improvements in production of peptides and peptide synthetase can be made by further analysis and understanding of biosynthetic processes.

3.2. Cell-free biosynthesis 3.2.1. Gramicidin S and peptide


Considerable effort has been spent at the M . I . T Cambridge and the T. U. Berlin to utilize this gramicidin S enzyme system of 2 multienzymes (280.000 and 100.000 daltons). Procedures have been designed for high production of enzymes in batch fermentation [16, 17] and by continuous fermentation. Aust et al. [18] have shown that enzyme yield is increased by maintaining a minimal dissolved oxygen level by oxygenenriched aeration in minimal as well as complex media. The beginning of the enzyme production phase is characterized by a significant rise in dissolved oxygen concentration corresponding to a decline in respiratory oxygen consumption. As has been shown for the peptide [19], enzyme formation is also under phosphate control [20], High phosphate concentrations decrease the enzyme level several fold. Both multienzymes are soluble, but they could also be isolated in a membrane-bound form [15]. Peptide synthesis proceeds from the constituent amino acids and M g A T P 2 (Scheme 3). Crude enzyme preparations obtained by ammonium sulfate precipitation or polyethylene glycol/dextran phase extraction have been used in batch and hollow fiber reactors [21]. Two main problems have been studied: A T P utilization and enzyme stability.

Application of Multienzyme Systems in the Production of Peptide Antibiotics

2 2 2 2 2

Phe Pro Val + 10 M A T P 2 " Orn Leu


cyclo (DPhe-Pro-Val-Orn-Leu) 2

(M = Mg, Mn or Ca) Scheme 3: Gramicidin S formation

In batch reactors the nonproductive degradation of ATP has been observed [22]. No specific inhibitors of this side reaction have been found. Partial purification, and control of M g 2 + and M g A T P 2 - concentrations, however, have been found to achieve an efficient ATP utilization [23], The regeneration of ATP [24] during synthesis has been demonstrated with acetyl phosphate, immobilized acetate kinase and the endogenous adenylate kinase contained in the enzyme preparation [21]. The efficiency of regeneration and ATP utilization in these experiments was low. In an economic analysis of enzymatic gramicidin S production [22], the importance of enzyme stability has been emphasized. Half-lives of enzyme preparations are in the range of 1 h at 37 °C in the absence of substrates. So far it has been possible to improve the stability to a half-life of 20 hours by the addition of polyethyleneglycol and EDTA. In a study of inactivation mechanisms [25], it has been concluded that in vitro inactivation probably results from the action of hydrolytic enzymes such as proteinases or 4'phosphopantetheine hydrolase, rather than thermal inactivation. Inactivation kinetics of crude and purified enzyme preparations have been evaluated by Arrhenius plots (Fig. 8). While crude preparations are inactivated in first order reactions in a process with an activation energy of 12 kcal/mol, the purified synthetase shows two



* 103

Fig. 8 Estimation of Arrhenius energies of inactivation processes of gramicidin S-synthetase (GS 2). While in crude extracts a fast process with an E A of 12 Kcal/mol (top) operates, a purified enzyme displays a two-phase curve. In the high temperature region ( > 42 °C) fast thermal inactivation is dominating (E A 150 Kcal/mol), while the low region slope suggests hydrolytic processes (E A 12 Kcal/mol). The denaturation or déstabilisation constant K d is defined by K d = In 2/t 1 / 2 , with t 1 / 2 = half-life.



such processes with 150 kcal/mol and 12 kcal/mol, respectively. In the high temperature region ( > 42 °C) thermal inactivation is apparently predominant (high activation energy). Half-lives dependent on thermal stability of 130 h at 37 °C and approximately one year at 30°C can be predicted. In a search for stabilizers, their use has been shown to be dependent on enzyme purity. EDTA (5 mM) may be effective in stabilizing a purified enzyme, but destabilized a crude preparation. Most efficient has been the addition of the relevant substrate amino acids and ATP. No activity loss has been detected within 5 hours at 37 °C. Currently inactivation processes are studied with reference to these conditions. Some evidence has been collected for a new type of product inhibition, the gramicidin S induced destabilization of its synthetase [21]. Although these results seem to be related to crude preparations, the varying yields in synthesis of analogs of gramicidin S remain to be explained. Replacing Phe by Tyr decreases the initial rate to 40%,'and product accumulation stops at 30% of the concentration obtained for gramicidin S. Replacement of Val by Leu [26] proceeds with a high initial rate (86%), but only 5% of product is accumulated. A possible interpretation is a product-specific inhibition of enzymes. To investigate the capacity of gramicidin S-synthetase, a number of amino acid analogs have been incorporated into the decapeptide as well as cyclodipeptide, which is formed in the absence of the third amino acid, or by enzyme fragments defective in this function. The synthesis of various peptide analogs seems to be the most interesting application of the enzyme systems (Table II). Initial rates of formation of substituted Table II

Synthesis of cyclodipeptides by gramicidin S-synthetase

amino acids used

rate of formation C-AB





phenylalanine phenylalanine phenylalanine phenylalanine phenylalanine phenylalanine

3,4-dehydroproline azetidin-2-carboxylic acid hydroxyproline y-thioproline allo-4-hydroxyproline sarcosine

/?-phenyl-/?-alanine 5 -methy 1-tryptophan 6-methyl-tryptophan 7-methyl-tryptophan tryptophan D-tyrosine tyrosine DL-o-tyrosine DL-m-tyrosine O-methyl-tyrosine /?-2-thienyl-DL-alanine

proline proline proline proline proline proline proline proline proline proline proline

* All rates are compared to the rate of gramicidin S formation


rate of formation of Gramicidin S-analogue Peptide C-(A-B-Val-Orn-Leu) 2 100*

91 73 1 179 8.4 22

85 58 15 12.5 10.5 0.7

2 2 2 1 16 7 7 4 5 32 21

2 1 «



T C £

/ /


C / S I



1 0

0-2.5 mM A






1 V






[VAL(mM)]- 1

1 5

0-53 mM


1-25 mM



[ M g A T P 2 - (mM)]~ 1

Fig. 5 Evaluation of substrate binding pattern and dissociation constants of valine and ATP by the papain derived valine fragment GS 2 P1. Intersection points left to the ordinate point to a random binding of both substrates. K d of valine and M g A T P 2 " are 0.45 mM and 0.60 m M respectively, compared to 0.4 m M and 1.25 m M in active GS2. Reaction conditions were 20 m M sodium phosphate, pH7.2, 0.5 m M sodium pyrophosphate, 10 m M MgCl 2 ,10 m M DTE, and 1 m M EDTA, at the indicated concentrations of ATP and valine.


M. Altmann, H.V.Döhren, A. El-Samaraie, R. Kittelberger, M . P o r e , H. Kleinkauf

[ LEU (mM)]"1

[MgATpa- (mW)]"1

Fig. 6 Evaluation of substrate binding pattern and dissociation constants of leucine and A T P by the papain derived leucine fragment G S 2 P 2. Conditions and conclusions are as in Fig. 5. The observed dissociation constants are 1.5 m M and 1 . 4 m M for levcine and M g A T P 2 ~ respectively, compared to 0.3 and l . l O m M for G S 2 .










60 mm.

j® jpP g









10» t v






Fig. 7 Cleavage of the multienzyme G S 2 by papain treatment, as followed by S D S - P A G E . Incubation has been carried out with 3% protease (papain/GS 2) at 30°C in phosphate buffer p H 7.2. At the indicated times, samples have been withdrawn, treated with iodoacetamide and heated with 1% SDS to 95 °C for 2 minutes. Electrophoresis was performed according to Laemmli in a 7% gel, but without stacking gel. In a preliminary interpretation of the cleavage pattern G S 2 (1) is split into (2) and possibly the small fragment (3), or the fragments (4), (6) and (5), (7), respectively. Then (2) is cleaved via (4) and (5), and fragments (4) and (5) are split. More detailed study of the fragments (8)—(12) is needed however.

Limited Proteolysis: Studies on the Multienzyme G S 2 of Gramicidin S-Synthetase


3.2. Trypsin cleavage Trypsin degradation of GS 2 can be effectively prevented by trypsin inhibitor (Fig. 8). The inactivation kinetics again show the ornithine site to be most sensitive. To demonstrate the active site directed cleavage, substrate protection with amino acids has been studies. In the case of valine and ornithine, complete protection is observed. It is obvious that the loss of biosynthesis reaction catalyzed with the complementary multienzyme GS 1 is most rapidly lost. Half lives have been estimated to 5 and 1 minute at 0 or 13 °C respectively. This loss of activity cannot be correlated with a loss of activation functions. In such a case each multienzyme having lost a single function is inactive. The addition of all activation functions lost cannot account for the loss of biosynthesis activity. This is supported by the finding that there is no substrate protection by M g A T P 2 - or the combined amino acids of this overall reaction. This site of trypsin action remains to be established. To detect conformational changes of GS 2 with temperature the kinetics of inactivation have been followed at 0, 13, 27.5 and 37 °C. A summary of estimated half lives is given in table III. No loss of leucine activation is detected at 0°C, while inactivation occurs at 13 °C. A further investigation of this finding is the temperature dependence of

Fig. 8 Protection of G S 2 from trypsin degradation. A : Rapid loss of biosynthesis activity (circles) is protected completely by a 5 fold excess of trypsin inhibitor (square). B-E: Loss of amino acid dependent ATP-PP, exchange (open circles) is partially protected by addition of 2.5 m M of the corresponding amino acid. All preincubations have been carried out at 37°C with 1% trypsin, the reactions have been terminated with trypsin inhibitor. Activities have been plotted on a relative log scale.

Table III Action of trypsin on activation reactions catalysed by G S 2 - temperature dependence of half lives (T 1 / 2 ) or reactions

Amino acid activated Pro Val Orn Leu * no loss detected

0 420 1520 340 N.D.*

Temperature 27.5 13 37 130 95 50

6 15 7 22

37°C 6 18 2.1 11


M.Altmann, H.V.Döhren, A.El-Samaraie, R.Kittelberger, M.Pore, H.Kleinkauf


3,5 T-1 C. in»")


Fig. 9 Leucine activation by GS 2. Temperature dependence of the amino acid dependent ATP-PP, exchange reaction. The biphasic Arrhenius plot indicates two conformations of this active site. This is consistent with the trypsin resistance of this site at 0°C.

leucine dependent ATP-PP; exchange reaction (Fig. 9). There is indeed a biphasic Arrhenius plot indicating two different conformations of this site. Evaluation of inactivation constants in Arrhenius plots indicate no trypsin detectable change of other activation sites between 0 and 27.5 °C, although this could be expected from temperature dependence studies (3). A cleavage pattern of the unprotected multienzyme is shown in Fig. 10.

4. Fragment analysis Generally the analysis of a nicked polypeptide may be complicated by association of fragments requiring chemical modification or detergents for dissociation. In such a case the correlation of a residual activity with a fragment will be difficult. For the multienzyme GS 2 this has not been observed. Fragments have been separated on DEAE-cellulose and characterized by correlation of activity and protein species detected by SDS-polyacrylamide gel electrophoresis (4). Since each cleavage leads to two fragments detectable under denaturing conditions, the maximum number of fragments is produced when all sensitive bonds are hydrolysed with equal velocity. The number of fragments (F) can then be calculated from the number of cuts (n) by : F = n+ t