Handbook of Industrial Mycology

MYCOLOGY SERIES

Editor

J. W. Bennett
Professor Department of Cell and Molecular Biology Tulane University New Orleans, Louisiana
Founding Editor

Paul A. Lemke

1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke 2. The Fungal Community: Its Organization and Role in the Ecosystem, edited by Donald T. Wicklow and George C. Carroll 3. Fungi Pathogenic for Humans and Animals (in three parts), edited by Dexter H. Howard 4. Fungal Differentiation: A Contemporary Synthesis, edited by John E. Smith 5. Secondary Metabolism and Differentiation in Fungi, edited by Joan W. Bennett and Alex Ciegler 6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy 7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin and Michael J. Leibowitz 8. Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, edited by Sally A. Leong and Randy M. Berka 9. The Fungal Community: Its Organization and Role in the Ecosystem, Second Edition, edited by George C. Carroll and Donald T. Wicklow 10. Stress Tolerance of Fungi, edited by D. H. Jennings 11. Metal Ions in Fungi, edited by Günther Winkelmann and Dennis R. Winge 12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas O. Mountfort and Colin G. Orpin 13. Fungal Genetics: Principles and Practice, edited by Cees J. Bos

14. Fungal Pathogenesis: Principles and Clinical Applications, edited by Richard A. Calderone and Ronald L. Cihlar 15. Molecular Biology of Fungal Development, edited by Heinz D. Osiewacz 16. Pathogenic Fungi in Humans and Animals: Second Edition, edited by Dexter H. Howard 17. Fungi in Ecosystem Processes, John Dighton 18. Genomics of Plants and Fungi, edited by Rolf A. Prade and Hans J. Bohnert 19. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts, edited by James F. White Jr., Charles W. Bacon, Nigel L. Hywel-Jones, and Joseph W. Spatafora 20. Handbook of Fungal Biotechnology, Second Edition, edited by Dilip K. Arora 21. Fungal Biotechnology in Agricultural, Food, and Environmental Applications, edited by Dilip K. Arora

Additional Volumes in Preparation

Handbook of Industrial Mycology

edited by

Zhiqiang An

Marcel Dekker

New York

Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recommendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-5655-X This book is printed on acid-free paper. Headquarters Marcel Dekker 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 Distribution and Customer Service Marcel Dekker Cimarron Road, Monticello, New York 12701, U.S.A. tel: 800-228-1160; fax: 845-796-1772 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright  2005 by Marcel Dekker. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Foreword

The economic importance of fungi is almost impossible to exaggerate. Not only are they integral to the great ecological cycles of the natural world, and not only do their mycorrhizal associations provide the foundation for agriculture and forestry, they are also essential to biotechnology. In fact, modern biotechnology has its roots in fungal fermentations. Yeasts have been used since antiquity in brewing and baking, while molds have been used to improve the flavor of cheese and in the production of Asian condiments such as soy sauce and miso. During the twentieth century, industrial fermentations were developed for production of organic acids, diastatic enzymes, plant growth hormones, natural and semisynthetic antibiotics, antitumor agents, and immunomodulators. The commercial development of penicillin, the first and most important of the clinically useful antibiotics, laid the groundwork for much of what has come after, especially the technology for growing filamentous fungi in submerged culture. With new discoveries, it has become apparent that the potential applications of fungal metabolism are greater than we once suspected. The original intent of the Marcel Dekker mycology series was to establish ‘‘a niche for publication of work dealing with all aspects of mycology.’’ A volume on Industrial Mycology is long overdue. Biotechnologists have made great progress in discovering how to manipulate yeasts and molds at the biochemical and molecular level. They have exploited two major physiological characteristics of fungi: their ability to degrade simple and complex polymers and their ability to biosynthesize a huge variety of complex secondary metabolites. Fungi can be engineered to produce many heterologous proteins and they remain an important source of novel drugs, including anticancer drugs and immune function regulators. Several species (e.g., Saccharomyces cerevisiae, Aspergillus nidulans and Neurospora crassa) are superb models. This new volume covers both theoretical and practical aspects of industrial mycology with emphasis on the dramatic advances in the application of bioinformatics, molecular biology, ‘‘reverse genetics,’’ combinatorial chemistry, metabolic engineering, genomics, proteomics, and cognate fields to the manipulation of fungi. Collectively, these techniques have altered what is possible in the discovery laboratory. Divided into eight sections and 26 chapters, Industrial Mycology is a comprehensive guide to the new mycology of the 21st century. It describes how fungi contribute directly
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to the contemporary pharmaceutical industry, and how they can be manipulated in order to contribute more in the future. Dr. Zhiqiang An, editor, has done an excellent job of bridging the traditional gulf between academic and industrial studies of fungi. In addition, he has brought together a stellar group of international experts to write the chapters. As a result of his efforts, Industrial Mycology presents up-to-date information on more than two dozen separate topics, thereby providing an invaluable reference for anyone involved in applied mycology. This book will guide researchers so that they can apply new scientific approaches for using fungi to solve many real world problems. It is intended to inspire as well as to inform. The pharmacological potential of fungi is immense and awaits further exploitation. J. W. Bennett Department of Cell and Molecular Biology Tulane University New Orleans, Louisiana, U.S.A.

Preface

Fungi are nutrition-absorptive eukaryotic organisms found in every ecological niche. Because of their simplistic and rapid life cycles, yet complex genetics, metabolism, and morphology, fungi have been studied in the laboratory as model systems (such as Saccharomyces cerevisiae, Aspergillus nidulans, and Neurospora crassa ) in every aspect of the biological sciences. The economic importance of fungi is equally impressive, with uses in the food and beverage industries, agriculture, the pharmaceutical and agrochemical industries, and medicine. The significance of fungi as both laboratory model organisms and industrial processes is reflected by the large number of specialized disciplines dedicated to the study of mycology, such as plant pathology, medical mycology, food mycology, environmental mycology, and industrial mycology. This book provides an overview of recent developments in industrial mycology, with emphasis on discovery of bioactive metabolites, biocatalysts, and the underlying genetics of fungi. Fungi are of interest to the pharmaceutical and agrochemical industries because of the diverse bioactive metabolites produced by these organisms. Since the treatment of bacterial infections using partially purified penicillin two-thirds of a century ago, bioactive fungal metabolites have strongly influenced the development of the modern pharmaceutical and agrochemical industries. Mevinolin, cyclosporin A, -lactam antibiotics, pneumocandins, ergotamine, strobilurins, and mycophenolic acid are examples of revolutionary pharmaceuticals and agrochemicals that have a fungal origin. Chapter 3 provides a comprehensive review of the bioactive fungal metabolites discovered to date. The isolation of sordarins is one example to illustrate the complexity of the bioactive metabolite discovery process (Chapter 11). In spite of the success of bioactive fungal metabolites as pharmaceuticals and agrochemicals, fungi remain an essentially untapped source of medicines because only a small fraction of the vast fungal kingdom has been explored for bioactive metabolite production. The potential for discovering new bioactive metabolites from fungi is unlimited. The industrial discovery of bioactive fungal metabolites is a complex, integrated, but somewhat empirical process. Major steps in this process include: 1) collection and identification of natural material (Chapter 6); 2) chemical extraction (Chapter 8); in vitro assay for biological activities (Chapter 10); 4) isolation and structural elucidation of the active components from the extracts (Chapter 8); 5) characterization of the in vivo biologiv

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cal efficacy and toxicity profiles; and 6) strain and fermentation improvement for higher production titer and reduction of undesirable side products (Chapters 19 and 20). Although one of most critical steps in fungal natural products drug discovery, in vivo biological efficacy and toxicity profiling of fungal metabolites is not addressed in this book because it is mostly a mammalian clinical issue, and not a mycological one, which is the focus of this book. Industrial fungal bioactive metabolite discovery has been largely an empirical process and this empirical approach has been and will continue to be effective. However, recent advances in the genetics of microbial secondary metabolite biosynthesis, genomics, and metabolic engineering will play an ever-increasing role in facilitating fungal bioactive metabolites discovery. This book attempts to provide readers with comprehensive discussions on the genetics of fungal secondary metabolism (Chapters 12B18). Another major advance in fungal bioactive metabolite discovery lies in detection and prediction methods, such as chemometric and genetic methods (Chapter 9). Genetic engineering will also help to expand the recovery of bioactive metabolites and enzymes from unculturable and difficult-to-grow fungi by expressing secondary metabolite-encoding and enzymeencoding genes and gene clusters in heterologous hosts (Chapter 7). To serve as a stand-alone reference book, overviews of industrial mycology (Chapter 1), the fungal kingdom (Chapter 2), fungal cell cycle control (Chapter 4), and fungal genetic transformation (Chapter 5) are included. Several chapters addressing other industrial mycology topics are also included: industrial fungal bioconversions (Chapter 21), metabolomics (Chapter 22), engineering of fungal metabolic biosynthetic pathways (Chapter 23), heterologous protein and enzyme expression in fungi (Chapter 24), mycotoxin (Chapter 25), and fungi as biocontrol agents (Chapter 26). Finally, I would like to express my gratitude to Dr. Joan Bennett for the opportunity to contribute to the Mycology series, and I am indebted to the expert authors who agreed to contribute to this endeavor. I also want to thank Ms. Anita Lekhwani and Joseph Cacciottoli of Marcel Dekker, Inc., for their dedicated assistance throughout the project. Zhiqiang An

Contents

Foreword Preface Contributors I. Mycology, Industrial Mycology, and Fungal Biology

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1. Industrial Mycology: Past, Present, and Future Arnold L. Demain, Javier Velasco, and Jose L. Adrio 2. Phylogeny of the Fungal Kingdom and Fungal-Like Eukaryotes Gerald Bills, Joseph W. Spatafora, and Meredith Blackwell 3. Biological Activities of Fungal Metabolites ´ Fernando Pelaez 4. Cell Cycle Regulation in Morphogenesis and Development of Fungi Xiang S. Ye 5. Agrobacterium-Mediated Transformation of Filamentous Fungi Jan S. Tkacz, Arlene M. Dahl-Roshak, Nageatte Ibrahim, and Philip S. Paress II. Fungal Secondary Metabolites Discovery

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6. Fungal Germplasm for Drug Discovery and Industrial Applications Toru Okuda, Katsuhiko Ando, and Gerald Bills 7. Expression of Cosmid-Size DNA of Slow-Growing Fungi in Aspergillus nidulans for Secondary Metabolite Screening Zhiqiang An, Guy H. Harris, Deborah Zink, Robert Giacobbe, Ping Lu, Rajinder Sangari, Gerald Bills, Vladimir Svetnik, Bert Gunter, Andy Liaw, Prakash S. Masurekar, Jerrold Liesch, Steven Gould, and William Strohl

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8. The Isolation and Structure Elucidation of Fungal Metabolites Guy H. Harris 9. PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers in High-Throughput Screening for Bioactive Compounds from Fungi Marc Stadler and Veronika Hellwig 10. Screening for Biological Activity in Fungal Extracts Marvin Schulman and Christine D. McCallum 11. Sordarins: Inhibitors of Fungal Elongation Factor-2 ´ Juan Manuel Domınguez and Julio J. Martin III. Biosynthesis of Fungal Secondary Metabolites

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12. Secondary Metabolite Gene Clusters Yongqiang Zhang, Heather Wilkinson, Nancy Keller, and Dimitrios Tsitsigiannis 13. Genomics and Gene Regulation of the Aflatoxin Biosynthetic Pathway Michael S. Price and Gary A. Payne 14. Indole-Diterpene Biosynthesis in Ascomycetous Fungi Emily J. Parker and D. Barry Scott 15. Loline and Ergot Alkaloids in Grass Endophytes Christopher L. Schardl, Jimmy D. Blankenship, Martin J. Spiering, and Caroline Machado 16. Biosynthesis of N-Methylated Peptides in Fungi Till Hornbogen and Rainier Zocher 17. Molecular Genetics of Lovastatin and Compactin Biosynthesis C. Richard Hutchinson, Jonathan Kennedy, Cheonseok Park, Karine Auclair, Steven G. Kendrew, and John Vederas 18. The Aspergillus nidulans Polyketide Synthase WA Isao Fujii, Akira Watanabe, and Yutaka Ebizuka IV. Fermentation, Strain Improvement, and Bioconversion

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19. Pneumocandin B0 Production by Fermentation of the Fungus Glarea Iozoyensis: Physiological and Engineering Factors Affecting Titer and Structural Analogue Formation Neal Connors and David Pollard

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20. Strain Improvement for the Production of Fungal Secondary Metabolites Prakash S. Masurekar 21. Fungal Bioconversions: Applications to the Manufacture of Pharmaceuticals Michel Chartrain and Michel Sturr V. Metabolic Engineering

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22. Metabolomics Jonathan Arnold, H.-B. Schuttler, D. Logan, D. Battogtokh, James ¨ Griffith, B. Arpinar, S. Bhandarkar, S. Datta, K. J. Kochut, E. Kraemer, J. A. Miller, A. Sheth, G. Strobel, T. Taha, B. Aleman-Meza, Jereme Doss, LaTreace Harris, and Abassi Nyong 23. Metabolic Engineering of Fungal Secondary Metabolite Pathways Guang Yi Wang, R. David Laidlaw, Jessica H. Marshall, and Jay D. Keasling VI. Heterologous Protein and Enzyme Expression in Fungi

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24. Heterologous Protein Expression in Yeasts and Filamentous Fungi Ningyang Zhang, Dayna L. Daubaras, and Wen-Chen Suen VII. Mycotoxin

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25. Toxigenic Fungi and Mycotoxins Jeong-Ah Seo and Jae-Hyuk Yu VIII. Fungi in Biological Control

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26. Fungal Secondary Metabolites in Biological Control of Crop Pests Xingzhong Liu and Shidong Li Index

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Contributors

Jose L. Adrio Puleva Biotech, Granada, Spain B. Aleman-Meza University of Georgia, Athens, Georgia, U.S.A. Zhiqiang An Merck Research Laboratories, Rahway, New Jersey Katsuhiko Ando Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., Tokyo, Japan Jonathan Arnold University of Georgia, Athens, Georgia, U.S.A. B. Arpinar University of Georgia, Athens, Georgia, U.S.A. Karine Auclair Department of Chemistry, McGill University, Montreal, Quebec, Canada D. Battogtokh University of Georgia, Athens, Georgia, U.S.A. S. Bhandarkar University of Georgia, Athens, Georgia, U.S.A. ´ ´ Gerald Bills Centro de Investigacion Basica, Merck Sharp & Dohme de Espana, S.A., ˜ Madrid, Spain Meredith Blackwell Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, U.S.A. Jimmy D. Blakenship Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, U.S.A.
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´ Spain Jereme Doss Clark Atlanta University. Rahway.S. New Jersey. New Jersey. Daubaras Schering-Plough Research Institute. New Jersey. New Jersey. Georgia.S. Tokyo.A. Rahway. Union. U.A. Juan Manul Domınguez GlaxoSmithKline Research and Development. U. U.S. Japan Isao Fujii Graduate School of Pharmaceutical Sciences. Yutaka Ebizuka Graduate School of Pharmaceutical Sciences. S. U. Dayna L. Veronika Hellwig PH-R Enabling Technologies Natural Product Research.S. U. Merck Research Laboratories. Germany Till Hornbogen Institut fur Chemie AG.). Merck Research Laboratories.S.S. Richard Hutchinson Kosan Biosciences. Neal Connors Department of Biocatalysis and Fermentation Development. U.A.A.S. S.A. . Bayer HealthCare AG. Japan Robert Giacobbe Merck Research Laboratories. U. Atlanta. U. Rahway.A. Atlanta. Georgia. Altanta. Drew University. The University of Tokyo.A. U.S.S.A. Georgia.S. Athens.xii Contributors Michel Chartrain Department of Bioprocess Research and Development. Rahway. James Griffith University of Georgia. Arlene M. U. New Jersey. Harris Merck Research Laboratories. U. U. U. Rahway.A. California.A. Inc. Tokyo. LaTreace Harris Clark Atlanta University. Georgia. New Jersey.I. Datta Georgia State University. Steven Gould Merck Research Laboratories. U. New Jersey.S. Hayward.. New Jersey. Berlin..A. Germany ¨ C. Demain Charles A. Biochemie und Molekulare Biologie.A.S. Dahl-Roshak Merck Research Laboratories.A.S. Madrid. Bert Gunter Merck Research Laboratories. Rahway. The University of Tokyo.A. New Jersey. Arnold L. Madison. Dana Research Institute (R. Rahway. Wuppertal. Tech¨ nische Universitat Berlin.S.A.S. Guy H.E.

Kentucky.A.A. Kochut University of Georgia. Spain ´ Prakash S.S. New Jersey.A. Caroline Machado Department of Plant Pathology. Logan Clark Atlanta University. Berkeley. Masurekar Merck Research Laboratories. Berkeley. Keasling Department of Chemical Engineering. U.S. U. New Jersey.Contributors xiii Nageatte Ibrahim Merck Research Laboratories. Chinese Academy of Sciences. Beijing. Wisconsin. David Laidlaw Department of Chemical Engineering.S. K. Athens. U. U. Institute of Microbiology.A.A. Beijing.S. Jay D.S. California. Marshall Department of Chemical Engineering. U.S.S.A. U.A. University of Wisconsin-Madison. Ping Lu Merck Research Laboratories. Xingzhong Liu Key Laboratory of Systematic Mycology and Lichenology.S. U. U.S. Shidong Li Key Laboratory of Biocontrol Resources and Utilization. Athens. Inc. U. Chinese Academy of Agricultural Sciences.. Georgia. Kendrew Biotica Technology. Atlanta. Julio J. New Jersey. University of California at Berkeley. People’s Republic of China Andy Liaw Merck Research Laboratories. Madrid.A. J. Rahway. R.S. U. New Jersey.A.A .A. Steven G.S. California. Rahway.A. Berkeley.A. U.S. New Jersey. S. University of California at Berkeley.A.. Martın GlaxoSmithKline Research and Development. Rahway. Rahway. United Kingdom Jonathan Kennedy Kosan Biosciences. Cambridge. Rahway. Rahway. Ltd. Christine McCallum Merck Research Laboratories. Biological Control Institute. California. Kraemer University of Georgia.. E. University of California at Berkeley.S.S. New Jersey. U. Nancy Keller Department of Plant Pathology. Jerrold Liesch Merck Research Laboratories. Georgia. University of Kentucky.A. Georgia. People’s Republic of China D. Jessica H. U. U. Hayward.A. Lexington. U. Madison.S. California.

U. U.S. and Molecular and Environmental Toxicology Center. Rahway.S. Athens.A. New Zealand Gary A. U. New Jersey.S. North Carolina. Christopher L. New Jersey. North Carolina State University. ˜ S. Madrid.A. Wisconsin. New Jersey.A. Athens. Spatafora Department of Botany and Plant Pathology. Georgia. Payne Department of Plant Pathology. U. Miller University of Georgia.S.A.A. South Korea Emily J. Oregon State University. A.A. University of Wisconsin. Kentucky. U. Athens.A. Marvin Schulman Merck Research Laboratories.A.S. Raleigh. Rahway. H. New Zealand Jeong-Ah Seo Department of Food Microbiology and Toxicology. A. Rahway.xiv Contributors J. North Carolina. Massey University.A. Barry Scott Institute of Fundamental Sciences and Institute of Molecular BioSciences. Toru Okuda Tamagawa University Research Institute. Georgia. .S. Abassi Nyong Clark Atlanta University. North Carolina.A.S. Joseph W.A.A.S. College of Sciences. Massey University. Spain David Pollard Department of Biocatalysis and Fermentation Development. Palmerston North. Georgia. U. Japan Philip S. Rahway.S.S. Price Department of Plant Pathology. Oregon. Schuttler University of Georgia. U. Palmerston North. Food Research Institute. U. Schardl Department of Plant Pathology. Corvallis. ´ ´ ´ Fernando Pelaez Centro de Investigacion Basica. Tokyo. Yongin.. U. Atlanta.A.S. Merck Sharp & Dohme de Espana. U. College of Sciences.S. Raleigh. Parker Institute of Fundamental Sciences and Institute of Molecular BioSciences. Georgia. New Jersey. Cheonseok Park Department of Food Science and Technology. Kyunghee University. Lexington. Michael S. State University. U. Paress Merck Research Laboratories. U. Madison. U. ¨ D. Sheth University of Georgia. Merck Research Laboratories.A. Rajinder Sangari Merck Research Laboratories.S.-B. University of Kentucky.

U. U.S. Bayer HealthCare AG. New Jersey. California. Wisconsin. Georgia.S.S. Edmonton. New Jersey.A. College Station. Alberta. Indiana. Rahway. U. Xiang S. Rahway. Canada Javier Velasco Puleva Biotech.Contributors xv Martin J.A. U. U. U.A.S.A. William Strohl Merck Research Laboratories.A.S. U. Georgia. University of Kentucky. T. Germany G. Lilly Research Laboratories. New Jersey. University of California at Berkeley. Granada.S. University of Wisconsin. U. Athens. New Jersey. Lexington.A. U. Spain Guang Yi Wang Department of Chemical Engineering. Ye Cancer Research.A. Wuppertal. University of Wisconsin.A. Food Research Institute and Molecular and Environmental Toxicology Center. Texas A&M University. Madison. Madison. Union. John Vederas Chemistry Department. Rahway.A. Michael Sturr Department of Bioprocess Research and Development. Eli Lilly and Company. Kentucky. University of Alberta. Strobel University of Georgia. Athens. . Jan Tkacz Merck Research Laboratories. Indianapolis.S.S.S. Japan Heather Wilkinson Department of Plant Pathology.A. Wen-Chen Suen Schering-Plough Research Institute. U.A. U.A.S. The University of Tokyo.S. New Jersey. Vladimir Svetnik Merck Research Laboratories. Merck Research Laboratories. Texas. Marc Stadler PH-R Enabling Technologies Natural Product Research. U. Berkeley. Rahway.S.A.S. Dimitrios Tsitsigiannis Department of Plant Pathology. U. Wisconsin. Tokyo. Taha University of Georgia. Akira Watanabe Graduate School of Pharmaceutical Sciences. Jae-Hyuk Yu Department of Food Microbiology and Toxicology. Spiering Department of Plant Pathology.

U. Rainier Zocher Institut fur Chemie AG. Wisconsin. University of Wisconsin-Madison. U. Biochemie und Molekulare Biologie. Tech¨ ´ nische Universitat Berlin.S. New Jersey.A. Rahway. Germany . Deborah Zink Merck Research Laboratories. Yongqiang Zhang Department of Plant Pathology.A.A. U.S. New Jersey. Union.S. Madison.xvi Contributors Ningyan Zhang Schering-Plough Research Institute. Berlin.

Today. JAVIER VELASCO and JOSE L. wine (30 million). Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. and glycolipids.S. Spain 1. lipids. Background The versatility of fungal biosynthesis is enormous. INTRODUCTION 1. citric acid (900 thousand). Drew University. amounts to almost 30 billion dollars and includes about 160 antibiotics. beer was produced in Sumeria. Dana Research Institute (R.E. New Jersey. 1 . The use of yeasts dates back to ancient days.A. such as the production of enzymes. Granada.1. vitamins.C. ADRIO Puleva Biotech. polyhydric alcohols. 1). some having markets of over 1 billion dollars per year. Before 7000 B.I. Milk has been made into Kefyr and Koumiss using Kluyveromyces in Asia for many centuries. The antibiotic market.C. Present. which includes fungal products (Fig.S. These are beer (60 million). Other important pharmaceutical secondary metabolites produced by fungi are hypocholesterolemic agents and immunosuppressants. Some of these products are produced commercially while others are potentially valuable in biotechnology. and baker’s yeast (600 thousand).1 Industrial Mycology: Past. pigments. and Future ARNOLD L. Fungi are also used in many other industrial processes.C. In addition to the multiple reaction sequences of fermentations. U. polysaccharides.. and ancient Rome had over 250 bakeries which were making leavened bread by 100 B. single cell protein and fodder yeast (800 thousand). fungi are producers of five leading fermentation products in terms of tons per year worldwide. Madison.). DEMAIN Charles A. Wine was made in Assyria in 3500 B.

Today. 166. These are becoming essential to the fine chemical industry in the production of single-isomer intermediates. . (From Ref. vaccines. The molecular biotechnology industry has made a major impact in the business world. Figure 1 The mold Penicillium notatum growing in a flask of culture medium for the production of penicillin in the early days of the antibiotic age. Molecular manipulations have been added to mutational techniques as means of increasing titers and yields of microbial processes and in discovery of new drugs. and monoclonal antibodies) having a market of 15 billion dollars. fungal biology is a major participant in global industry. The best is yet to come as fungi move into the environmental and energy sectors. biopharmaceuticals (recombinant protein drugs.) fungi are extremely useful in carrying out biotransformation processes.2 Demain et al. Recombinant DNA technology. has also produced a revolution in agriculture and has markedly increased markets for microbial enzymes. which includes yeasts and other fungi as hosts.

The main reason for the use of microorganisms to produce compounds that can otherwise be isolated from plants and animals or synthesized by chemists is the ease of increasing production by environmental and genetic manipulation. the fermentation microbiologist desires a ‘‘wasteful’’ strain that will overproduce and excrete a particular compound that can be isolated and marketed. On the other hand. Thousand-fold increases have been recorded for small metabolites. usually the active ones. (3) a facility to adapt to a large array of different environments. and (5) an ability to make specific enantiomers. or even cells.1. Although microbes are extremely good in presenting us with an amazing array of valuable products. but the species used depends on the substrate employed. Overview Microorganisms are important to us for many reasons. they probably will not be made commercially by chemical synthesis. the microbiologist searches for organisms with weak regulatory mechanisms. they can compete efficiently with other forms of life and survive in nature. or a polysaccharide that can be depolymerized to a fermentable sugar. The microbiologist is actually modifying the regulatory controls remaining in the original culture so that its ‘‘inefficiency’’ can be further increased and the microorganism will excrete tremendous amounts of these valuable products into the medium. Alcohols Ethyl alcohol is a primary metabolite that can be produced by fermentation of a sugar. whereas Kluyveromyces . Regulatory mechanisms have evolved in microorganisms that enable a strain to avoid excessive production of its metabolites. (2) a tremendous variety of reactions that microorganisms are capable of carrying out. (4) the ease of genetic manipulation. Thus. they tend not to overproduce their metabolites. respectively. but the principal one is that they produce things of value to us.2. which facilitates the rapid uptake of nutrients required to support high rates of metabolism and biosynthesis. For the foreseeable future. 2. they usually produce them only in amounts that they need for their own benefit. a development program is begun to improve titers by modification of culture conditions.Industrial Mycology: Past. Once a desired strain is found. mutagenesis. in cases in which normal chemical synthesis yields a mixture of active and inactive enantiomers. which we usually separate into metabolites essential for vegetative growth and those inessential—that is. These may be very large materials such as proteins. to increase productivity. During the screening stage. allowing a culture to be transplanted from nature to the laboratory flask or the factory fermentor. or they can be smaller molecules. thus. Saccharomyces cerevisiae is employed for the fermentation of hexoses. where it is capable of growing on inexpensive carbon and nitrogen sources and producing valuable compounds. and recombinant DNA technology. The importance of the fermentation industry resides in five important characteristics of microorganisms: (1) a high ratio of surface area to volume. nucleic acids. Yeasts are preferred for these fermentations. Most natural products are made by fermentation technology because they are complex and contain many centers of asymmetry. Present. to modify structures and activities. and Future 3 1. and to make entirely new products. The power of the microbial culture in the competitive world of commercial synthesis can be appreciated by the fact that even simple molecules such as lactic acid are made by fermentation rather than by chemical synthesis. PRODUCTION OF FUNGAL PRIMARY METABOLITES 2. both in vivo and in vitro. carbohydrate polymers. primary and secondary metabolites.

11]. brewing. The pharmaceutical industry uses approximately 16% of the available supply of citric acid. Citric acid exerts negative feedback regulation on its own biosynthesis in A. noncaloric. Erythritol can also be produced from sucrose by Torula sp. Another selective tool is resistance to high concentrations of citrate and sugar [10. whereas in Brazil ethanol produced from cane sugar amounted to 12. With regard to beverage ethanol. titers reaching about 100 g/L. and diabetic-safe sweetener erythritol but to decrease growth [5]. Consequently. Ethanol is mainly manufactured by fermentation at a level of 4 million tons per year.86 g/L/hr and 45% conversion). and a stabilizer in a variety of foods. the major portion (80%) of the sugar is converted to citric acid. 2. and has low toxicity. with a yield of 50% and a productivity of 1. Another factor contributing to high citric acid production is the low pH optimum (1. niger resistant to low pH ( 2) are improved citric acid producers [9].6 billion gallons in the United States in 2000 [1]. but good fermentation processes are available [3]. (1. Aspergillus niger has an inherent capacity to excrete organic acids during aerobic growth.5 billion L/yr being used either as a 22% blend or as a pure fuel [2]. top.000 tons per year are made by fermentation. ethanol is being substituted as a blend to raise gasoline’s octane rating. Organic Acids The market for citric acid amounts to $1. a rate of 2. sake yeast. Previous processes were done with Aureobasidium sp. Osmotolerant yeast strains (Candida glycerinogenes) can produce up to 137 g glycerol of per liter with a yield of 65% based on consumed glucose and a productivity of 32 g/L/day [4]. which amounts to 12% of the country’s need. it is widely used in the food and pharmaceutical industry. With special yeasts.67 g/L/hr [7]. Strain improvement by genetic manipulation and protoplast fusion has contributed superior strains for the above processes. and baking industries each year. In 4 to 5 days. the fermentation can be continued to alcohol concentrations of 20% by volume.and bottom-fermenting brewing strains (varying in the degree of flocculation occurring during fermentation). may be utilized if lactose or pentoses. In China. Such a high concentration slows growth and the fermentation ceases. fragilis or Candida sp. The fungi used for alcoholic products are wine yeasts (including special flocculent strains for the production of champagne and film-forming strains for the production of flor sherry).6 billion pounds [8]. It is employed as an acidifying and flavor-enhancing agent. a buffer in jams and jellies. niger at concentrations above 10 g/L. these concentrations are attained only after months or years of fermentation. 10.0). is palatable. are the substrates. However.8 g/L/hr and 41% conversion from glucose [6]. Osmotic pressure increase was found to raise volumetric and specific production of the noncariogenic.7–2. Production of glycerol is usually done by chemical synthesis from petroleum feedstocks. Because of the elimination of lead from gasoline in the United States. with a production level of 1. . Production by a Candida magnoliae osmophilic mutant yielded 187 g/L. approximately 10% to 12% ethanol by volume is obtained within 5 days.2.4 billion per year. About 2 million tons of yeast are produced for the distilling. Mutants of A. Production of ethanol from starchy crops (mainly corn) reached 1. Citric acid is easily assimilated. respectively. an antioxidant for inhibiting rancidity in fats and oils. Under optimum conditions. (48% yield) and the osmophile Trichosporon sp. at 200 g/L after 120 hours.4 Demain et al. and distiller’s strains for alcohol production from cereal starch. some 60 million tons of beer and 30 million tons of wine are produced each year.

3.Industrial Mycology: Past. The market for carotenoids was $750 to $800 million in 1999.85 per pound. and succinic acids. Rhizopus arrhizus produces 97 g of fumaric acid per liter [14]. In the nutritional area.23]. utilis requires no added vitamins.000 tons per year [8]. fruits. C. Present. such as sulfite waste liquor (a byproduct of the paper industry) or wood hydrolysate. lactic. Its ability to grow on cheap substrates not suitable for most other yeasts has made it the organism of choice for this purpose. more importantly. Itaconic acid is used as a co-monomer in resins and synthetic fibers and is also used in coatings.000 tons per year and sells for $4 per kilogram. which synthesize riboflavin in concentrations greater than 20 g/L. and vegetables. 2.-linoleic acid and. Crude substrates. and a production level of 40. Rhizopus oryzae is favored for production since it makes stereochemically pure L-( )-lactic acid. and its price is $1. The production of the vitamin F group (polyunsaturated fatty acids) is mainly by fermentation of different strains of the fungi Mortierella isabellina and Mucor circinelloides. colorants of flowers. thickeners. are used for its propagation. the content of methionine of the methylotrophic yeast Candida boidinii has been markedly increased (from 0. are able to produce dihomo. and grows at 37 C. itaconic. Other Applications Candida utilis is the most widely used species for the production of feed yeast for animal diets. and ADM) is over 3000 tons per year [21]. Eremothecium ashbyii and Ashbya gossypii [20].62 g/g of glucose using Torulopsis glabrata [13]. pyruvic. Pyruvic acid production amounted to 69 g/L in a 56-hour fermentation. Gluconic acid has a market of $93 million. The global market for lactic acid is more than 100. They include vitamin A precursors. which can accumulate up to 5 g/L of -linoleic acid [18].000 tons per year [12]. Aspergillus terreus is the main producer of itaconic acid. can utilize ammonium or nitrate nitrogen. a titer of over 100 g/L. a price of $0. malic. Lactic acid was made at one time by chemical synthesis but this process has been totally eliminated in favor of fermentation. essential for all forms of life. Vitamins Carotenoids are pigmented natural products with antioxidant activity. but today fermentation is the major route. Riboflavin overproducers include two yeastlike molds. tartaric. arachidonic acid in concentrations of 7 to 11 g/L [22. It is made by fungi (Aspergillus sp. and a productivity of over 2 g/L/hr. Two related species.5 to 9 mg/g dry cell weight in the intracellular pool and from 6 to 16 mg/g as total cellular methionine) by mutation to ethionine resistance .4. but certain Candida sp. Mortierella alpina and Mortierella alliacea.) at 15. and Future 5 Other valuable organic acids include acetic.05 per pound [8]. gluconic. Riboflavin (vitamin B2) was produced commercially for many years by both fermentation and chemical synthesis [19]. BASF. Itaconic acid has an annual market of $68 million [17]. Annual production of riboflavin by the three most prominent producing companies (Roche. with a yield of 0. adhesives. fumaric. can grow on many carbon sources (including cellobiose and D-xylose). About 100 tons of -carotene are annually produced by chemical synthesis as well as fungal (Blakeslea trispora) and algal (Dunaliella salina and Dunaliella bardawil) fermentation [18]. and binders [15]. It is produced from raw corn starch at 1 g/L/hr and a concentration of 76 to 80 g/L with a 55% yield [16]. 2. and are used in cosmetics and foods. It is produced anaerobically with a 95% (w/w) yield based on charged carbohydrate. produce 42 g/L. From 120 g/L of glucose. Synthetic processes are not competitive with the fungal process.

Applications of pullulans include use as a flocculator of clay slimes in hydrometallurgical processes. lactis. in coating and packing material for foodstuffs and pharmaceuticals. lactis for hydrolysis of lactose from milk or whey. Some microbial strains produce very high concentrations of extracellular enzymes (e. A number of yeast products are useful in the flavor industry. and in the manufacture of special fibers and fabrics. (2) screening procedures are simple and thousands of cultures can be examined in a reasonably short time. strains of Aspergillus produce 20 g/L of glucoamylase [28]). Ethyl acetate and acetaldelyde are produced by C. (2) plant and animal proteases by Aspergillus protease for chill-proofing beer and meat tenderization. as well as their instability at low pH [27]. fragilis. Yeast enzymes extremely useful in industry include invertase from K.. In the last decade. their high degree of substrate specificity (which reduces side-product formation). baking. PRODUCTION OF ENZYMES Enzymes are valuable in manufacturing because of their rapid and efficient action at low concentrations under mild pH values and temperatures. These shifts include the partial replacement of (1) amylases of malted barley and wheat by amylases from Aspergillus in the beer.g. shear. Additional reasons for using microbial cells as sources of enzymes are as follows: (1) enzyme fermentations are quite economical on a large scale due to short fermentation cycles and inexpensive media. Enzyme levels can be increased by environmental and genetic manipulations. In spite of attractive viscosity and gel properties for possible use as food additives. and Sporobolomyces adorus [25]. Saccharomyces fermentati. and monoterpenes are formed by Ambrosiozyma monospora. fragilis or K. (3) pancreatic proteases by Aspergillus for leather bating and in detergent preparations. and -galactosi- . and textile industries. their low toxicity. The major application of microbial enzymes has been the use of bacterial glucose isomerase in conjunction with fungal -amylase and glucoamylase to convert starch to mixtures of glucose and fructose known as ‘‘high-fructose corn syrup’’ [29]. in adhesives. and (3) different species produce somewhat different enzymes catalyzing the same reaction. [24]. and (4) rennet (chymosin) by Mucor for cheese manufacture. and S. and heat. cerevisiae for candy and jam manufacture. Several species of Hansenula produce copious quantities of extracellular phosphomannans [26] from glucose under aerobic conditions. fungal enzymes have been increasingly used for applications that traditionally employed plant and animal enzymes. utilis. Saccharomyces carlsbergensis. allowing flexibility with respect to operating conditions in the reactor. and the ease of stopping their action by mild treatments. -galactosidase (lactase) from K. and biosynthetic enzymes have been increased several hundred-fold. Terpenes and lactones are produced by K.6 Demain et al. Fermentation yields of pullulan from starch hydrolysates in 10% concentration have been reported to be as high as 75% [26]. Thousand-fold increases have been recorded for catabolic enzymes. application of these water-soluble gums has been hampered because of their sensitivity to salts. A number of extracellular polymers from fungi have shown potential utility in foods or are used in industrial processes. 3. The attractiveness of microbial polysaccharides is due in part to their differential stabilities and physical behaviors in solution. A second example is the production of the glucan pullulan by strains of the dimorphic yeastlike fungus Aureobasidium pullulans. esters and other volatiles are made by Kluyveromyces lactis.

2%. and European enzyme market—mainly made up of protease.1. The market continued to increase and reached $2 billion in 2000. Only about 10% of the 2000 to 3000 enzymes described in the literature were commercially available. 1.000 antibiotics known in 1995.S. textiles. Inulinase from various yeasts has the capability to produce fructose in high concentration from inulin in Jerusalem artichokes and chikory. and L-phenylalanine from trans-cinnamic acid. pesticides. Fungi can be used for a number of bioconversions [32]. Other bioconversions include n-tetradecene to l-tetradecene and phenol degradation. this situation improved. and microbial enzymes accounted for 400 million of that total.6 billion in 1998 [31]. toxins. and (5) effectors of differentiation [34]. fungi. They have no function in growth of the producing cultures. secondary metabolites are usually produced after growth has slowed down. However. 45% (of which starch processing represented 11%). In nature. 3%. oxidation of hydroxyl groups.Industrial Mycology: Past. The U. Also. carlsbergensis for crystallization of beet sugar.36]. These include the natural penicillin G and the biosynthetic penicillin V with a combined market of $4. insects and plants. The search for new antibiotics continues in order to combat evolving pathogens. In batch or fed-batch culture. divided into the following application areas: food. A great many of these secondary metabolites are produced by fungi. 22% could be produced by filamentous fungi [35. In the pursuit of effective antibiotics. Of the 12. other medicinals. are produced by certain restricted taxonomic groups of organisms. and parasites. detergents. Antibiotics Antibiotics are defined as low-molecular-weight organic natural products made by microorganisms that are active at low concentration against other microorganisms. 34%. leather. microbial rennet. L-lysine can be produced from L-aminocaprolactam. viruses. they have tremendous economic importance. (3) competitive weapons against other bacteria. which have a market of $11 billion. and conversions of alkaloids and xenobiotics. and the semisynthetic cephalosporins. As a group that includes antibiotics. the ability to combat tumors. and are usually formed as mixtures of closely related members of a chemical family. many semisynthetic penicillins. 4. FUNGAL SECONDARY METABOLITES Microbially produced secondary metabolites are extremely important to our health and nutrition [33]. Present. many of the new products are made chemically by modification of natural antibiotics in a process called ‘‘semisynthesis. 11%. (4) agents of symbiosis. and previously susceptible bacteria and fungi that have developed resistance. and improved safety and potency. 4. and over half of these sold for more than 1 million dollars per pound [30]. Other desirable properties of new antibiotics include improved pharmacological properties. naturally resistant microbes. and the market reached $1. amoebae. The main problem that retarded rapid development of enzyme technology in the 1980s was the poor availability of enzymes for large scale application testing. glucoamylase. not even including diagnostic and therapeutic enzymes. and pulp and paper.’’ The .4 billion. including ketoreduction. amylase. and glucose isomerase—amounted to 700 million dollars in 1987. and Future 7 dase from S. ester hydrolysis and hydrogenation of double bonds in steroids. functioning as (1) sex hormones. (2) ionophores. and animal and plant growth factors. The most important are the -lactam antibiotics. secondary metabolites are important for the organisms that produce them.

37].1. 4. This compound was originally discovered in .2. respectively. pravastatin and others [40]—which act as inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Similarly. Pharmacological Agents In the early years. but a recent successful nonactinomycete molecule is Taxol (paclitaxel). Brown et al.2. Hypocholesterolemic Agents Only 30% of the cholesterol in the human body comes from the diet. Sales of cyclosporin A have reached $1. Antitumor Agents Most natural anticancer agents are made by actinomycetes.g. mevinolin) in broths of Monascus ruber and A. the regulatory and rate-limiting enzyme of cholesterol biosynthesis in liver. zearelanone) before they were put to work for medicine and agriculture [38]. Discovery of immunosuppressive activity led to its use in heart. such as the -lactam peptide antibiotics and others [36. The remaining 70% is synthesized by the body. Endo et al. [42] discovered compactin in broths of Penicillium citrinum. The ester is called mycophenolate mofetil (CellCept ) and is a prodrug that is hydrolyzed to mycophenolic acid in the body. [44] independently discovered the more active methylated form of compactin known as lovastatin (monacolin K. A very old broad-spectrum fungal antibiotic. Food and Drug Administraiton (FDA) in 1987. the major pharmaceuticals used for noninfectious diseases were strictly synthetic products. 4. mainly in the liver. The global market for finished antibiotics reached $35 billion in 2000. Immunosuppresive Agents Cyclosporin A was originally discovered as a narrow-spectrum antifungal peptide produced by the mold Tolypocladium nivenum (previously Tolypocladium inflatum) [39]. As new lead compounds became more and more difficult to find. Lovastatin exists in two forms. [41] discovered the first member of this group.. liver. 4. Despite the testing of thousands of synthetic compounds. microbial broths filled the void and microbial products increased in importance in therapy of nonmicrobial diseases. but its 2-morpholinoethylester was approved as a new immunosuppressant for kidney transplantation in 1995 and for heart transplants in 1998. 4. major therapeutics for nonmicrobial parasitic diseases in animals came from the screening of chemically synthesized compounds followed by molecular modification.S. compactin (ML-236B) as an antibiotic product of Penicillium brevicompactum. One huge success has been the fungal statins—including lovastatin (mevinolin).8 Demain et al. and kidney transplants and to the overwhelming success of the organ transplant field. Many of the following fungal compounds were first isolated as poor or toxic antibiotics (e. and later Endo [43] and Alberts et al.2. gibberellins. was never commercialized as an antibiotic. All members of the group are substituted hexahydronaphthalene lactones.5 billion. cyclosporin A) or as mycotoxins (ergot alkaloids. Lovastatin was approved by the U. only a few promising structures were uncovered. The majority (90%) is the acid (open) form. Many people cannot control their cholesterol at a healthy level by diet alone but must depend on hypocholesterolemic drugs. mycophenolic acid.2. Statins have a very large market of $15 billion. antibiotic market includes about 160 antibiotics and derivatives. the minor part is the inactive lactone.4.2. which is active. terreus.

They are produced at a level of over 25 tons per year and have a market of $100 million. produced by Gibberella zeae (syn. and stem elongation [53]. and serotonin and the contraction of smooth muscles of the uterus. The yeast Phaffia rhodozyma has become the most important microbial source for the production of the carotenoid astaxanthin [56]. Taxol promotes tubulin polymerization and inhibits rapidly dividing mammalian cancer cells [47]. and reduce by half the time required to obtain lettuce and sugar beet seed crops.Industrial Mycology: Past. Feeding of pen-reared salmonids with a diet containing this yeast induces pigmentation of the white muscle [57. cerebral circulatory disorder. improve malt quality. In addition. Fusarium graminearum) [51]. REGULATION OF FUNGAL SECONDARY METABOLISM The intensity of secondary metabolism can often be increased by addition of limiting precursors such as phenylacetic acid in the production of penicillin G. Ergot alkaloids are not the only successful example. It is amazing but true that these ‘‘poisons’’ are now used for angina pectoris. and Future 9 plants [45] but is also produced by the fungus Taxomyces andreanae [46]. is an estrogen. Some of the ergot alkaloids also possess antibiotic activity.58] 5. is produced by Gibberella fujikuroi and is the cause of the ‘‘foolish rice seedling’’ disease of rice [52]. seed germination. They are isoprenoid growth regulators controlling flowering. reduction in bleeding after childbirth. with amino acids present in the fermentation media [55]. hypertonia. The watersoluble red pigments. Gibberellins are used to speed up barley malting. The genus Gibberella is responsible for two others. and prevention of implantation in early pregnancy [49. Present. monascorubrin and rubropunctatin. Pigments Fermentation of Monascus purpureus on rice to prepare koji or ang-kak (red rice) has been used as a traditional Chinese food and medicine since 800 AD [54]. Most secondary . a member of the phytotoxic mycotoxin group known as the gibberellins. These mycotoxins were responsible for fatal poisoning of humans and animals (ergotism) throughout the ages after consumption of bread made from grain contaminated with species of Claviceps.3. are produced by reaction of the orange pigments. and is authorized for food use in China and Japan. hypertension. In 2000. Zearelanone. wine. Taxol sales amounted to over $1 billion for Bristol-Myers Squibb. and fish. Among their physiological activities are the inhibition of action of adrenalin. and its reduced derivative zeranol is used as an anabolic agent in cattle and sheep. Gibberellic acid. yet this is true for the ergot alkaloids.50]. 4. soy bean cheese. increasing both growth and feed efficiency. Mycotoxins as Sources of Useful Agents It is difficult to accept the fact that even poisons can be harnessed as medically useful drugs. inhibition of prolactin release in agalactorrhea. 4. The fungus is used for preparing red rice. meat. It is approved for breast and ovarian cancer and is the only commercial antitumor drug known to act by blocking depolymerization of microtubules.4. monascorubramine and rubropunctamine. representing 10% of the company’s pharmaceutical sales and its third largest selling product [48]. serotonin-related disturbances. migraine headache. increase the yield of vegetables. uterine contraction. This pigment is responsible for the orange to pink color of salmonid flesh and the reddish color of boiled crustacean shells. noradrenalin. zearelanone and gibberellins.5.

sucrose Noninterfering Carbon Source Sorbitol. galactose. the former is used first to produce cells.10 Demain et al. acetylhydrolase Target . Regulation by the Nitrogen Source Nitrogen regulation is of wide significance in microbiology.g. organic acids Lactose Expandase. starch). Polysaccharides (e. oligosaccharides (e. known as the ‘‘idiophase. sterigmatocystin [60] by Aspergillus nidulans. The control of enzyme synthesis is generally exerted by the intracellular nitrogen pool. mannitol Sucrose. and trichothecenes [61] by Fusarium sporotrichiodes. soybean oil. the ‘‘second-best’’ carbon source is used for the production phase. and oils (e.2. metabolites are formed via enzymatic pathways rather than by a ribosomal mechanism. glycerol Glucose Glucose. galacto se Polyols. ammonium salts). fructose. Whether chromosomal or plasmid-borne. The genes encoding the enzymes involved in secondary metabolism are usually chromosomal. or as parts of modules of large multifunctional polypeptides carrying out a multitude of enzymatic steps.g.1. but little to no secondary metabolite is formed.. Regulation by the Carbon Source Glucose. As a result. often interferes with the formation of secondary metabolites (Table 1). usually an excellent carbon source for growth. the secondary metabolism genes are often clustered. 5. There is also a more specific type of control in which a particular amino acid (or biosynthetic group of amino acids) represses and/or inhibits production of a secondary metabolite because the primary metabolite(s) and the idiolite are derived from the same branched pathway and the amino acid(s) exerts negative feedback regulation on the biosyn- Table 1 Carbon Sources Interfering with Secondary Metabolism Idiolite Benzodiazapine alkaloids Cephalosporins Ergot alkaloids Penicillin Interfering Carbon Source Glucose Glucose. After the rapidly assimilated compound is depleted. Clusters of fungal biosynthetic genes have been found encoding biosynthetic genes for production of penicillins and cephalosporins [59]. complex fermentation media often include a protein source and defined media include a slowly assimilated amino acid as the nitrogen source to encourage high production of secondary metabolites.. lactose). free or complexed. such as in the cases of polyketide synthases and peptide synthetases.g. In media containing a mixture of a rapidly used and a slowly used carbon source. The enzymes occur as individual proteins. methyloleate) are often preferable for fermentations yielding secondary metabolites [34]. Information concerning the mechanism(s) underlying the negative effect(s) of ammonium and certain amino acids on industrial processes is scarce. but not necessarily as single operons. affecting both primary metabolism [62] and secondary metabolism [63].. Many secondary metabolic pathways are negatively affected by nitrogen sources favorable for growth (e.g. Processes subject to nitrogen source regulation are shown in Table 2.’’ 5..

and Future 11 Table 2 Nitrogen Sources Interfering with Secondary Metabolism Idiolite Aflatoxin Alternariol Bikaverin Cephalosporin Gibberellic acid Macbecin Patulin Penicillin Trihydroxytoluene Interfering Nitrogen Source Nitrate Nitrate.3. whereas the ultimate product is not. which is caused by lysine inhibiting homocitrate synthase [64]. p-aminobenzoate. which slow down assimilation of the carbon source mannitol and allow it to remain for a longer period. 5. Zinc stimulates aflatoxin production. Regulation by the Phosphorus Source Regulation by phosphorus sources. Present. includes both specific and general controls. Although only little is known about the mechanism of general phosphate control of secondary metabolism. L.Industrial Mycology: Past. glutamine L-tryptophan. Mg2 inhibited pigment synthase action. 5. there is a strong possibility that phosphate regulation also works by affecting enzyme activities such as phosphorylation by protein kinases and dephosphorylation by phosphoprotein phosphatases [65]. an enzyme involved in the formation of the penicillin precursor. An example is the negative effect of lysine on penicillin synthesis. Because biosynthetic intermediates of certain pathways are phosphorylated. urea Glycine NH4+. In general. and Fe2 via stimulation of synthase action rather than an effect on induction or enzyme stabilization. as occurs in the formation of bikaverin. and manganese increases patulin production via an effect on transcription [66].-aminoadipic acid. Phosphate also appears to interfere in many secondary metabolic pathways not known to have phosphorylated intermediates. which are suboptimal for growth. anthranilate NH4+ NH4+. and cephalosporin C.4. L-lysine NH4+ Noninterfering Nitrogen Source NH4+ L-asparagine. L-lysine NH4+. phosphate-sensitive fermentations have to be conducted at low levels of free phosphate (usually below 10 M). phosphatases are sometimes required in biosynthesis. . A rather specific negative effect of inorganic phosphate arises from its ability to inhibit and/or repress phosphatases. L-glutamate. Mn2 . On the other hand. Studies by Lin and Demain [67] have shown red pigment formation by Monascus to be increased by Zn2 . L-arginine L-glutamate thetic pathway before the branch point. Optimal production of pneumocandins by Zalerion arboricola occurs under magnesium-limiting conditions [68]. thus extending the duration of antibiotic production. Regulation by Metals Metals often exert control over secondary metabolism. ergot alkaloids.

is inhibited. an O-methyltransferase. Broad domain control employs the positively acting nitrogen regulatory gene areA [78] and/or a negatively acting carbon repressor gene. creA [79].5. These effectors are often precursors. which has an in vivo half-maximal lifetime of 7 hours [80]. fujikuroi produces bikaverin at a growth rate of 0. Stimulatory precursors that are also inducers include tryptophan for dimethylallyltryptophan synthetase in ergot alkaloid biosynthesis [69].01 h 1 [75].12 Demain et al. Fungal Regulatory Genes Clustering of fungal genes is not common except in cases of assimilation of certain nutrients (e. Derepression also occurs when the growth rate is reduced by other means. dimethylallyl tryptophan synthetase. nitrate) and production of secondary metabolites [76]. Regulation of pathways in fungi (mainly studied in A. whereas in the pathway to mycophenolic acid. and phenylacetate for the phenylacetate uptake system in Penicillium chrysogenum involved in penicillin G formation [73]. when growth slows down and formation of the first enzyme. Induction of Secondary Metabolite Synthases In a number of secondary metabolic pathways. urticae is caused by decay of the initial enzyme.72]. 5. it appears that low growth rate is more important than nitrogen limitation [74]. Control by Growth Rate Growth rate control is important in secondary metabolism and may be the overriding factor in the cases where nutrient limitation is needed for production of secondary metabolites. In the production of patulin by Penicillium urticae. is derepressed.g.8. Feedback Regulation The role of feedback regulation in controlling secondary metabolism is well known. cyclase and expandase in the cephalosporin pathway of Acremonium chrysogenum [71.9. 6-methylsalicylic acid synthase. . proline. ethanol. The resultant 6-methylsalicylic acid induces transcription of genes encoding later enzymes in the pathway.7. Enzyme Decay Production of secondary metabolites eventually stops due to feedback regulation (see above) and decay of the synthase(s). 6-methylsalicylic acid synthase. Ergot alkaloids inhibit the first enzyme of their biosynthetic pathway. Narrow domain regulation usually involves a positively acting pathway-specific regulatory protein containing a zinc binuclear cluster: CX2CX6CX6CX2CX6CX2.6.. the shift from the growth phase (trophophase) to idiophase occurs at the point of nitrogen source depletion. 5. Thus.aminoadiphyl)-L-cysteine-L-valine synthtase (ACVS).05 h 1 but forms gibberellin at 0. Cessation of patulin production by P. nidulans) can be narrow or broad domain regulation [77]. the final enzyme. 5. methionine for -(L. Many secondary metabolites inhibit or repress their own biosynthesis. primary metabolites increase production of the final product. and one has to determine whether the effect is merely due to an increase in precursor supply or includes induction of one or more synthases of the biosynthetic pathway. G. 5. 5. quinate. phenylalanine in benzodiazapene alkaloid formation [70].

99]. cerevisiae are (1) an efficient and tightly regulated methanol promoter (AOX1).Industrial Mycology: Past. Aspergillus niger [84]. human superoxide dismutase [92].105]. MOLECULAR FUNGAL BIOTECHNOLOGY The development of molecular biology techniques has provided new ways to use yeasts and molds as microbial cell factories for the production of food ingredients and heterologous (especially mammalian) proteins. After the discovery in 1969 that some yeast species can grow on methanol [95]. water-miscible. One of these species. due to shorter chain lengths of N-linked high-mannose oligosaccharides. This yeast can be grown rapidly in simple media and to a high cell density. S. Despite these successful examples. and Future 13 6.89]. cerevisiae is considered to be a safe host for the production of pharmaceutical proteins. However. S.100]. Mammalian genes cloned and expressed in S. interest in using methanol as a pure. and relatively inexpensive substrate increased rapidly [96.97]. Several species of fungi have that status and are currently being used for large-scale production of recombinant proteins [81]. 12 g/L of tetanus toxin fragment C [106]. 4 g/L of secreted human serum albumin [104]. nidulans. human hemoglobin [91]. A. usually up to 20 residues lacking the terminal -1. and Neurospora crassa [85]—and sequencing of three others are in progress (A. (3) integration of multicopies of foreign DNA into chromosomal DNA. fumigatus. and absence of strong and tightly regulated promoters. and interleukin-6 [81]. and its genetics are more advanced than any other eukaryote [87].100.3-linked mannose residues that could cause antigenic response in patients. pastoris [87. and 0. (2) less extensive glycosylation. and (5) highdensity growth and straightforward scale-up [99.104] include 6 to 10 g/L of tumor necrosis factor [101. Pichia pastoris. cerevisiae is sometimes regarded as a less than optimal host for large-scale production of mammalian proteins because of certain drawbacks such as hyperglycosylation (which results in the addition of long chains of N-linked high-mannose oligosaccharides to the recombinant protein). presence of -1. Present. Among the advantages of this methylotrophic yeast over S. Schizosaccharomyces pombe [83]. yielding stable transformants [87. Intracellular or extracellular recombinant products made in P.103]. human epidermal growth factor [90]. Four fungal genomes have been sequenced—S. The choice of the host strain for protein production is made on the basis of production yields and regulatory issues. which yields alcohol oxidase at 30% of soluble protein. cerevisiae [82]. one of the main drawbacks for this excellent expression system is the non-GRAS status of P.98. 1.5 g/L of human insulin-like growth factor [108]. (4) ability to secrete high levels of foreign proteins. has become one of the most extensively used expression systems [87. 6. and Candida albicans) [86]. it can secrete heterologous proteins into the extracellular broth. The most commercially important yeast recombinant process has been the production of the genes encoding surface antigens of the hepatitis B virus resulting in the first safe hepatitis B vaccine [93.25 g/L of the envelope protein of HIV-1 [107].98. 4 g/L of intracellular interleukin-2 [104]. although some products made by this yeast are being evaluated in phase .94]. Host strains are usually chosen from among those which have attained the so-called GRAS (Generally Recognized as Safe) status by the FDA. pastoris.3-mannose linkages [100–102].1. Production of Recombinant Pharmaceutical Polypeptides Since it is a food organism. cerevisiae include human interferon [88. especially for fungi used in the food industry.

N-linked glycosylation is more conserved [87].118]. Levels of production of nonfungal proteins are low compared with those of homologous proteins. efficient secretion signals [117.. erythropoietin and human chorionic gonadotropin [hGC]).121].122]. and 1. A. polymorpha is also highly expressed and tightly regulated. The three glucose residues and one mannose are removed. elucidation of the role of secretion-related chaperones and foldases [131–134]. Although O-linked glycosylation in yeast is quite different from that in higher eukaryotes. and Trichoderma reeseii cellobiohydrolase I (cbh1) are genes cloned with this strategy [118]. Expression levels of about 8 to 9 mg/L of intracellular hepatitis B middle surface antigen [113].118. and gene fusions with a gene that encodes part of or an entire well-secreted protein [81. gene fusion) remains the first choice in attempts to produce nonfungal proteins in fungal hosts. transcription. A.125]. a thrombin inhibitor from the medicinal leech Hirudo medicinalis. For many proteins that have pharmaceutical applications. or pharmacokinetics [138].123].. In yeast recombinant proteins. translation. and therefore tight regulation of the MOX promoter is lost in the high-glucose conditions usually used for high-biomass fermentations [112]. awamori 1.5 g/L of secreted hirudin are achieved with this methylotrophic yeast. proper folding (e. awamori -amylase (amyA and amyB). Several recent studies have shown the fungal secretory pathway to be a limiting factor in heterologous enzyme production. and 250 mg/L human lactoferrin [127]. The last-named strategy (i.-endoxylanase (exlA). One example is recombinant hirudin. 1 g/L of human serum albumin [114].. introduction of a large number of gene copies [116. secretion or extracellular degradation [81. Studies on screening for mutant strains with altered secretion properties using green fluorescent protein as reporter [130]. including the construction of protease-deficient strains [120. Several factors that influence production act at different levels (i. a core oligosaccharide unit (GlcNAc2Man9Glc3) is present at the endoplasmic reticulum [139].14 Demain et al. as well as in mammalian polypeptides.5 g/L of secreted product was obtained [109]. tissue.e.g.4. Different strategies have been developed to overcome these problems. A. giving enzyme levels up to 37% of total cell protein [110]. Glycosylation is species. N-glycosylation is necessary for stability. The development of molecular techniques for production of recombinant heterologous proteins in filamentous fungi is laborious and contrasts markedly with the success achieved in yeasts. Heterologous gene expression in the methylotropic yeast Hansenula polymorpha is similar to that of P. kinetic studies on protein secretion [135]. pastoris. use of strong fungal promoters. and . III clinical trials. The promoter of the methanol oxidase gene is used to express foreign genes. The ability to introduce or delete genes remains difficult although some advances in transformation have been recently reported. niger or Aspergillus awamori glucoamylase (glaA). Higher concentrations have been obtained for some of these proteins after mutagenic treatment of highly producing strains. in which 1. 2 mg/L of human lysozyme [126]. Fusion has resulted in levels of secreted proteins of 5 mg/L of human interleukin6 [124. One major difference is that expression of the MOX gene is significantly derepressed in the absence of glucose or during glucose limitation [111]. and effect of hyphal branch frequency [136] are examples of the work being carried out to understand this complex process.e. pastoris. Almost all eukaryotic excreted polypeptides are glycosylated. and cell-type specific [137]. the MOX gene in H. such as human lactoferrin (5 g/L [128]) and chymosin (1 g/L) [129].117–119]). including restriction enzyme–mediated integration [115] or Agrobacterium tumefaciens-Ti plasmid–mediated transformation [116]. As with AOX1 in P.

Although the number of heterologous fungal enzymes approved for food applications is not very large. and chemical industries because they (1) are stable in organic solvents. To be suitable. recombinant fungi are one of the main sources of enzymes used for industrial applications. and in the interesterification of fats and oils to produce modified acylglycerols. the list is continuously increasing [145]. food. Further improvement was done by parasexual recombination. one from Rhizomucor miehi and another from Thermomyces lanuginosus. and starch processing industries are recombinant products [144]. There are two fungal recombinant lipases currently used in this industry. niger var awamori amounted to about 1 g/L after nitrosoguanidine mutagenesis and selection for 2-deoxyglucose resistance [129].g. Mammalian proteins show two different types of oligosaccharide side chains: low-mannose residues (GlcNAc2Man3) plus additional residues of galactose. biomedical sciences. Little research has been carried out on glycosylation in molds. and Future 15 processing of the core oligosaccharide continues in the Golgi.3. and (4) exhibit a high enantioselectivity [148–150]. oryzae [146]. and leather processing. many of the recombinant food-grade proteins are of fungal origin [119. (3) do not require cofactors. or peroxidases [151]. Novo-Nordisk was the first company to launch a product (Lipolase ) containing a recombinant fungal lipase. 6. Recombinant yeast proteins usually show highmannose side chains (GlcNAc2Man2–6).2 g/L [147]. oxidases. Recombinant Commercial Enzymes Besides the production of some pharmaceutical heterologous proteins (see above).146]. Brewing and Baking Beer wort contains barley -glucans that reduce the filtrability of beer and lead to precipitates and haze in the final product. oryzae genome for overproduction [151. industrial cleaners. fucose. Microbial lipases have a tremendous potential in areas such as food technology. resulting in a strain producing 1. where they can be combined with proteases. (2) possess broad substrate specificity. Production of this bovine protein in recombinant A. both being produced in A. The glycosylation of a protein can differ depending on factors such as the medium in which the cells are grown.. lipases should be alkalophilic and able to work at temperatures above 45 C and pH values of about 10. Over 60% of the enzymes used in the detergent. Lipases are also very important in the detergent industry. where elongation may take place in further addition steps. There is one exception in which the donor strain is not another fungus—calf rennin (chymosin) used for cheese making. They are extensively used in household detergents. they should be capable of functioning in the presence of the various components of washing product formulations such as oxidants or surfactants. where there is divergence between yeasts and higher eukaryotes. and sialic acid or a mixture of high-mannose and complex-type oligosaccharides [140]. It was developed by genetic and protein engineering of the Humicola lanuginose lipase gene introduced into the A.2. lipases are commonly used in the production of different products (e. Present. in the production of desirable flavors in cheeses. although hyperglycosylation does not seem to occur and low-mannose side chains are formed [141–143]. The gene coding for endoglucanase was transferred . Besides. fruit juices and baked foods). In the food industry.152]. 6.5 g/L from parents producing 1.Industrial Mycology: Past. Because of the low yields achieved with nonfungal proteins (see above).

and the engineered yeast strain efficiently hydrolyzed the -glucans [153]. is in progress [162.158]. Similiar technology has created starch-utilizing S. a recombinant baker’s yeast was approved for breadmaking in the United Kingdom. Recently. which normally follows a 1-week fermentation stage [156]. The engineered strain outperformed Saccharomyces diastaticus [155]. The first is the development of alternative hosts. Brewing yeasts have been modified by recombinant DNA technology so that they produce A. The resulting beer suffered no loss of quality and flavor. Major advances have been achieved in the antibiotic field. Other Products Thaumatin. the recombinant yeasts were unfortunately not used because of fear of consumer reaction. a protein from the plant Thaumatococcus danielli with an intense sweetness (about 3000 times sweeter than sucrose). 7.5. thereby increasing production of the final products. an impressive improvement in yield (up to 14 mg per liter) has been obtained in A. especially those which have already been given . FUTURE PROSPECTS The past few years have been a period of great progress using filamentous fungi as cell factories. and high levels of ethanol were produced. Ninety-five percent of the carbohydrates in the 25% starch substrate was utilized. Production of Secondary Metabolites by Recombinant Fungi Production of new fungal metabolites by application of recombinant DNA technologies is of great interest. awamori was cloned and expressed stably in polyploid distiller’s yeast. In 1990. niger var awamori by use of stronger promoters and higher gene dosage [123]. niger amyloglucosidase and break down unfermentable dextrins for light beer production [154]. 6.16 Demain et al. Brewing yeasts have been engineered to produce acetolactate decarboxylase from Enterobacter aerogenes or Acetobacter aceti. Successful expression of thaumatin was achieved in Penicillium roqueforti and A. Continued progress in the area of metabolic engineering has led to overproduction of limiting enzymes of important biosynthetic pathways.163]. Development of fermentation processes for the production of -carotene in the food-grade yeast C. cerevisiae has been achieved by cloning and expression of a muscle bovine lactate dehydrogenase. reesei to brewer’s yeast. the genes amplified by recombinant DNA technology were melibiase and maltose permease. Production of lactic acid in S. niger var awamori [159] at yields of 2 to 7 mg/L. has been recently approved as a food-grade ingredient (E-957). cerevisiae strains and wine yeast strains that produce lower acidity and enhanced flavor. containing the carotenoid biosynthetic genes from the bacteria Erwinia uredovora and Agrobacterium aurantiacum. Despite these economic advancements. utilis. a high level of glucoamylase being secreted. Production of the sweetener xylitol has also been improved by transforming the XYL1 gene of Pichia stipitis encoding a xylose reductase into S. reaching productivities of 11 g/L/hr [161]. There are four major fronts in which work is currently under way.4. 6. This enzyme eliminates both diacetyl and the requirement for a 3.to 5-week flavor maturation period. The glucoamylase gene from A. especially in the area of cephalosporins [157. cerevisiae [160]. Lower acidity and enhanced flavor in wine has been achieved by transformation of wine yeast with the gene encoding the malolactic conversion enzyme from Lactobacillus delbrueckii. from T.

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Completion of the sequence of Saccharomyces cerevisiae. milkatae.000–39. INTRODUCTION At the time of writing.S. representing both the phylogenetic breadth of fungi and some of the most medically and economically significant species are in progress or in the planning stages [1]. Oregon State University. 1. roughly 70% as many as Drosophilia melanogaster (14. Louisiana. bayanus. Baton Rouge. Madrid.A. SPATAFORA Department of Botany and Plant Pathology. S. N. ˜ S. Louisiana State University. Spain JOSEPH W. and about 30% as many as Homo sapiens (21. U.S. Merck Sharp & Dohme de Espana.A. For example. and Schizosaccharomyces pombe.000 genes. U. MEREDITH BLACKWELL Department of Biological Sciences. crassa is estimated to have around 10. 50% as many as Caenorhabditis elegans (20. Corvallis.2 Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes GERALD BILLS ´ ´ Centro de Investigacion Basica. Saccharomyces cerevisiae. was a landmark in molecular biology and has enabled scientists to globally study eukaryotic gene expression and gene function. high-quality draft genomic sequences of five ascomycete yeasts. Sequencing projects for at least another dozen species of fungi. The sequences of these fungi reveal organisms that are remarkably similar in genomic complexity. S.. organization.000 genes). and function to other eukaryotes.000 genes). have been completed. Oregon. crassa are without obvious counterparts in public databases. paradoxus. S. Neurospora crassa. About 4000 of the estimated proteins of N.A. and the first filamentous fungus. which 27 .000 genes). one of man’s oldest domesticated partners.

organisms formerly classified as fungi (bold text). cerevisiae or Sch. except for carotenoids and melanin pigments. The basic backbone phylogeny based on phylo- Figure 1 Phylogenetic position of the true Fungi. Among the 1400 genes that have counterparts with other eukaryotes are genes associated with a biological clock. three nonribosomal peptide synthases. and many core signaling pathways for cellular functioning.28 Bills et al. and other major lineages of eukaryotic organisms. and EF-1 ] in addition to rDNA. Genomic analyses of Neurospora crassa have identified some seven polyketide synthases gene clusters. crassa have no counterpart in S. more than half the genes of N. light sensing. reveals that our knowledge of secondary metabolites of fungi and how to elaborate and engineer their production is still in its infancy.000 species of ascomycetes [2]. The complete or partial gene clusters of secondary metabolite biosynthetic pathways have now been characterized for only several dozen fungi. Surprisingly.) . -tubulin. While genomic scientists have been opening the doorway to the realm and function of the fungal genome. considering these are only three species among the estimated 150.4]. mycologists have been deciphering the evolutionary histories within the fungal kingdom and other fungal-like organisms and defining their placement among the eukaryotes. The tree was based on sequences of four protein-coding regions [ -tubulin. hinting at the potential genetic diversity in fungi. The finding of such extensive biosynthetic capacity in a fungus that is generally thought to be devoid of secondary metabolites. (Adapted from Ref. 5. actin. may be a function of general lack of fungal genomic data and points to the enormous potential for new gene discovery in fungi. pombe. Unparalleled progress has been made in the last decade toward developing a phylogenetic classification of fungi [3. and several genes associated with diterpene biosynthesis.

1). hyphae. Basidiomycota. deriving from among several independent eukaryotic lineages. The origin of the fungi seems to be correlated with the origin and diversification of land plants. and other Eukaryota. Chytridiomycota. exclusive of slime molds and oomycetes. One important finding has been that ‘‘fungi’’ are polyphyletic (Fig. Most phylogenetic studies to date have been performed on nucleotide data determined from the nuclear rDNA. still are unclear (Fig. However. the early divergences within the filamentous ascomycota (Pezizomycotina) and macrosporocarp-producing basidiomycota (Hymenomycetes) are poorly understood. Could symbioses between green algae and fungi predate the evolution of land plants and their mycorrhizal symbioses? What is the origin of dikaryotic fungi? When do we find the first organized sporocarps and fungal tissues? . the ‘‘crown fungi’’ (crown fungi Glomales. 2). The utilization of SSU rDNA has redefined fungal systematics for mycologists and allowed them to approach many long-argued questions in fungal systematics. 1. genetic analyses using DNA characters was developed relatively early. What are the origins of the fungi? Are choanoflagellates the ancestors for fungi? Where is the origin of DAP lysine biosynthesis in the fungal ancestry? Can evolution of key structures (flagella. A well-defined and monophyletic group of ‘‘true fungi’’ (Figs. 2). and Ascomycota) of the Kingdom Fungi. The limitations of single gene phylogenies have been recognized.) be traced? Can the morphology and trophic relations of the ‘‘first fungus’’ be inferred? What were the earliest divergence and radiation events within the Kingdom Fungi? The relationships among the terminal clades.Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 29 Figure 2 Relationships among the monophyletic grouping of the crown or terrestrial fungi (Glomales. Basidiomycota). Ascomycota. Furthermore. and fundamental questions regarding the fungal evolution will be solved by use of additional genes and taxa. and their morphologies have converged. is considered a kingdom-level taxon [5–8]. the Zygomycota. the relationship of the Glomales with the ascomycete/basidiomycete clade is tenuous and sampling of plant-associated zygomycetes is incomplete. etc.

The next section summarizes our current state of knowledge of the phylogeny of true fungi (Kingdom Fungi) and fungal-like organisms placed in several distinct eukaryotic clades (Fig. of which roughly 120. 2. [5] analyzed deduced amino acid sequences from four protein-coding regions [ -tubulin. a group that includes fungi and fungal-like organisms (Fig.5 million species is generally accepted as working estimate for the number of fungi [10. The analyses identified and strengthened several branches.g. To avoid the constant changing of formal names of higher taxa during ongoing phylogenetic research. rather than formal taxa. as well as highlighting taxa that have produced historically important bioactive molecules.11]. but because the relationships of the fungi and their relatives are still unresolved or not included in all analyses. Zygomycota. comprising the traditional phyla Ascomycota. as opposed to phylogenetic models. representing the earliest evolutionary divergences. but an encouraging recent trend is the increasing use of additional genes and incorporation of biochemical and ultrastuctural characters to help resolve conflicting trees based on single genes and analytical approaches (see Assembling the Fungal Tree of Life website http://ocid.30 Bills et al. Baldauf et al. Classifications should reflect phylogenetic relationships. Referral to any two or more classification systems. has become a practical alternative [12]. indicates that they are in a state of flux. Until now. -tubulin. The numbers of fungi have been consistently revised upward from the numbers of known described species. 1). and elongation factor 1-alpha (EF-1 )] in addition to rDNA to improve resolution of the deep branches of the eukaryote tree.. and this convention has been adopted for this discussion of higher fungal taxa (e. Another important realization during the last decade is that the dimensions of the fungal kingdom in terms of the diversity of species are enormous. Basidiomycota. the grouping of dictyostelid and plasmodial slime molds. and the missing fungi must be discovered.4). 1): 1. including the Fungi-Microsporidia link. see section 3.g. the use of clade names.13]. and Chytridiomycota. FUNGI AMONG THE EUKARYOTES Multiple genes and data partitioning have been used to develop a hypothesis of the relationships of the large diverse group of eukaryotes. and fungi and fungal-like eukaryotes that are important pathogens or model organisms in biochemistry and genetics. and it is generally suspected most fungi still await formal description and naming. identified. In addition to the relatively sparse numbers of taxa included in the trees. Baldauf et al. and incorporated in order to derive a definitive phylogenetic framework. The number of 1.org/research/ aftol). and the remote basal position of the acrasid slime molds. additional support has been derived from correspondence of nonmolecular traits (e. flagellation and arrangement of mitochondrial cristae). No single phylogenetic reconstruction to date has included more than about 800 fungi and fungal-like organisms [9]. 1).000 currently known species [10]. taxonomic coverage across higher taxa has not been thorough. In some cases. easily rivaling the numbers of other speciose groups. fungal phylogenetic studies relied heavily on ribosomal DNA. are a monophyletic group of ‘‘true Fungi. actin. such as the insects..000 species have been validly named [10.5 million fungal taxa [11] are daunting. classification schemes may vary in their inclusiveness.’s broadview study of fungi and fungal-like organisms arrived at several important conclusions about the fungi (Fig. which represents less than 1% of the 120.nacse. Estimates of up to 1. The fungi.’’ .

Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 31 2. The traditionally defined zygomycetes are polyphyletic. has . although still sparsely sampled.24]. and the Glomales constitute the monophyleytic ‘‘crown fungi’’ and are the most-derived clade within the Kingdom Fungi. 2. current studies have shown that the classification is inadequate (Figs. and Ascomycota [3. 1. A second traditional zygomycete group. brown algae. but rDNA has clearly established their position among the fungi [7. The Chytridiomycota (estimated 800 species). Zygomycota. which is consistent with a choanoflagellate-like ancestor. Basidiomycota. Myxomycetes. the Glomales. Chytridiomycota.17]. Chytridiomycota (Zoosporic Fungi) The Chytridiomycota (Figs. Microsporidia. Fig. 3) have been defined traditionally by the presence of a single posteriorly inserted smooth flagellum. Acrasid slime molds are distant from other slime mold groups. 7. Basidiomycota. however. and dictyostelids may constitute a monophyletic group.g. The appearance of earliest members of the crown fungi on Earth likely coincided with the origin and diversification of terrestrial plants [19–22]. however.15]. The dictyostelid and plasmodial slime molds. In the past some mycologists doubted whether the Chytridiomycota were true fungi. 3. Modern analyses of the SSU rDNA do not support the phyla Chytridiomycota and Zygomycota as monophyletic groups. KINGDOM FUNGI Historically. diatoms. however. 2). are defined by the presence of a meiospore known as a zygospore and the lack of a flagellum. 3.1. The zygomycetes (approximately 1000 species). chrysophytes) form a clade. these two taxa form a monophyletic lineage with the Metazoa (animals). form a monophyletic group that together with certain amoebae are the sister group to Fungi. Phylogenetic analyses indicate that some chytrid lineages occupy the most basal branch of Kingdom Fungi. 4. The Microsporidia are a sister group to Fungi. protostelids. Oomycetes and other groups with a heterokont or derived flagellar condition (e. is not monophyletic. The Glomales are important in the evolutionary history of land plants. is a sister clade to the Ascomycota/Basidiomycota clade [17–20]. The ‘‘crown’’ groups predominate in all terrestrial environments and are critical to the function of life on Earth. and Metazoa. the Ascomycota.. this large group is a sister to the clade made up of ciliates and Apicomplexa (sporozoa). The Ascomycota and Basidiomycota are the two largest phyla of the Kingdom Fungi and together constitute over 95% of all known fungi [15]. hyphochytrids. 3. 5. Plasmodiophorales belong to a clade of protozoans near alveolates [14]. taxon sampling remains inadequate.23. The monophyly of Chytridiomycota.6. and the two groups may intergrade or diverge at several points [16. occurring in a clade with predaceous vahlkampfiid amoebae. Together. the monophyletic Fungi have been classified in four phyla. it is estimated that 80% of all plant species are associated with these arbuscular mycorrhizaforming fungi. currently defined by the primary ancestral character state of a smooth posterior flagellum. linked by a choanoflagellate-like common ancestor. 6.

The terminal clades (orders) of the Zygomycota and Chytridiomycota are generally well supported. Neocallimastigales. and four groups of Chytridiales in distinct clades. not been upheld by subsequent studies. and Chytridiales—were established on the basis of zoospore ultrastructure [26]. Monoblepharidales. possibly from a blastocladialean ancestor. based on analyses of nuclear rDNA and EF-1 data. However. Additional chytrid diversity likely will be uncovered when more chytrids with unique zoospore types are included in phylogenetic analyses [16]. The complete genomic sequencing of Batrachochytrium dendrobatidis has been proposed due to its implication in the worldwide amphibian decline [1]. Evolutionary models based on zoospore morphology are congruent with mitochondrial phlyogenies [27]. consisting of the orders Blastocladiales. including Batrachochytrium dendrobatidis. Five orders—Blastocladiales. including increased taxon sampling. The phylogenetic analysis distinguished four monophyletic clades. but their relationships are poorly resolved. do not fall within any of the defined groups on the basis of DNA characters. Some chytrids with unique zoospore morphologies have not been classified in previously existing orders. Spizellomycetales. consistent with the groupings based on zoospore ultrastructure. The discovery of anaerobic rumen-inhabiting chytrids (Neocallimasticales) has been .32 Bills et al. and Spizellomycetales. Neocallimastigales. Monoblepharidales.25]. although chytrid groups are basal in phylogenetic trees [16. the relationships among the orders and chytrid clades remain to be resolved [16]. Some important metabolites and enzymes of the Zygomycota are indicated to the right. As a group. Figure 3 Major clades of the phyla Chytridiomycota and Zygomycota. the chytrid associated with amphibian decline. the relationship of the clades to each other has been tested using SSU rDNA characters [16]. but several of these. excluding the Glomales. More recently. they represent the loss of the flagellated stage.

lipases.29. EF-1 gene exons. however. and Zoopagales [16.28]. was the lack of support for the traditional families of the order. Their reduced morphological features have inaccurately portrayed the genetic diversity of the group. 3. Their fast growth and high enzyme activity have made them popular for use as agents of organic molecule biotransformations. as mentioned above. perhaps. The conflict could be caused by an accelerated rate of sequence evolution in SSU rDNA of zygomycetes relative to other fungi and resulting long-branch attraction in phylogenetic analyses employing the gene [16]. In particular. Glomales The Glomales (Figs. Basidiobolus ranarum commonly is associated with insects and amphibians in terrestrial habitats. However.30]. [31] used a greatly expanded molecular data set of SSU rDNA. have been important in the food processing industry for their production of amylases. Blastocladiella emersonii and Allomyces species (Blastocladiales) have been important model organisms for studying biochemical and cellular processes in zoosporic fungi. excluding Amoebidiales). These true anaerobic fungi have been the most intensively studied chytrids because of their significant participation in rumen digestion and the potential for applications of their cellulolytic enzymes. especially Rhizopus and Mucor species. species of Basidiobolus have a rocketlike forcible spore release mechanism that differs from the remaining species of Entomophthorales. LSU rDNA. 3). B.2. the larger families Mucoraceae. with certain species in the Entomophthorales. and Pilobolaceae were polyphyletic. Thamnidiaceae. and alcohols. The relationships among the monophyletic lineages. have not been well supported in most cases (Fig. Some species of the Mucorales. 2.3. ranarum falls within the zygomycetes (Fig. The Basidiobolus example is worth highlighting as yet another persistent conflict between genetic and morphological data. An early molecular study [20] placed the Glomales in . most species of Basidiobolus share capilliconidia. it has been placed within the core chytrid group in analyses using rDNA [17. however.33]. distinctive animal-dispersed diaspores. and 11 morphological traits to produce a tree providing a new view of the Mucorales but somewhat contradictory to morphology-based phylogenetic hypotheses. Zygomycota The Zygomycetes comprise a monophyletic group if the Glomales and. and several members of the Mortierellales were identified as a basal outgroup. In trees based on analysis of . These include Entomophthorales.Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 33 considered one of most significant mycological discoveries of the 20th century. Mucorales. Although this species lacks flagella. Kickxellales (excluding Spiromyces). In addition. are excluded. Basidiobolus ranarum (Entomophthorales). The striking result of this and subsequent studies [32. peptidases.31]. carotenoids. Several traditional zygomycete lineages are considered to be monophyletic.29. O’Donnell et al. 3) [8].25. Morteriellales. the group has been estimated to be associated with 80% of the world’s plant species.and -tubulin genes. Basidiobolus has a distinctive spindle pole body (nucleus-associated organelle) with microtubular structure reminiscent of centrioles that are known only among chytrids [3. including transformations among intermediates in cortisone synthesis. 3) comprise fungi with an arbuscular mycorrhizal (AM) habitat and. 3. Compared with members of the Entomophthorales. which have cannonlike mechanisms. organic acids. Dimargaritales. rennins. however. Trichomycetes (Harpellales. A monophyletic grouping of Mucorales was supported.

Other major taxa of the Taphrinomycota include Pneumocystis carinii.g. and disease (e. but rather represent a series of basal lineages in phylogenetic trees based on nuclear SSU rDNA.g. and Taphrinomycotina (Archiascomycetes.4. The largest order of the Taphrinomycota is the Taphrinales.2... and this placement has been reinforced by subsequent studies [17–19]. 3. Saccharomycotina (Saccharomycetes. Candida albicans. and immunological reactions against monoclonal antibodies are consistent with molecular evidence. a monophyletic clade basal to the Ascomycota-Basidiomycota clade. 4). A surprising finding of the study was the discovery of two previously unrecognized lineages represented by the genera Archaeospora and Paraglomus [19. filamentous.36] (Fig.000 described species [15]. and Saitoella complicata.37–39].g. The Ascomycota is composed of three major groups or classes. The Saccharomycotina are supported as a monophyletic clade and probably share a most recent . mostly filamentous. which includes about 100 species of plant pathogenic fungi [41]. Saccharomyces cerevisiae. Two major lineages exist within the Glomales. 3. Ascomycota The Ascomycota is the largest phylum of the Kingdom Fungi. Certain species are inseparably linked to human civilization and have been domesticated for food beverage production (e. one includes Glomus as the sister to Gigaspora and Acaulospora [19]. including Pezizomycotina (Euascomycetes. a facultative human pathogen) and mutualists and endosymbionts of arthropods and animals [44].g. Saccharomyces cerevisiae). Most fungi used in industrial processes and as biotechnological and molecular biological tools belong to the phylum.34 Bills et al. Ascomycetes are characterized by the production of meiospores (ascospores) within sac-shaped cells (asci). despite many superficial morphological similarities shared with Glomales. and Trichophyton rubrum). Candida albicans. Saccharomycotina Most fungi that biologists consider as ‘‘true yeasts’’ are members of the Saccharomycotina (Saccharomycetes). Pneumocystis carinii..g. fatty acid profiles. Relationships of the major groups within each of these classes are still tenuous.1. diverged outside of the Glomalean lineages. and possibly sporocarp-producing species. most are represented by well-supported terminal clades that are linked by poorly supported more basal nodes [9..4. an asexual yeast. Penicillium chrysogenum). Studies involving multiple independent loci are needed to test the validity of the Taphrinomycota concept. a paraphyletic assemblage of basal taxa) [35. sporocarp-producing and mitosporic or conidial forms). Species of the class are ubiquitous in all environments and habitats.4. medicine (e.43]. The ascoma-producing earth tongue genus Neolecta also appears to be grouped among the Taphrinomycota [42. They are among the most commonly encountered fungi and have transformed human civilization through food (e. the causal agent of pneumocystis pneumonia.34] which. The Taphrinomycota are not strongly supported as a monophyletic group. Taphrinomycota The Taphrinomycota (Archiascomycetes) are recognized almost solely on the basis of phylogenetic analyses of rDNA sequence data [35. Differences in mycorrhizal morphology.40] and include yeastlike. with approximately 32.36. 3. baker’s and brewer’s yeast). the fission yeasts (Schizosaccharomyces pombe and its relatives). the true yeasts). whereas others function as mammalian pathogens (e..

whereas others. like Ascoidea spp. from analyses of nuclear rDNA and RNA polymerase II.3. Some important metabolites and enzymes are indicated to the right. however.4. alternating between filamentous and unicellular growth. and Pezizomycotina.Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 35 Figure 4 Major clades of the phylum Ascomycota. The group shares a number of ultrastructural features. Numerous species exhibit dimorphism. sporocarp-producing taxa and their equally diverse asexual lifecycle phases (Coelomycetes and Hyphomycetes). common ancestor with the Pezizomycotina [37. 3. and the major lineages and orders of the Pezizomycotina polytomy. display predominantly filamentous growth [46] and species of Cephaloascus-producing ascophores. Pezizomycotina The Pezizomycotina (Euascomycetes) are the largest class of Ascomycota and include the major lines of filamentous.45]. which have been interpreted as ascogenous hyphae [47]. including unique details of nuclear division and ascospore delimitation. The dominant phases of the lifecycle are yeast cells that lack the development of ascogenous hyphae and sporocarps. including the three classes Taphrinomycotina. The Pezizomycotina perhaps are the . Saccharomycotina..

The most basal lineage of the apothecial fungi. etc. the starting material for the antifungal drug Cancidas (Merck) [59]. the nomenclature for the major groups within the Pezizomycotina has been in constant flux. The apothecium in these fungi can vary from cup shaped to earth tongue to hysteriiform. along with other types of nucleotide sequence data (e. Gyromitra. the nuclear SSU rDNA. and animals. however. Convergent evolution in ascus morphology has been apparently common. sometimes called the inoperculate discomycetes.36 Bills et al. most successful group of fungi. by analyses involving numerous independent loci [5]. It has been postulated that the poorly resolved base of the Pezizomycotina clade was the result of a radiation event in which many of the major clades of the class originated over a relatively short time [37.52]. pathogenic. Morchella. mycorrhizae.38. and saprobes of litter and woody debris. the Pezizomycotina are characterized by numerous well-supported terminal clades. and numerous familial revisions have been proposed recently [57]. therefore. In the past few years. the remainder of the euascomycete diversity emanates from a large polytomy [52]. whereas saprobic lineages are able to decompose essentially all known organic substrates.61].).39.. and mutualistic with plants. mycologists have tended to interchangeably use both formal and informal designators that refer to major groups [3]. The Pezizales are the next basal lineage of the class [37. however. an obscure clade of nematophagous ascomycetes [55.g. depending on the species. The order is grossly polyphyletic. the order is not strongly supported as monophyletic. hold great promise toward resolving many components of the euascomycete polytomy [5. which is characterized by an exposed fertile layer of asci (hymenium). particularly among mechanisms of ascus dehiscence [48–51]. These morphologies have undergone repeated losses and gains during the evolutionary history of the fungi. These hypotheses are based on a single gene. as an order.60. As with other higher groups of fungi. The primitive sporocarp morphology of the Pezizomycotina appears to be that of the apothecium [9. Moreover.g. .56]. They include numerous macroscopic forest fungi (e. it includes numerous independent lineages of truffles [57] and. These fungi include most of the most notorious mycotoxin-producing fungi. EF-1 ). Traditional classifications have been based primarily on the morphology of sporocarps (ascomata) and asci. The Pezizales are characterized by the production of operculate asci. with nutritional modes ranging from parasitic. Alternative classifications have been proposed [54] with the ultimate goal of accurately reflecting monophyletic clades of the Pezizomycotina. probably contains the majority of ectomycorrhizal species of ascomycetes. algae. Molecular phylogenetic studies have shown that most traditional taxa based on ascomatal morphologies (e.31].g. as well as the lion’s share of fungi that produce bioactive metabolites. Helvella.. Analyses of nucleotide and predicted amino acid data from RNA polymerase II [58].37. or at least resolvable. cleistothecia. apothecia.. which produces pnuemocandin B0. and perithecia) do not represent natural groups. however. but the relationships of the major clades still await definitive resolution. -tubulin.38].56] and are possibly the best known and the largest group of apothecial fungi. The Helotiales are another major group of apothecial and anamorphic fungi and include endophytes. and similar hypotheses for the major groups of eukaryotes [53] later proved erroneous. The Helotiales. as well as that of the Pezizomycotina. with representatives found across the ‘‘backbone’’ of the Pezizomycotina tree [56. Also found among the Helotiales is the fungus Glarea lozoyensis. includes the genus Orbilia. In most phylogenetic analyses of the SSU rDNA. are distinguished from the operuculate Pezizales because the asci lack the operculum. plant pathogens.

and Chaetothyriales—because the majority of the species belong to these orders and most of molecular phylogenetic analyses have been performed with these taxa. aflatoxin and patulin). in general biology. Among them are important anamorphic fungi that produce important medicines (e. Pyrenomycete (Sordariomycetes) is another outdated class name that is now used to designate a clade.. they are not supported as a member of the Dothideales [67]. rather than a specific ascomatal morphology. and their status as a weakly supported monophyletic group is sensitive to taxon sampling [66]. Plectomycetes. with at least two independent origins of ascostromatic development [39. The Dothideales are largely characterized by the lack of paraphysoids [63.. which are possibly more closely related to the Pleosporales and Pyrenomycetes. the Chaetothryriales are more closely related to the plectomycetes in analyses of both SSU rDNA [39. Furthermore. it has been hypothesized that the Plectomycetes are a highly derived lineage. Dothideales. the asci of many species are bitunicate asci and display a ‘‘jack-in-the-box’’ mode of dehiscence [65]. chitin synthetases [23]. is also supported as a member of the plectomycete clade and is the only known ascomycete truffle lineage outside of the Pezizales [74].g. whose origin was associated with the loss of a lichenization [62]. however. valley fever caused by Coccidioides immitis.. a genus of ectomycorrhizal false truffles.73]. a finding consistent with the synapomorphy of sterile cells (pseudoparaphyses) interspersed among the asci [63. Furthermore.72.68]. Rather. characterized by evanescent apical pseudoparaphyses [69.71] and chitin synthetases [23].66. have been limited to the clade of Eurotiales and Onygenales. Elaphomyces. and the second clade comprised the Arthoniales. have been demonstrated [38]. In an analysis of nuclear SSU rDNA. and athlete’s foot caused by Trichophyton rubrum). and RNA polymerase II [58] have strongly supported the Eurotiales/Onygenales relationship. Here we will focus on three orders—Pleosporales. The Chaetothyriales.Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 37 The other major group of apothecial ascomycetes includes the lichenized species of the Lecanorales (some 7000 species). they often are considered the quintessential mutulistic symbioses. The pyrenomycetes exhibit a wide breadth of macromorphology and micromorphology and include most ascomycetes that produce flask-shaped ascomata or perithecia with unitunicate asci. life-threatening diseases (e. The loculoascomycetes are fungi that with ‘‘ascostromatic development. and problem mycotoxins (e. The Eurotiales and Onygenales include numerous species that produce closed ascomata (cleistothecia) and relatively simple.67]. and Claviceps purpurea.g. evanescent asci (protunicate asci). .g. Tolypocladium inflatum. Phylogenetic analyses of nuclear SSU rDNA [48]. an obsolete taxon formerly referring to fungi with a completely closed ascomata [48. The Pleosporales are supported as a monophyletic clade [66]. Within the pyrenomycetes are found the historically important fungi. lung disease caused by Histoplasma capsulatum. One lineage consisted of the Lecanorales sensu lato. In addition. and the integration of the two in phylogenetic analyses and classifications remains a major goal in fungal systematics [62]. Fewer phylogenetic studies have been performed on lichenized ascomycetes than nonlichenized species.’’ in which ascogenous hyphae are produced in preformed locules within a stroma [63. which produces cyclosporine A. Inclusion of all three orders in a monophyletic Loculoascomycetes is rejected by the data. the source of the ergot alkaloids. The plectomycete clade includes an unusually high concentration of fungi that are of human interest.68].64]. two major clades of lichenized ascomycetes. are supported as a monophyletic group. which contain most taxa that have been considered plectomycetes. aspergillosis caused by Aspergillus fumigatus. penicillin and lovastatin).70]. indicating two independent origins of the lichen symbiosis. and lichenization has been independently lost more than once in both these lineages [62].

Phyllachorales.g. Modern interpretations include the classes Urediniomycetes (the rusts and relatives).g. which represent multiple and repeated losses of the perithecial neck and ostiole [48. shelf fungi. The orders Diaporthales.g. Phylogenetic studies have revealed that the Phragmobasidiomycetes are not monophlyetic and that the septate basidium is probably an ancestral character state for the Basidiomycota [83–86].90]. 5) [85..93.94]. the class contains important genetic model organisms (e.50. Although most species produce perithecial ascomata. 3. including many of the common macroscopic forest fungi (e.2). Ustilagiomycetes Fungi of the Ustilaginiomycetes (smuts) are characterized by the production of a diploid overwintering spore. Molecular analyses point to a greater degree of convergent evolution among macromorphology and micromorphology than was previously appreciated. toxins. and Xylariales—rival the Eurotiales in their number and complexity of their secondary metabolites.81]. However.5. Furthermore. and the Exobasidiomycetidae includes the Doassansiales.g. Sordariales.75–80]. as are many of the ordinal relationships within each of them [83.85. Basidium morphology has been the focal point of past classifications of the Basidiomycota with fungi possessing septate basidia classified in the Phragmobasidiomycetes (Heterobasidiomycetes) and fungi with nonseptate basidia classified in the Homobasidiomycetes (Holobasidiomycetes) [82. and Hymenomycetes (mushrooms and relatives) in the Basidiomycota (Fig. because of the teliospore [89. chestnut blight (Cryphonectria parasitica) and dogwood blight (Discula destructiva)].83]. Neurospora crassa and Podospora anserina). the pyrenomycetes clade also possesses numerous lineages of cleistothecial fungi (formerly placed in the plectomycetes). neither the Phragmobasidiomycetes nor the Teliomycetes are supported as monophyletic taxa in molecular phylogenetic analyses [84. Because of certain similarities in morphology and parasitic relationships with plants..88]. the teliospore and the overall the ‘‘smut’’ morphology (i. The phylum is characterized by meiospores (basidiospores) that are borne upon club-shaped cells or basidia. Entylomatales.. Also. 3. and dimorphic lifecycles with a saprobic yeast phase and a pathogenic filamentous state (see section 3. [e.51. and degradative enzymes.86]. puffballs). the Ustilaginomycetidae includes the Ustilaginales and Urocystales.5.. Georgefish- . Sordariales.87. the teliospore. Both groups have been the subjects of controversial and conflicting classifications [84].38 Bills et al.5.e.91]. basidiospores produced in a darkly pigmented sooty mass) are yet more examples of convergent evolution in morphology among fungi [93]. Hypocreales.. Exobasidiales. Basidiomycota The Basidiomycota is the second largest phylum of Kingdom Fungi (approximately 23. or in the Phragmobasidiomycetes. Lulworthiales.1. infamous forest pathogens. mushrooms. Ustilagniomycetes (the smuts). The relationships of the three classes within the Basidiomycota are still unsettled.000 described species) [15]. Some groups of the Pyrenomycetes—especially the Hypocreales. members of this group often have been confused with some of the Urediniomycetes. and Xylariales constitute the clade [48. Microascales. Metarhizium anisopliae and Beauveria bassiana).51. and virulent insect pathogens (e. Ophiostomatales. Traditional classifications placed the two groups in the Teliomycetes. Ultrastructural studies and molecular phylogenetic analyses support the Ustilagniomycetes as possessing three major clades or subclasses with the following orders: the Entorrhizomycetidae includes the Entorrhizales. Halosphaeriales. based largely on septate basidia [82.

and the genus Mixia [92. Atractiellales. Urediniomycetes The Urediniomycetes is a large group of dimorphic and yeastlike fungi that includes the Uredinales and Septobasidiales of the Urediniomycetidae. Many taxa that were once thought to belong to the Ustilaginales clade (e. Urediniomycetes. More derived autoecious species exist on a single host with a reduced number of distinct stages. The Septobasidiales are an interest- . In the filamentous forms. and Tilletiales [94].95]. Urediniomycetes share the trait of a septate mycelium with simple pores that lack any associated flaring of the cell wall surrounding the pore (dolipore septum) or associated pore membranes (parenthosomes) [84].Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 39 Figure 5 Major clades of the Basidiomycota. and Hymenomycetes and their respective major orders from analyses of SSU and LSU rDNA sequences.96]. Some important metabolites and enzymes are indicated to the right. Microbotryum) have been convincingly demonstrated to be derived members of the Urediniomycetes [93. Erythrobasidium-clade.2. including the three classes Ustilaginiomycetes. Graphiolales.g. 3. The most elaborate lifecycles are those of the macrocylic heteroecious rusts. Microbotryomycetidae. The Uredinales contain plant parasites that exhibit some of the more complex lifecycles known among fungi.5.. requiring two distantly related plant hosts to complete their lifecycle. Microstromatales. Agaricostilbomycetidae. erales. and possessing up to five distinct spore stages. Malasseziales.

1) are divided into two large subgroups [5]. and two groups of slime molds (see section 5). The most derived clade of the Hymenomycetes includes those homobasidiomycetous taxa with nonseptate basidia and no yeast phase in their lifecycle. the crust (corticioid) morphology is found in all eight of the major clades and the cantharelloid clade contains four basidiocarp types. For example.. are members of the Hymenomycetes. 3. this character state is likely not without convergence [83]. however. animals. basidiomycetous yeasts are polyphyletic with the species found in no fewer than three clades of the Urediniomycetes. all of which contain multiple basidiocarp and hymenophore morphologies. with particular emphasis on the spore-producing tissues or hymenophore. KINGDOM STRAMINIPILA (HETEROKONT ZOOSPORIC FUNGI) The eukaryotes (Fig. including the Erythrobasidium clade and Agaricostilbum clade [96].97]. shelf fungi) familiar to most people.5. the fewest of any of the eight clades [83]. The other large group of eukaryotes is divided into two subgroups. and insect symbioses. including polyporoid. with the imperforated form being ancestral for the class.g. on either side of the pore. plant pathogens. jelly fungi.99]. one of these consists of the plants (including green algae) and the other subgroup. The organisms are characterized by zoospores with anteriorly or laterally biflagellate cells with one flagellum bearing tubular tripartite hairs and the other being smooth (whiplash). mushrooms. the dolipore septum. contains algae with chlorophylls a and c. and an assortment of ‘‘protozoan’’ groups. cantharelloid. These classifications have been rejected by numerous phylogenetic analyses of molecular data from both the nuclear and mitochondrial genomes [88. and Sporidiobolus [93. a finding consistent with these characteristics being ancestral ones for the Basidiomycota. and gomphoid-phalloid clades. The Microbotryomycetidae is a heterogenous group of species that is recognized mostly on molecular characters and includes smutlike species of Microbotryum and yeastlike species of Rhodotorula. As mentioned already. euagaric. in the Tremellales is found the human pathogen Filobasidella neoforman (anamorph Cryptococcus neoformans).96]. hymenochaetoid. An estimated eight major clades exist within the Hymenomycetes. Traditional classifications of the homobasidiomycetous fungi relied largely on basidiocarp morphology. several groups of heterotrophs that previously were classified as fungi. The general theme that has resulted from these studies is that the overall basidiocarp morphology has been subject to repeated episodes of convergent and divergent evolution and that it is not a phylogenetically informative trait at higher taxonomic levels.g. 4. litter and wood decay. However. The orders Ceratobasidiales and Tulasnellales. which include plant pathogenic (e. Also. thelephoroid. The clade is unified by a unique mycelial septum structure. perhaps closely related to the Auriculariales [92. which involves the flaring of the cell wall near the pore of the septum and a membrane structure.98. Rhodosporidium. This clade includes the fungi with complex macroscopic fruiting bodies and the trophic modes ranging among mycorrhizae. Rhizoctonia) and saprobic species. The parenthosome may be perforated or not. depending on the clade. the Straminipila. Many of these taxa possess septate or deeply divided basidia and a dimorphic lifecycle with a yeast phases. ing group of specialized symbionts of scale insects. one of these groups includes fungi. russuloid. Hymenomycetes The Hymenomycetes comprise the conspicuous fleshy fungi (e. in some instances one (the .3.40 Bills et al. bolete.. the parenthosome.

Chromista and Heterokonta. For many years. Tubular hairs are sometimes found as ornamentation of certain zoospore cyst walls.2. and cytochrome oxidase (cox II) [109–112]. algae and animals. chrysophytes) but also heterotrophs (oomycetes. Within the kingdom other name changes have occurred. may be included in Peronosporomycetidae [110]. although it is unclear if the heterotrophic straminipiles are monophyletic [107. Phylogenetic hypotheses for oomycetes generally are in agreement with two major clades defined.g.116]. One debatable point is the placement of Sapromyces and. are common terrestrial organisms associated with decaying vegetation. and pesticide sensitivity that distinguished straminipiles from true fungi [100.Phylogeny of the Fungal Kingdom and Fungal-like Eukaryotes 41 smooth one) or both flagella have been lost secondarily during evolution. small subunit rDNA. AND DICTYOSTELIDS: PLASMODIAL AND CELLULAR SLIME MOLDS Many cell biologists and biochemists are familiar with common soil-dwelling cellular slime molds because of the extensive body of work carried with Dictyostelium discoideum [113–115].106]. Recent trees have been based on morphology and biochemistry and three genes. alternatively. or ‘‘true’’ or plasmoidial slime molds. 1) [5. a group of taxa with obligately fermentative respiration. Oomycota The oomycetes are common in both terrestrial and aquatic environments. the primarily marine labyrinthulids and thraustochytrids have been placed among the straminipiles. In an analysis of EF-1 amino acid sequences. 4..106]. confirmation of their distant phylogenetic position from fungi and similarity to certain algal groups based on molecular data was expected [102–105]. hyphochytrids. MYXOMYCETES. The Saprolegniales appear to be a monophyletic group within the oomycetes. the myxomycete Physarum polycephalum was placed as a sister group to several . PROTOSTELIDS. large subunit rDNA. Some members are causal agents of devastating crop diseases. emphasizing morphological and biochemical traits. The myxomycetes (Myxogastridae). golden brown algae. which in the past were roughly equivalent terms. Rhipidales are possibly an independent lineage within the oomycetes of equilavent rank to Saprolegniomycetidae and Peronosporomycetidae or. The name Straminipiles (also known by the variants ‘‘stramenopiles’’ and ‘‘straminopiles’’) has undergone numerous changes and conflicting usages [102. were used before the widespread adoption of Straminipiles. and Plasmopara viticola (downy mildew of grapes). diatoms. 5.101]. brown algae. In addition to the apparent sister taxa Oomycota and Hyphochytriomycota. Saprolegniomycetidae (water molds) and Peronosporomycetidae (plant and animal parasites).104. the order Rhipidiales. and labyrinthulids and thraustochytrids). therefore. most notably the use of Peronosporomycetes as a class name for the monophyletic grouping of Oomycota and Hyphochytriomycota [102. In this kingdom level group are found not only photosynthetic organisms with chlorophylls a and c (e.108]. Phylogenetic information on various slime mold groups indicates that plasmodial and cellular slime molds constitute a monophyletic group (Fig. Phytophthora cinnamomi (damping off disease of many plants). which traditionally have been grouped with the fungi. mycologists recognized that these organisms were distinct from true fungi. such as Phytophthora infestans (late blight of potato). such as flagellation. Their trophic modes range from saprobes to virulent pathogens of plants. cell wall composition. Therefore.

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fungal compounds also have been described as inhibitors of nearly 100 different 49 . immunosuppressants. these references are not included in Fig. and antibacterial activities are reported most frequently from fungal metabolites. Madrid. A total of 718 nonredundant references were surveyed. Additionally. 1. Antifungal. which have become extremely diversified during the course of evolution. is really astounding. The results of a survey in the literature for fungal metabolites with biological activity reported from 1993 to 2001 are summarized in Fig. Merck Sharp & Dohme de Espana. nonribosomal peptides. and terpenoids. Overall. more than 60 references were found during the same period for fungal compounds without any reported biological activity. a few report new biological activities identified for previously known metabolites. plus combinations of these). contributing significantly to the welfare of mankind and to the spectacular rise in life expectancy observed in the second half of the century. The vast majority of the references relate to newly discovered compounds.3 Biological Activities of Fungal Metabolites ´ FERNANDO PELAEZ ´ ´ Centro de Investigacion Basica.A. as reported to date. ˜ S. more than half of all the fungal metabolites described in this period showed one or more of these activities. and cholesterol-lowering agents derived from fungal compounds have been used in the clinic during the past 50 years. accounting for more than 1500 metabolites or families of metabolites. it is not surprising that the number of targets hit by fungal compounds. 1. displaying a broad array of biological activities. Spain 1. However.. Antibiotics. In view of this remarkable structural diversity. antitumor. The amazing range of chemical structures observed for fungal metabolites is derived from a relatively small number of basic metabolic pathways (mainly polyketides. antifungals. INTRODUCTION Fungal metabolites have had an extraordinary impact on the quality of human life during the 20th century.

50 ´ Pelaez .

Aspergillus spp. The prevalence of systemic fungal infections has rised significantly during the past decade. it is impossible to go over all the compounds discovered or biological activities reported even in this limited period of time.Biological Activities of Fungal Metabolites 51 types of enzymes and receptors or as effectors of different biological activities measured in whole cell based assays. Bioorganic and Medicinal Chemistry Letters. Tetrahedron Letters.. Journal of Organic Chemistry. these were counted as just one reference.. Some of them. Serious invasive fungal infections caused by Candida spp. A total of 718 references were surveyed describing new metabolites with biological activities.. 1. Phytochemistry. 1). The journals screened included Journal of Antibiotics. and other pathogens represent an increasing threat to human health. and transplant patients). Organic Letters. It seems plausible that one of the reasons why antibiotic activity (in its broader sense) stands out as the major type of biological activity reported has to do with the modest resources necessary to test for non–mode-of-action antibacterials. Emphasis has been given to compounds that have recently progressed beyond discovery to subsequent phases of development (e. Journal of the American Chemical Society. Cryptococcus neoformans. as shown in Fig. the scientific or medical impact. Mycotoxins. when compared with the resources required to check for other biological activities needing more sophisticated techniques (enzymatic assays.). ANTIFUNGAL Fungal infections in humans range from superficial conditions of the skin (e. fungal metabolites that produce animal or human toxicity. mainly due to the more frequent prescription of broad-spectrum antibiotics. antifungals. marked increases in immunocompromised people (AIDS. especially those produced by fungi growing on grains and food. or cytotoxic activities. as well as to those that have prompted additional research regarding their mechanism of action.. 2.g. life-threatening diseases. the use ← Figure 1 Distribution of biological activities reported for fungal metabolites in the period 1993 to 2001. The selection has been based on criteria such as the number of compounds reported with a given biological activity. References from these and other sources were also obtained by searching in the databases Biosis. This chapter presents an overview of the most relevant biological activities with potential therapeutic relevance (mainly for human health) of fungal metabolites described during the last decade. cancer. and Current Contents. Likewise. Embase. in vivo activity in animal models. The biological activities and metabolites are arranged by therapeutic areas. and the Proceedings of the Natural Academy of Sciences. and these in turn have been sorted according to the number of references found for each.g. phytotoxins. Tetrahedron. ringworm and athlete’s foot) and nails (onychomycoses) to disseminated. represent important public health problems. medicinal chemistry. and the stage of development. have been studied extensively. although also frequently reported as fungal metabolites (Fig. When a metabolite was described in several different papers. Natural Products Letters. . Journal of Natural Products. etc. They will not be treated in this chapter unless they show any additional biological activity of potential therapeutic interest. For reasons of space. will not be reviewed here. clinical development).

the number of antifungal compounds for which the mechanism of action is known is small. glucan synthesis is the only process of cell wall synthesis that has been used successfully for the development of a new drug product.7].or (1. An astounding number of fungal metabolites with antifungal activity have been described in the literature over the last 10 years.3). and an aging patient population [1]. Glucan is a polysaccharide built of glucose monomers linked by (1.1. The pneumocandins. cyclic hexapeptides N-acylated with an aliphatic chain of different length [6]. Comparatively speaking. The semisynthetic echinocandin Cancidas is a substituted aza-derivative of pneumocandin B0 (Fig. The compound has been shown to be effective in animal models of disseminated candidiasis. a metabolite produced by Penicillium spp. have been successfully used to develop an antifungal drug recently launched in the market. The most classic examples of inhibitors of glucan synthesis are the echinocandins. an echinocandin derivative with a sulfate ester moiety in the hexapeptide nucleus. many members of the family have been discovered in diverse fungi [6. Since the first echinocandin was discovered in the early 1970s. Cell Wall One of the targets for novel antifungals under active investigation is the fungal cell wall. and Pneumocystis carynii pneumonia [7. Clinical trials have demonstrated that caspofungin is well tolerated and is efficacious in the treatment of esophageal candidiasis [8. Cancidas has recently been approved in the United States and in several other countries for its use against invasive aspergillosis in patients refractory or intolerant to other therapies. Most antifungal compounds described from fungi have been discovered by using non–mode-of-action based screens. a type of echinocandins derived from the fermentation of the fungus Glarea lozoyensis. and . the development of new antifungal agents. and it is an essential component of the cell wall. Structural variants arise from different substitution patterns on the hexapeptide ring or in the fatty acid side chain. Other echinocandin-like antifungal agents in clinical trials are micafungin (FK463). These antifungal agents show some limitations. Antifungal agents acting on this target are expected to be inherently selective and should have fungicidal features that make them particularly attractive for clinical development. the most recent drug based on a fungal metabolite that has reached the market at the time of preparation of this manuscript is the antifungal agent Cancidas (caspofungin acetate. aspergillosis. and its mode of action remains unclear even today [4]. Actually.bonds.8]. These will be the subject of the remainder of this section. guaranteeing many of its physical properties [5]. 2. At present. such as the significant nephrotoxicity of amphotericin B and the emerging resistances to azoles. Only a limited number of antifungal agents (polyenes and azoles) currently are available for the treatment of lifethreatening fungal infections.52 ´ Pelaez of central venous catheters. Although lipid formulations of polyenes and new triazoles have shown improved characteristics over the original compounds. Griseofulvin.6). which has been used in the clinic for several decades to treat skin infections by fungal dermatophytes. preferably with novel mechanisms of action. Merck). discovered in 1939. is an urgent medical need [1–3]. was discovered using this strategy.9] as well as in invasive candidiasis [10] and aspergillosis [7. 2). by simply looking for compounds able to inhibit the growth of a target yeast or filamentous fungus.8]. that is. Inhibitors of glucan synthesis have been shown to possess antifungal activity in vitro as well as in vivo in many different animal models.

Pneumocandin B0. an inhibitor of respiratory chain. papulacandin A. and enfumafungin. . Strobilurin. a protein synthesis inhibitor.Biological Activities of Fungal Metabolites 53 Figure 2 Antifungal compounds. glucan synthesis inhibitors. Sordarin.

and inositol phosphoceramide synthase. Besides echinocandins and the like. cyclodecapeptides with two lipophilic tails [11]. all of them produced by fungi. Furthermore. Although these compounds were inactive or only weakly active in vivo in a mouse model [7]. The papulacandins (Fig. Fig. minimoidin was discovered as an inhibitor of the fatty acid elongation pathway.. mainly due to their limited potency in animal models [14]. cyclic depsipeptides lacking a long lipophilic radical [13]. they represent a new paradigm in the search for antifungal compounds with this mode of action. 2). Three key enzymes in the pathway have been targeted in the search for novel antifungals: serine palmitoyltransferase. neither papulacandins nor any of their relatives have been developed as drugs. Fumonisin B1 and australifungin inhibit ceramide synthase. Sphingofungins. Ceramide has been implicated as a component of an essential cell-signaling pathway in Saccharomyces [17]. and the clavariopsins.2. Inhibition of sphingolipid synthesis results in growth inhibition and cell death [16]. Sphingolipid Synthesis Although present in relatively small proportion in the fungal cytoplasmic membrane. which has a terphenyl head group and a C5 chain [3. A series of related compounds have been discovered over the years. viridiofungins. This polar moiety can be a glycoside (as in enfumafungin and ascosteroside. and an unnamed sphingosine-like metabolite isolated from Colletotrichum acutatum inhibit serine palmitoyltransferase. other cyclic peptides have been described as glucan synthesis inhibitors. to date.19]. Inhibitors of these three enzymes have been isolated from fungi [7. 2) are glycolipids. and potential toxicity. Although a number of steps in the human and fungal sphingolipid biosynthetic pathway are similar. 2. However. such as the arborcandins. and they could be useful as the basis for the development of improved drugs. limited whole-cell or in vivo activity. application of this idea to antifungal therapy is not an easy task due to the eukaryotic nature of fungi and therefore the high .3. ceramide synthase. there are several enzymes found exclusively in fungi. only two other types of glucan synthesis inhibitors are known: the papulacandins (and related compounds).54 ´ Pelaez anidulafungin (LY303366. Sphingolipids also are involved in the synthesis of glycosylphosphatidylinositol anchors in Saccharomyces. thus making sphingolipid synthesis a potential target for antifungal therapy. However. Protein Synthesis Protein synthesis has always been considered to be one of the more attractive targets in the development of antimicrobial agents. discovered in the late 1970s. besides cyclic lipopeptides.7]. Although some of these compounds showed remarkable potency (e. and aureobasidins and khafrefungin inhibit inositol phosphoceramide synthase.g. 2. and the acidic triterpenes. khafrefungin and minimoidin). The success with the lipopeptide class of glucan synthesis inhibitors has prompted interest from the industry in the search for other structural types with improved features over the echinocandins (especially because of their lack of oral absorption). with 12 amino acids and a 3-hydroxypalmitoyl moiety [12]. none of them have been subjected to further development due to issues of solubility.16. V-echinocandin). sphingolipids are essential for cellular functions. a succinate (as in arundifungin) or a sulfate-derivative aminoacid (as in ergokonin A). However. and they appear to be the major repository for very-long-chain fatty acids in fungi [18]. the compound FR901469. The most recently discovered class of glucan synthesis inhibitors are triterpenes containing a polar moiety [15].

a protein secretion inhibitor. hypocrellin A. an angiogenesis inhibitor. an antimitotic agent. TNP-470. clavaric acid. a cell sensitizer. . a cytotoxic compound. rhizoxin. PKC inhibitors.Biological Activities of Fungal Metabolites 55 Figure 3 Antitumor compounds: irofulvene (HMAF). ( ) balanol and calphostin C. an inhibitor of ras FPTase. brefeldin A.

24]. ANTITUMOR Although there is a substantial number of antitumor agents derived from natural products in clinical use (doxorubicin.56 ´ Pelaez similarity between the fungal and mammalian protein synthesis machineries. etc. sales of azoxystrobin (Heritage) alone amounted to $415 million in 1999. have been isolated from diverse fungal species [7].4. The strobilurins and the related oudemansins are metabolites produced by many species of basidiomycetes belonging to the genera Strobilurus. 2). among the existing soluble translation factors. a substantial number of fungal metabolites have been reported to have either cytotoxic activity against tumor cell lines. and C. trifloxystrobin.). and/or cytotoxic to mammalian cell lines.). Recent publications from the groups at Merck and Glaxo demonstrated that sordarins are potent and selective inhibitors of translation in fungi. and it has been reported that these compounds may also be phytotoxic.22]. tropicalis. This has allowed their development as commercially useful antifungal agents. Antifungals in Agriculture Outside the area of human health. The most important family of antifungal agents acting at this level is the sordarins. krusei. C. fungal metabolites that have been used to develop several agricultural antifungal products (azoxystrobin. Crepidotus. This target is not fungal-specific. sharing the aglycone moiety of sordarin. 2) was isolated by scientists at Sandoz from fermentations of Sordaria araenosa [21]. Compounds in this class inhibited in vitro translation in C. etc. albicans. parapsilosis. acting by stabilizing the fungal EF-2–ribosome complex [20. make the sordarins a promising series for clinical evaluation. glabrata. kresoxim-methyl. despite their mode of action against a ubiquitous target. they show very low oral toxicity in mammals. C. The potent broad-spectrum in vitro activity and the fact that some of the sordarins have shown oral efficacy in animal models are significant advantages of these compounds. A derivative of illudin S. Some of the most relevant compounds are reviewed in this section. neoformans to varying degrees but were inactive against C. Early studies revealed that sordarin targeted a protein involved in the translation of mRNA. C. Several structurally related compounds. because it is functionally distinct from its mammalian counterpart [20]. produced by basidiomycetes from the genera Omphalotus and Coprinus [25]. elongation factor 2 (EF-2) has been identified as a suitable antifungal target. However. 3. depending on the derivative [24]. kefyr. 2. as well as by the ascomycete Bolina lutea [23. However. or target signal transduction mechanisms mediating cell proliferation. Sordarin (Fig. HMAF. has shown good activity in animal models of cancer either alone or in combination with other antitumor agents [26]. These compounds are inhibitors of the respiratory chain by virtue of binding to mitochondrial cytochrome b. MG114. taxol. The illudins are a group of sesquiterpene antibiotics often reported as antibacterial and antitumor agents. Fig. mention must be made of the strobilurins (Fig. insecticidal. and others. and C. These advantages. This is an important market. This compound has been tested in Phase . However. irofulvene (6-hydroxymethylacylfulvene. together with additional pharmacokinetic and toxicological data. vinca alkaloids. Oudemansiella. 3). none have been obtained from fungi. Mycena. for reasons that are as yet unclear.

which were competitive with either Ras or farnesyl pyrophosphate. and subsequent rational chemical synthetic design. and some preussomerins [40]. a macrocyclic lactone produced by Rhizopus chinensis. A series of related compounds derived from computer-based similarity searches using clavaric acid and an alkaline hydrolytic product.35]. to test the hypothesis that the lack of clinical efficacy in early trials could be influenced by inadequate exposure to the drug [37]. It was clear from the analysis that the design and synthesis of more potent inhibitors can be achieved and selectively directed to either substrate binding site on the enzyme. but they have not been tested in humans. exhibited IC50 against FPTase in a range from 0. produced by an undetermined Phoma species. cylindrols. The compound TAN-1813. and several others. the enzyme that catalyzes this prenylation. Although its mode of action is not clear. we have identified as many as eight families of inhibitors of FPTase derived from fungal fermentations: chaetomellic acids. oreganic acid. have been shown to inhibit growth of tumor cells. The compound is an antimitotic agent that inhibits tubulin polymerization by binding to the same site as vinca alcaloids [31].04 to 100 M. One of the enzymatic steps identified early on as essential in cell malignancy was prenylation of Ras protein.Biological Activities of Fungal Metabolites 57 II clinical trials in a variety of cancer models. such as phomopsin A from Phomopsis leptostromiformis [31]. the compound showed some activity in a study on non–small-cell lung cancer [36]. Clavaric acid has been used as a template for obtaining more effective inhibitors. Nonetheless. and one of the very few natural FPTase inhibitors reported with this mechanism of action. extensive efforts have been made by a number of groups to identify potentially useful antitumor agents by using screens based on biochemical processes related to the cascade of events mediating cell proliferation. The total synthesis of rhizoxin has been described recently [33]. was shown to induce the morphological reversion of . clavarinone. but the results were disappointing [34. both leukemias and solid tumors models. Normal and oncogenic Ras proteins are posttranslationally modified by a farnesyl group that promotes membrane binding. barceloneic acid. The most interesting of all these compounds was clavaric acid [40. Clavaric acid was the only reversible inhibitor of FPTase competitive with Ras peptides found in our screening program. irofulvene has been shown to promote caspase-mediated apoptosis in tumor cells by a mechanism involving the activation of Erk 1/2 and JNK 1 kinases [30]. Rhizoxin showed good activity in vivo against various preclinical murine models. A number of inhibitors of this enzyme are in clinical trials [39]. as well as in vincristine. the compound inhibited Ras-processing in NIH3T3 ras-transformed cells that express Ha-Ras. and other cancer types. FPTase inhibitors reported by other groups include the andrastins and kurasoins [43].41] (Fig. TAN-1813 [44]. without manifesting any toxicity at the doses tested. clavaric acid. Other fungal compounds have been described with the same mechanism of action as rhizoxin. In our own screening program of microbial natural products. but published results are not encouraging [27–29].and doxorubicin-resistant leukemia lines [32]. or the related compound ustiloxin A from Ustilaginoidea virens [38]. 3). kampanols. Rhizoxin has been tested in clinical trials in phase II in breast cancer. Another antitumor fungal compound that has been tested in humans is rhizoxin. melanoma. More importantly. More recent studies in humans have evaluated the use of rhizoxin in more prolonged dosing schedules. fusidienols. Modest changes in the structures of these derivatives dramatically changed their inhibitory activity [42]. Inhibitors of farnesyl protein transferase (FPTase). Although most of the clinically useful compounds used to treat cancer have been discovered as simply cytotoxic compounds.

and Penicillium sp. Inhibitors of HDAC are known to have detransforming activity—that is. For instance. and reducing the expression of angiogenic factors such as VEGF. diheteropeptin. including gene expression. the most characteristic being cyclic tetrapeptides containing an epoxyketone moiety. allowing the passage of DNA segments through these breaks before resealing. Thus. DNA topoisomerases are essential enzymes in the control of DNA topology and the multiple processes linked to DNA metabolism. This suggests that these compounds might be useful anticancer agents also due to their antiangiogenic properties [48]. HC toxin. Several fungal metabolites have been described as potent HDAC inhibitors. an alkaloid from Camptotheca acuminata. replication. These enzymes produce transient breaks in the DNA backbone. also from Paecilomyces sp. These compounds produce the irreversible inhibition of HDAC at nanomolar concentrations by covalently binding to the enzyme through the epoxide [49. Type I topoisomerases produce single-strand breaks. A plethora of cultured tumor cell lines have been shown to be susceptible to HDAC inhibitors. The potential of some HDAC inhibitors as therapeutic agents is being tested in clinical trials [47]. [58]. is a less potent irreversible inhibitor [45]. chlamydocin. whereas type II can simultaneously cleave both strands. discussed below as an antiparasitic agent [50]. it also has been reported that HDAC inhibitors have antiangiogenic activity in carcinoma mouse models. showing a potency comparable to other inhibitors. The resulting compounds are also reversible inhibitors.46]. although there may be other factors involved in the mode of action of these compounds [47].. such as trapoxins [49]. and others [52]. upregulating p53 and other tumor suppressor genes. are selective for type I [55. Structurally related fungal tetrapeptides lacking the epoxyketone also have been identified as HDAC inhibitors. Eukaryotic DNA topoisomerases are the target of several antitumor drugs already on the market or in advanced stages of development. the most relevant being saintopin and related naphthacenediones.56]. with potencies in the subnanomolar range [54]. discovered by screening using calf thymus DNA topoisomerase I. inhibition of HDAC has been shown to result in upregulation of some genes and downregulation of others. and others [50. Saintopin is an antitumor antibiotic produced by Paecilomyces sp. a reversible HDAC inhibitor isolated from Streptomyces hygroscopicus [53]. in some cases without noticeable side effects [47]. The synthesis of p21WAF1 (an inhibitor of cyclin-dependent kinases) is the effect most consistently observed. and studies using rodent models for cancer have shown that these compounds reduce the growth of tumors and metastases in vivo. whereas topotecan and other derivatives of camptothecin. and recombination. and the topopyrones. such as apicidin [46]. Acetylation and deacetylation of histones is known to play a critical role in the regulation of transcription in eukaryotic cells. Saintopin induces both topo I– and II–mediated DNA breaks. Hybrid structures have been synthesized combining the tetrapeptide moiety of trapoxin and moieties similar to the hydroxamic acid of trichostatin A. Structurally related molecules with the same mode of action also have been reported. The latter are . and this could explain the G1 arrest and G2/M block frequently observed in cells treated with HDAC inhibitors.52]. Besides their antitumor effect. they are able to induce morphological reversion from oncogen-transformed to normal cells [45. doxorubicin targets type II topoisomerase.51]. such as camptothecin [57].58 ´ Pelaez NIH3T3 cells transformed with K-ras and has antitumor activity against human tumor models in mice [44]. such as UCE1022. Several fungal metabolites have been described that target mammalian DNA topoisomerases. a linear epoxide. Depudecin. One of the most promising antitumor targets studied in the recent years is histone deacetylase (HDAC). isolated from Phoma sp.

produced by species of Penicillium and Ascochyta [23]. brefeldin A has been shown to affect the cell cycle and to induce apoptosis in cancer cells. Brefeldin A (Fig. isolated from an actinomycete [69]. but were inactive or much more weakly active on serine/threonine kinases such as PKC [70]. Studies on its mode of action have revealed that brefeldin is an inhibitor of protein secretion. is a submicromolar inhibitor of EGF-TK. the effect of these polycyclic quinones is due to the formation of hydrogen peroxide in the cells after photoactivation. differentiation. Hypocrellin derivatives are being studied for the potential treatment of colorectal and breast cancers and nonmalignant diseases. 3) are pigments derived from Hypocrella bambusae and Shiraia bambusicola. including antifungal. and actinic keratoses..65]. Hypocrellins (Fig. the compound shows pharmacokinetic properties that preclude its use in the clinics (poor oral bioavailability and rapid clearance from plasma). was originally isolated as an antibiotic in 1958. blocking traffic from the endoplasmic reticulum to the Golgi apparatus but not vice versa.68]. A number of oncogenes have been identified that encode for activated protein kinases. Small molecules or monoclonal antibodies inhibiting the tyrosine kinases associated with growth factor receptors such as EGF.68]. Furthermore. and antitumor effects [62]. two fungi used in traditional medicine in China and which have been thoroughly studied for their ability to sensitize cells to light and ultrasonic waves [60]. but later was established to have several other activities. some of these are derivatives of staurosporine. competitive with respect to both ATP and the substrate peptide. with potencies in the low micromolar range.67]. and this has prompted interest in its development as an anticancer agent [64. Apparently. A number of fungal metabolites have been described in the last decade as protein kinase inhibitors. as well as low aqueous solubility [66]. antiviral. showing selectivity for this kinase against other tyrosine and serine/threonine kinases [71]. Protein kinases are a large family of proteins playing critical roles in signaling pathways regulating cellular functions such as cell growth. which leads to apoptosis via activation of caspase-3 [61]. resulting in the disassembly of Golgi complex. Several inhibitors of the protein kinase C (PKC) also are being tested in humans. Inhibitors of cyclin-dependent kinases (CDKs). This compound. A family of resorcylic acid lactones structurally related to the antifungal agent hypothemycin has been reported as potent and selective inhibitors of MEK (MAPK/ERK kinase). and PDGF have been already launched (e. the paeciloquinones. Gleevec) or are in advanced stages of clinical development [39. the kinases controlling the cell cycle. and of the protein kinases involved in the signaling cascade of the Ras pathway (RAF. The compound BE-23372-M. emodin. and other related compounds [70] are inhibitors of EGF-TK.Biological Activities of Fungal Metabolites 59 very potent (comparable to camptothecin) and specific topoisomerase I inhibitors and also show antiviral activity against herpesvirus [59]. 3) is one of the fungal metabolites that have been the subject of extensive research in the last decade (a simple Medline search for the word brefeldin from 1993 to date produced 1709 references). For instance. isolated from Rhizoctonia solani. These compounds also inhibited other tyrosine kinases such as vAbl and c-Src kinase. including psoriasis. nematocidal. MEK) also have reached clinical trials [39. A number of brefeldin A prodrugs have been synthesized to address these issues [62. These compounds also inhibited the growth of several human epithelial cell lines and showed . and apoptosis. VEGF. These effects appear to be mediated by the inhibition of guanine nucleotide-exchange proteins for ADP-ribosylation factors that are essential in vesicular trafficking ([63] and references therein). However. acne. and this suggests that protein kinase inhibitors may have therapeutic value in the treatment of cancer.g.

It has been suggested that the teratogenic effect of secalonic acid D could involve PKC inhibition [86]. an effect that resembles that of the hypocrellins. such as fibroblasts [90]. 3). also has been used to develop an antitumor agent. PKA. The physiological role of this enzyme. This effect seems to involve activation of p53. but whether these other analogs are also protein kinase inhibitors has not been disclosed [73]. The compound is an ATP competitive inhibitor exhibiting low nanomolar Ki values. Fumagillin. and this activity is dependent on exposure to light [79]. This mechanism was observed only in endothelial cells and not in other cell types. renal cell carcinoma. Obviously. Another interesting PKC inhibitor derived from a fungus is calphostin C (Fig. the discovery and industrial development of penicillin from Penicillium chrysogenum stands out as a hallmark in the . Calphostin C. ANTIBACTERIAL Antibiosis against bacteria was the first described biological activity of fungal metabolites that resulted in a therapeutically useful drug. which has been shown to be an inhibitor of PKC. the mechanism mediating this effect is not totally clear [81]. Finally. inhibits PKC by competing at the binding site for diacylglycerol and phorbol esters. This compound has been used extensively as a research tool in an astounding number of publications. its semisynthetic derivative TNP-470 (Fig. 4. and other kinases [85]. secalonic acid D is an acutely toxic and teratogenic mycotoxin [84] produced by Claviceps purpurea and Penicillium oxalicum. Its expression correlates with cell growth. Other hypothemycin analogues named as aigialomycins recently have been reported showing antimalarial activity and cytotoxicity against several tumor cell lines. Several fungal metabolites have been reported as inhibitors of PKC. which was isolated from Verticillium balanoides in 1993 [74]. Specifically. Recent work has revealed that TNP470 produces the accumulation of p21CIP/WAF. and brain. The target of fumagillin and its derivatives has been shown to be methionine aminopeptidase-2 (MetAP-2) [89]. Crystal structures of PKA complexed with balanol have been elucidated [76]. 3) has been subjected to extensive studies in diverse types of cancer and also regarding its mechanism of action. calphostin C has been reported to have several other effects. produced by Cladosporium cladosporioides. These compounds are potent antiangiogenic agents [88]. structurally related compounds discussed above as photosensitizers (Fig. TNP-470 is in clinical trials in Kaposi’s sarcoma. A substantial number of analogs have been reported showing selectivity for PKC versus PKA or vice versa [77. but it is expressed in both endothelial and nonendothelial cells. One of the most relevant ones is ( )balanol (Fig. Again. inhibiting neovascularization by arresting endothelial cell cycle in the late G1 phase. and subsequently from Fusarium merismoides with the name azepinostatin [75]. one of the cyclin-dependent kinases inhibitors. but has no effect on tyrosine kinases. Besides being a PKC inhibitor. so the selectivity of fumagillin and TNP-470 to inhibit the endothelial cell cycle may involve other factors. is still unclear. Calphostin C has been shown to induce light-dependent apoptosis in a number of tumor cell lines at nanomolar doses [80]. which removes amino-terminal methionine residues from peptides. breast. it inhibits also other serine-threonine kinases such as PKA (cAMPdependent protein kinase). 3). and from other fungi. including directly inhibiting phospholipase D at nanomolar concentrations [82] and blocking Ltype calcium channels [83]. and other cancers [91].78].60 ´ Pelaez antitumor activity in a mouse model [72]. 3). as described in section 6. an old antibiotic used successfully to treat microsporidial infections in the clinic [87]. leading to cell cycle arrest at the G1 to S transition.

5). Fig. a huge number of fungal metabolites with antibacterial activity have been described. the inhibitors of 3-hydroxymethylglutaryl-CoA reductase that are the most widely used cholesterol-lowering drugs on the market. lovastatin (Mevacor. In the past 50 years. showed a broad spectrum of antibacterial activity. Nearly all the new antibacterial agents described from fungi have been discovered by using non–mode-of-action assays—that is. but these were not specific for the bacterial enzyme. like penicillin. The few exceptions found in the literature in recent years include clerocidin and other fungal inhibitors of DNA gyrase [100. produced by Fusidium coccineum and other species [23]. being as potent or more than ciprofloxacin against gram-positive and anaerobic bacteria [100]. cephalosporin has been at the origin of several generations of semisynthetic derivatives (e. is a -lactam inhibitor of bacterial cell wall synthesis. cefotaxime. but it is also indicated for intestinal infection with Clostridium difficile [97] and for infections of different organs by Staphylococcus aureus and other gram-positive bacteria [98. 5.99].Biological Activities of Fungal Metabolites 61 Figure 4 Fusidic acid. an inhibitor of protein synthesis in bacteria. which inhibits protein synthesis in bacteria at the level of the elongation factor G [93]. Originally discovered from the species Acremonium chrysogenum in the 1940s.101]. ceftriaxone.. It is active against gram-positive bacteria. ceftazidime. Fusidic acid (Fig. Since the discovery of penicillin. a few other fungal-derived antibiotics have been taken to the market. cefaclor. The first natural statin introduced in the market. Cephalosporin. history of medicine. ATHEROSCLEROSIS Much of the prestige that fungi may have as an interesting source of pharmaceutically useful compounds has to be attributed to the statins. cephalexin. Clerocidin. but none of them could be developed as clinically useful antibiotics. by looking for compounds able to inhibit the growth of the target bacteria. cefixime). some of which are still among the best-selling antibiotics in the market today [92]. was . the most remarkable being cephalosporin and fusidic acid. cefuroxime. 4) is a bacteriostatic antibiotic. It is most frequently used topically to treat skin [95] and ophthalmic infections [96]. a metabolite isolated from Fusidium viride and other species. because they inhibited eukaryotic toposiomerase II as well.g. but lacks activity against gram-negative bacteria [94]. including anaerobic species.

in subjects with elevated or even average cholesterol levels. followed soon after. a squalene synthase inhibitor. simvastatin (Zocor). In the following years. pyripyropene A. Thus. A semisynthetic derivative.62 ´ Pelaez Figure 5 Cholesterol-lowering compounds: lovastatin. Several clinical studies have evidenced the benefits of statins in the prevention of coronary artery disease. an ACAT inhibitor. other natural (pravastatin) or totally synthetic statins (atorvastatin. zaragozic acid A. an HMG-CoA reductase inhibitor. discovered by Merck scientists in the late 1970s [102. the therapeutic class of the statins has had a tremendous impact in the lives of .103]. fluvastatin) from other companies reached the market.

Biological Activities of Fungal Metabolites 63 millions of patients at cardiovascular risk [104. Fungal inhibitors of CETP include a series of rosenonolactone derivatives isolated from Trichothecium roseum [118]. and for which fungal inhibitors have been reported. a few other inhibitors of squalene synthase have been isolated from fungi. is being evaluated [106]. toxicity issues [102] precluded their development as drugs. The zaragozic acids were discovered simultaneously by groups at Merck and Glaxo [108]. 5). the major regulatory enzyme for sterol biosynthesis. with IC50s in the picomolar range. Moreover. and inhibit cholesterol absorption in a hamster model [116]. Epi-cochlioquinone A is a structurally related compound from Stachybotrys bisbyi that also shows activity in vivo in rats [117]. and a number of targets have been used in industrial screening programs. At least one CETP inhibitor is now in clinical trials. but despite the initial excitement raised by these compounds. The zaragozic acids showed oral activity in animal models [108. A large number of natural analogs were directly isolated from many fungal species or produced by directed biosynthesis. some of which are submicromolar inhibitors of ACAT in vitro. such as Alzheimer’s disease. a large family of metabolites (18 members reported to date) isolated from Aspergillus fumigatus. Also. and alkylcitrates such as the viridiofungins [113] and the related compounds CJ-13. These compounds are potent inhibitors of squalene synthase. Other enzymes that have been suggested as potential targets for atherosclerosis. [119] and others. At least 10 families of compounds isolated from fungi have been reported as inhibitors of this enzyme. and diacylglycerol acyltransferase [122]. with IC50 values in the micromolar range.982 [114]. 5). rich in triglycerides. and they were subjected to extensive medicinal chemistry studies regarding structure–activity relationships [109]. such as schizostatin [111].115]. Besides zaragozic acids. 15lipoxygenase [121].105]. no further progress has been reported for any of them beyond the discovery step. as well as in the formation of cholesteryl ester storage droplets within cells residing in the vessel wall. most of them showing IC50s in the micromolar range. include ATP citrate lyase [120]. Fig. One of the most exciting discoveries of the early 1990s in the area was that of zaragozic acids (also called squalestatins. However. fungal compounds have been reported that are inducers of LDL receptor gene expression [123]. Another enzyme that has been extensively used as a target for the discovery of cholesterol-lowering agents has been acyl-CoA:cholesterol acyltransferase (ACAT).981 and CJ-13. among others. This protein plays a critical role in the process of reverse cholesterol transport from HDL to LDL. the head-to-head ligation of two molecules of farnesyl diphosphate. ACAT inhibitors have been reported to lower cholesterol accumulation in animal models and some of them are being tested in clinical trials [104. This enzyme participates in the assembly of very low density lipoproteins.110]. after showing efficacy in animal models [104]. The most interesting are the pyripyropenes (Fig. but all of them are relatively weak (IC50 10 M) and no further progress has been reported for any of them. Squalene synthetase catalyses the first dedicated step in sterol biosynthesis. an apiosporamide-related metabolite reported from Cytospora sp. It is therefore no surprise that this has been an area in which extensive efforts have been made by many groups. their potential to treat other disorders. . the bisabosquals [112]. which would be expected to have a protective effect against atherosclerosis. The cholesterol ester transfer protein (CETP) is another target that has been used to develop agents aimed at increasing high-density lipoproteins (HDL). but all of these were much weaker inhibitors than zaragozic acids. At least one synthetic inhibitor of the enzyme (BMS-187745) has been evaluated in humans in phase I [107].

there is no effective treatment. A number of human and animal parasitic diseases. fumagillin is an antiangiogenic agent. which appears to be a modulator of -aminobutyric acid receptors in nematodes.and ectoparasites). This compound has been used for the treatment of microsporidiosis. Other structurally related anthelminthic agents produced by Aspergillus spp. Paraherquamide (Fig. these HDAC inhibitors have been extensively studied for their additional potential as antitumor agents.. and they are able to inhibit the coccidial (Eimeria tenella) HDAC as well [50]. produced by Fusarium species. and coccidiosis. Semisynthetic derivatives of apicidin with potencies in the picomolar range have been reported [124]. However. cryptosporidiosis. given that the compound is very active against nematode strains resistant to ivermectin and benzimidazoles [126]. a microsporidian gill pathogen [125]. this toxicity is species-specific (it is not observed in ruminants). 6) is a broad-spectrum anthelminthic agent produced by Penicillium paraherquei. Fumagillin also has been tested successfully for the control of infections in rainbow trout by Loma salmonae. are caused by protozoa of the subphylum Apicomplexa. Nodulisporic acids (Fig. 6) are novel indole diterpenes that exhibit potent insecticidal properties. As already discussed. 6) was discovered as an antibiotic in the 1950s. although lacking anthelminthic efficacy. including malaria. Its potency against a range of parasites evaluated in cattle models is lower than ivermectin. and were discovered by screening for activity against the larvae of the blowfly . the gold standard of the area. shows remarkable insecticidal properties [128].7% of the references surveyed for the period from 1993 to 2001 reported compounds with this type of activity. Other structurally related fungal tetrapeptides that were known inhibitors of mammalian HDAC. such as HC-toxin. The mode of action of paraherquamide is not well understood. HIV-infected) patients. as well as in vivo activity against Plasmodium berghei malaria in mice. The cyclic tetrapeptide apicidin (Fig. Other interesting anthelminthic agents derived from fungi found in the recent literature include the depsipeptide PF1022A. and it causes chronic diarrhea. Microsporidiosis is produced by Enterocytozoon bieneusi. comparable to apicidin. and it is still possible that further structural refinement could lead to a more promising candidate [126]. toxoplasmosis. in a way different from ivermectin [126]. Apicidin exhibits a broad-spectrum in vitro activity against the Apicomplexa. ANTIPARASITIC Fungi are known to produce a good number of compounds with antiparasitic activity (both for protozoan and metazoan endo. whereas paraherquamide is a poor insecticidal agent.64 ´ Pelaez 6. It acts as a potent inhibitor (IC50 1–2 nM) of the parasite histone deacetylase (HDAC). also have potent antiprotozoal activity. as previously discussed. Some of the most interesting of these fungal metabolites are discussed here. with satisfactory results in clinical trials [87]. but it seems to be distinct from that of other major anthelminthic classes. are the aspergillimides. Unfortunately. malabsorption. and wasting in immunocompromised (e. Fumagillin (Fig. was identified by Merck scientists as a broad-spectrum agent effective against a range of apicomplexan parasites [50]. About 7. The mechanism of action of fumagillin seems to be inhibition of methionine aminopeptidase-2 [89].g. 6). Currently. and some derivatives have been taken to clinical trials as antitumor agents. In addition to its properties as an antiparasitic agent. but superior to other marketed products. the compound showed toxicity in canine models. which showed even higher potency than paraherquamide in rodent models [127]. and this precluded its development as a drug. and sclerotiamide which.

an HDAC inhibitor with antiprotozoan activity. in a similar fashion to ivermectin [126. most likely the anamorph of a Hypoxylon sp. with no adverse effects. paraherquamide.Biological Activities of Fungal Metabolites 65 Figure 6 Antiparasitic compounds: fumagillin. Nodulisporic acids are produced by a pantropical species of Nodulisporium. nodulisporic acid A. Nodulisporic acid A was about 10-fold more potent than ivermectin in a flea model. an activator of insect glutamate-gated chloride channels with insecticidal properties. apicidin. an anthelminthic agent. Nodulisporic acids exert their action by activating the insect glutamate-gated chloride channels. a methionine-aminopeptidase-2 inhibitor with activity against microsporidia. [130]. In vivo studies in dogs have shown that nodulisporic acid A exhibited potent systemic efficacy.131]. Lucilia sericata [129]. . Derivatives of these natural products with improved properties have been also reported [126].

arisugacin A. Ergotamine (Fig. Because of its strong vasoconstrictor effects. Inhibitors have been reported to kill Lucilia cuprina blowfly larvae in vitro. cyclic pentapeptides isolated from Gliocladium and Clonostachys spp.66 ´ Pelaez Chitinases.. Two fungal chitinase inhibitors have been reported recently. an inhibitor of acetyl cholinesterase. . 7). cuprina chitinase. The structure of the enzyme in complex with these two compounds recently has been disclosed [132]. NEUROLOGIC DISORDERS The most relevant clinically useful fungal compounds in the area of neurologic disorders are the ergot alkaloids. The compound was isolated in 1918 and has been used for the treatment of migraine since 1926. enzymes able to hydrolyze linear chitin polymers. respectively [132]. The compounds are nanomolar inhibitors of the L. and this should prove useful in the design and synthesis of derivatives with greater therapeutical potential. The mode of action of ergotamine has been the subject of a substantial Figure 7 Central nervous system disorders: ergotamine. 7. have been proposed as targets for the development of insecticidal and antimalarial drugs. it was used in traditional medicine to precipitate childbirth and control post-partum hemorrhage. the main alkaloid of the ergot fungus Claviceps purpurea. was the cause of mass poisoning in Europe in the Middle Ages due to eating bread from rye contaminated with sclerotia of the fungus [133]. without any specific alternatives for several decades thereafter. argifin and argidin. an antimigraine compound.

Although the drug has been used in the clinic for many years (as Cafergot. Inhibitors of the enzyme. some of the existing AChE inhibitors are known to have undesirable side effects. Arisugacins A (Fig. At therapeutically relevant doses. have been reported to improve cognitive function in Alzheimer’s disease patients [137]. due to its poor selectivity.Biological Activities of Fungal Metabolites 67 number of studies [133]. such as cabergoline (Fig. and xyloketals [141]. however. Other structurally similar ergot alkaloids have been used in the clinics. such as ergotoxine (Hydergine). with the introduction of the much more specific triptans. There is evidence that cholinergic functions decline in some brain areas in individuals affected by Alzheimer’s disease and in a variety of afflictions in which memory and cognitive function are impaired. a number of side effects have been reported that limit the applicability of this compound. but none of them have progressed from the discovery step. Tacrine. and for this reason it has been used to stop the production of milk and to treat acromegaly [134]. quinolactacins [138]. inhibitors of acyl-CoA:cholesterol acyltransferase.140]. the triptans. 7) and B and the related territrems are especially potent. for instance. However. the first AChE inhibitor in the clinic. However. All the inhibitors used in the clinic also inhibit butyrylcholinesterase (BuChE). However. an enzyme present in plasma. Other analogs have been developed more recently. and other brand names). rivastigmine. dopamine. it has been used to treat hyperprolactinaemia due to prolactinomas. Therefore. launched in 1995. and metrifonate. ergotamine has become a less preferable choice for clinicians and patients. and noradrenaline receptors. The compound is able to bind a diversity of receptors. discussed previously [116]. Furthermore. 7) and terguride. Finally. donepezil. ergotamine will keep its place in history as one of the most extensively used fungal compounds in the clinical practice. . a substructure of arisugacins has been used to prepare new synthetic analogs of tacrine that have improved selectivity for AChE vs. and highly specific for AChE versus BuChE [139]. such as arisugacins. and as such has been used to treat Parkinson’s disease since the early 1970s. a vasodilator to treat disturbances in peripheral blood circulation [134]. Bromocriptine (2-bromo-ergocryptine) is as semisynthetic derivative that is a potent dopamine agonist specific for the D2 receptor. the new synthetic antimigraine agents. The most obvious pharmacological effect of ergotamine is vasoconstriction. which represent 50% of all pituitary adenomas and 12% to 15% of all intracranial tumors [135]. In contrast. is known to induce hepatotoxicity.136]. Therefore. which are also dopamine agonists and are used for the treatment of Parkison’s disease and hyperprolactinaemic disorders [102. are more specific for 5-HT1B/1D versus other receptors. including several subtypes of the 5-HT. This has long been believed to be the basis for its antimigraine properties (although there are reports claiming that the effect is more due to inhibition of neurogenic inflammation and neuronal transmission). particularly marked in the carotid vascular bed. Arisugacins are structurally related to the pyripyropenes. Ergostat. its usefulness is still controversial. territrems [139. Also. 5-HT1B/1D and dopamine D2 receptors. more specific inhibitors of AChE would be expected to be attractive leads for the treatment of Alzheimer’s disease [138]. reports have been published that both support and refute its efficacy [133]. BiChE [142]. such as tacrine. It is also a reducer of prolactin and growth-hormone secretion. with IC50 values in the nanomolar range. Several fungal metabolites have been reported to be inhibitors of the enzyme. ergotamine appears to act mainly as an agonist of -adrenoceptors. enhancement of cholinergic neurotransmission by inhibiting acetylcholinesterase (AChE) has been considered as a therapeutical approach against Alzheimer’s and other diseases.

enzymes involved in the catabolism of phospholipids. The most potent fungal inhibitors of PLA2-II reported to date are the thielocins (Fig. Another compound. this effort has not yet resulted in a marketable drug of fungal origin. competitive with cyclic AMP at low micromolar levels. an extracellular phospholipase present in inflammatory regions. Fig. thielocins A1 and B3 showed activity in a rat carrageenan-induced pleurisy model [151]. Other targets evaluated for the development of new anti-inflammatory agents include several phospholipases (PL). INFLAMMATION Anti-inflammatory drugs represent a very important sector within the pharmaceutical market. including blockbuster drugs such as the coxibs. PLA2II in particular. which has been reported to be an inhibitor of calcium/calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE). For instance. It is assumed that compounds with this activity could have an application in an array of neurological disorders. Several other fungal compounds have been described as neuritogenic by screening for morphological changes in PC12 cells or other cell lines. and the effect was additive to that of NGF. produced by Stachybotrys parvispora. 8) has been shown to significantly attenuate TNF levels in plasma when administered orally to LPS-challenged mice [149]. such as synovial fluid of patients with rheumatoid arthritis. Therefore. Examples include a series of phomalactone analogs produced by a Phomopsis sp. much like nerve growth factor (NGF). also showed a protective effect against neuronal damage in primary cultures of neurones [143]. The compound. Ceramide production has been reported to increase in response to inflammatory stimuli. stachybotrin C was demonstrated to induce differentiation of PC12 cells into neuronlike cells. Inhibitors of nSMase would therefore be expected to interfere in the inflammatory response. showed the same effect on PC12 cells. the cyclooxygenase II inhibitors. also has been implicated in the inflammatory response. [147]. fungal inhibitors of the production of the proinflammatory cytokines tumor necrosis factor (TNF ) and/or interleukin-1 (IL-1 ) induced by stimuli such as the bacterial lipopolysaccharide (LPS) in intact macrophages or peripheral blood monocytes (a typical inflammatory response) have been reported repeatedly. isolated from Penicillium minioluteum. One of the O. Marasmiellus sp. but it did not show any protective effect on neurone primary cultures [144]. isolated from Thielavia terricola. and this subsequently triggers signaling pathways leading to cell proliferation and differentiation or to apoptosis. an enzyme catalyzing the hydrolysis of sphingomyelin to ceramide and phophocholine. produced by an Acremonium sp. Nonetheless. and inducing neurite formation in a neurally derived cell line [145]. at micromolar doses. and much less potent against the pancreatic type PLA2-I. or even in compounds moving beyond the early discovery phase. but no details on the mechanism of action have been provided. 8. Several fungal inhibitors . Neutral sphingomyelinase (nSMase). and diterpenes isolated from Oidiodendron griseum [148]. For instance. NG-061. a significant number of fungal metabolites have been reported with activity in targets related to inflammatory processes. all of them with potencies ranging in the submicromolar to micromolar range. Also.68 ´ Pelaez A number of fungal metabolites have been described that have some type of neurotrophic or neuritogenic activity. methyl-5-substituted pyridine-2-carboxylates from a basidiomycete. An exception is the compound PS-990. including TNF and IL-1 . with IC50 values in the nanomolar range [150]. has been shown to have proinflammatory activity. significant effort has been expended to find new anti-inflammatory compounds from fungal metabolites. griseum compounds (PR 1388. [146]. However.. 8). including dementia.

a Bruton’s tyrosine kinase inhibitor. produced by Phoma sp.Biological Activities of Fungal Metabolites 69 Figure 8 Antiinflammatory compounds: scyphostatin. a neutral sphingomyelinase inhibitor. The most interesting PAF antagonists obtained from fungi are the phomactins. it inhibited carrageenin-induced paw edema in rats [152]. terreic acid. PR 1388. of nSMase have been reported recently. PAF binds to diverse cell types. and hypotension. inducing a series of biological responses. leukocyte activation. thielocin B3. Derivatives of this compound have been prepared with . This compound is a selective micromolar inhibitor of nSMase. phomactin D (Fig. and much less potent versus acid SMase. phomactin D. such as platelet aggregation. an inhibitor of TNFI production. the most interesting being scyphostatin (Fig. a potential mediator of allergic and nonallergic inflammatory diseases. [153]. produced by Trichopeziza mollissima. It inhibited the production of prostaglandin E2 and IL-1 induced by LPS in human peripheral monocytes and. a phospholipase A2 inhibitor. 8). The most potent compound in the series. showed an IC50 in the submicromolar range in a platelet aggregation assay. when administered orally. 8). an inhibitor of PAF. There are several fungal metabolites reported that antagonize the platelet activating factor (PAF).

9) have been described. integracins [164]. without blocking many other cellular functions. As for other viruses. 8) has been reported more recently as an inhibitor of Bruton’s tyrosine kinase (Btk) in mast cells. This effort resulted in a significant number of novel compounds that were active in the micromolar range. Terreic acid (Fig. A promising target in the fight against HIV infection is the integrase. Two enzymes encoded by the viral genome. it could be used as a lead to generate Btk inhibitors with improved therapeutic profiles [158]. Examples of these are the mniopetals. it also has shown to be a specific inhibitor of the interaction between Btk and PKC. integric acid. integramides [163]. produced by Chrysosporium merdarium. and the semicochliodinols. Flutimide is a substituted 2. 9). Other fungal inhibitors have been described but were less potent and did not show activity in animal models [155]. several compounds inhibiting influenza virus replication in cells have been reported. such as 10-norparvulenone [167] and the compounds FR191512 and FR198248 (Fig. It inhibits protein synthesis by blocking the formation of leucyltRNA in sensitive bacteria. such as the interleukin-converting enzyme [156] or stromyelisin [157]. Another interesting anti-influenza compound is flutimide (Fig. Terreic acid was originally isolated in 1942 as an antibiotic from Aspergillus terreus. The mechanisms of action of these compounds have not been fully established. and is involved in the signaling cascades leading to exocytosis of pro-inflammatory cytokines in the allergic response [158]. Fungal inhibitors of other enzymes involved in the inflammatory process have been described. and this results in the recapitulation of the phenotype of mutations of Btk. structurally related to aspergillic acid. 9). The . which showed in vivo efficacy in a mouse model [168.. although at least the compound FR198248 seems to inhibit the stage of virus adsorption [169]. weak inhibitors of reverse transcriptase of HIV and other viruses [159]. the reverse transcriptase and the protease.70 ´ Pelaez enhanced potency [154]. and phomasetin [165]. which are submicromolar and relatively specific inhibitors of HIV protease [160]. 9. have been used to look for anti-HIV agents that have resulted in clinically useful drugs. which was discovered in a screening program seeking inhibitors of cap-dependent transcription of influenza virus [170]. although none of them showed improved properties compared to the parent molecule [166]. the enzyme that catalyzes the insertion of the viral DNA into the genome of the host cell. Although a number of fungal metabolites have been described as inhibitors of these two enzymes. However. The HIV pandemia raised the urgent need for antiretroviral drugs.169]. Btk is activated by cross-linking of high-affinity IgE receptor. such as integrastatins [162]. ANTIVIRAL Considerable effort has been dedicated to the search of antiviral agents from natural products by several research groups. Inhibitors of the enzyme are able to inhibit viral replication in cells [161]. Our group was involved in an intensive screening for HIV-1 integrase inhibitors from fungal natural products for several years. Although the compound cannot be used in the clinic because of its cytotoxicity. none has progressed beyond early discovery. and there are already some inhibitors in development as clinical candidates. metabolites related to the asterriquinones.6-diketopiperazine. metabolites produced by the basidiomycete Mniopetalum sp. such as equisetin and oteromycin [165]. Some derivatives prepared by chemical and enzymatic modification of integric acid (Fig. but none of them originated from natural products. plus some previously known compounds for which this biological activity had never been described.

cyclosporin soon became the most widely used immunosuppressive drug in the clinic [172]. Numerous natural members of the cyclosporin family have been reported [174]. Integric acid. an inhibitor of cap-dependent transcription of influenza virus. being ineffective versus other viral polymerases [170].Biological Activities of Fungal Metabolites 71 Figure 9 Antiviral compounds. an antiinfluenza agent. Cyclosporin suppresses immune responses by blocking the calcium-dependent signal transduction pathway resulting from the activation of Tcell receptors. 10. the most important of which is nephrotoxicity. IMMUNOSUPPRESSION One of the major medical breakthroughs of modern medicine is undoubtedly the development of the organ transplantation techniques. compound is produced by Delitschia confertaspora. thereby inhibiting the activation of helper T-cells and the production of interleukin-2 (IL-2). These toxicity . The success of these approaches was possible only after the discovery of immunosuppressive agents. it has several important side effects. The details on its mechanism of action have been extensively reviewed [173]. It specifically targets the endonuclease activity of the cap-dependent transcriptase of influenza A and B viruses. the discovery of cyclosporin A (Sandimmune. Although cyclosporin A is one of the few blockbuster drugs derived from a fungus. Flutimide. a new fungal species isolated from dung of dassie collected in Namibia [171]. an HIV integrase inhibitor. In this field. Neoral) and its application to renal transplantation in 1978 was an important milestone. FR198248.

had been discovered as an antibiotic in the first years of the 20th century (it was crystallized before penicillin). antiviral. such as tacrolimus. another immunosuppressant fungal metabolite reached the market. Figure 10 Immunosuppressants: mycophenolic acid and mizoribine. FTY720. inhibitors of purine biosynthesis. ISP-1. produced by Penicillium spp. antifungal. This compound. and other fungi [23]. 10) (see Bentley [175] for a comprehensive review on its history and properties). but its immunosuppressive properties were recognized only much later. . This has prompted the search for safer immunosuppressants with a different mechanism of action.72 ´ Pelaez issues are shared by other drugs with the same mode of action. an actinomycete metabolite. Some years after the development of cyclosporin. The compound shows antibiotic. mycophenolic acid (Fig. an agonist of S1P receptors. an inhibitor of serine palmitoyltransferase.

which showed activity in a rat skin allograft model and is thought to be an inhibitor of purine nucleotide biosynthesis [180]. FTY720 [184] (Fig. the mycestericins. Mizoribine (bredinin. and others. which. is a potent IMPDH inhibitor and has been extensively used in Japan for human renal transplantation and several autoimmune disorders [177. is a useful prodrug. T and B lymphocytes are especially sensitive to mycophenolic acid because. All these compounds are structurally related to the sphingofungins. unlike that compound. It is a nanomolar inhibitor of the enzyme inosine 5′-monophosphate dehydrogenase (IMPDH). psoriasis. mycophenolic acid is less toxic than other immunosuppressants. blocking sphingolipid biosynthesis and inducing apoptosis in an IL-2–dependent mouse cytotoxic T cell line [183]. Its potential in anti-HIV therapy has been recently suggested [176]. The mechanism of action of mycophenolic acid is different from that of cyclosporin or tacrolimus.Biological Activities of Fungal Metabolites 73 and antitumor activities in animal studies. The antifungal activity of all these compounds seems to be due to the inhibition of this enzyme. Analogues with similar activity. Clinical trials against several cancers showed only a poor response. an enzyme involved in the de novo biosynthesis of guanine.182]. It was subsequently reported that the compound is a picomolar inhibitor of serine palmitoyltransferase. Eimeria. the most interesting of the fungal compounds described as immunosuppressants in this decade is the metabolite ISP-I (Fig. Although the natural products were not suitable as clinical candidates. The compound was more potent than cyclosporin A in suppressing lymphocyte proliferation in several models. More than a dozen publications have appeared since 1993 reporting fungal metabolites with immunosuppressive activity (or at least targeting anyone of the steps involved in the immune response). and other parasitic protozoa. The compound usually is coadministered with other immunosuppressants. Food and Drug Administration for prevention of rejection in renal allograft recipients in 1995 and for heart transplantation in 1998. were described from one of the myriocin-producing strains [182]. several synthetic analogs were reported with improved profiles [182. 10) is another fungal metabolite with the same mode of action as mycophenolic acid. as discussed in section 2. . ISP-I was discovered as a potent immunosuppressant produced by Isaria sinclairii [181]. This inhibition results in impairment of DNA synthesis. almost lacking the capability to reutilize free purines generated in catabolic processes. One of these derivatives. 10). as well as activity against Trichomonas. 10). but it had earlier been isolated from other fungi as an antifungal agent. and the compound FR901483. they seem to be dependent on the de novo pathway of guanine synthesis. under the names of myriocin and thermozymocidin. However. However. mycophenolic acid was developed as a clinically useful immunosuppressant. A search for a derivative with improved oral availability led to the discovery of the 2-morpholinoethyl ester. due to their toxicity and solubility problems. Fig. Examples include the stevastelins [179]. showed good efficacy in animal models of transplantation as well as for several autoimmune disorders. it did not inhibit production of IL-2 [181. possibly as a result of the depletion of the GTP pool [175]. This compound. which inhibited lymphocyte activation and proliferation. the first of a family of fungal metabolites used for the development of a clinically useful immunosuppressant. and these studies were discontinued. It was approved by the U.184].S. Furthermore. produced by Eupenicillium brefeldianum.178]. The compound also prevents the glycosylation of adhesion molecules involved in the attachment and infiltration of lymphocytes and monocytes into sites of graft rejection. unlike other cell types. which have been reported to inhibit fungal serine palmitoyltransferase [7]. Mycophenolic acid also has been used experimentally for the treatment of rheumatoid arthritis and other autoimmune diseases such as lupus. as mycophenolate mophetil (CellCept).

Several nonpeptidic antagonists of ETA or dual ETA/B antagonists have been developed and are in clinical trials for the treatment of vascular and pulmonary hypertension [187]. Inhibition of lipid peroxidation activity in rat liver microsomes commonly has been used to search for compounds with free radical scavenging activity. such as myocardial and cerebral ischemia. or both. 11). G-protein coupled receptors that have sphingosine 1-phophate as their natural ligand. and others. Instead. a metabolite produced by Penicillium sp. reduced the chromosomal aberrations induced by paraquat. Finally. cancer initiation.. The list includes the spirodihydrobenzofuranlactams [188]. A number of compounds isolated from basidiomycetes have been reported with this activity. FREE RADICAL SCAVENGERS Peroxidative disintegration of cells and organellar membranes by free radicals has been implicated in the pathogenesis of a broad array of diseases. The activation of the S1P receptors has been correlated with the induction of lymphopenia in the animals. However. . no further progress has been reported. a phosphate ester metabolite of the compound that is naturally formed after administration in rats and mice has been shown to bind with high affinity (in the sub or low nM range) to at least four of the five members of the S1P receptor family. Although FTY720 itself is only a weak ligand of some of these receptors. All of these compounds showed IC50 values in the micromolar or submicromolar range. 7). and others. Rumbrin (Fig. HYPERTENSION Endothelins (ETs) are peptidic hormones produced by endothelial cells that have been recognized as having a significant role in the development of hypertension and other cardiovascular diseases. with Ki values in the micromolar range. rheumatoid arthritis. from Lenzitus betulina [193]. has been described recently to have strong antioxidant activity by stimulating the production of glutathione and also by directly scavenging free radicals. 11). was able to prevent cell death caused by calcium overload in cells exposed to toxic concentrations of a calcium ionophore [195]. such as the curtisians. the ergot alkaloid derivative cabergoline (Fig. Interestingly. 11. The retention of lymphocytes in the lymph nodes would inhibit the infiltration of lymphocytes into grafted organs [186]. At least nine families of fungal compounds have been described as antagonists of ETA. ETB. This could help to explain its neuroprotective properties observed in vivo [196]. free radical scavengers have been suggested to have a potential as protective agents against several diseases. p-terphenyls isolated from Paxillus curtisii [192]. which is used to treat Parkinson’s disease (see section 7). azaphilones [189]. because ETA is involved in vasoconstriction and ETB in vasodilation. which produce opposite effects. 12. the compound acts through the S1P (edg) receptors. oteromycin [190]. in addition to inhibiting lipid peroxidation. Therefore. the betulinans. which are mediated by the generation of superoxide anions [194].74 ´ Pelaez and has been tested successfully in clinical trials of renal allograft transplantation [185]. RES-1214 [191]. because this is not an inhibitor of serine palmitoyltransferase. ETA and ETB. the mode of action of FTY720 seems to be different from that of the natural analogs. and ageing processes. The effects of endothelins are mediated through two receptors. diabetes. atherosclerosis. a metabolite produced by Auxarthron umbrinum. Phenopyrrozin (Fig. all these antagonists showed only moderate affinity for the receptors.

a cathepsin B and L inhibitor. 12). L. such as the insulin-like growth factor receptor or the EGF receptor. free radical scavengers. This compound was discovered as an orally available insulin mimetic agent through a screening assay designed to detect small molecules activating the human insulin receptor tyrosine kinase [101]. are involved in a number of physiopathological processes. lysosomal cysteine proteases. It mimicked insulin in several biochemical and cellular . DIABETES One of the most exciting discoveries of the last decade concerning fungal metabolites was that of bis-demethyl-asterriquinone B1. acute pancreatitis. such as the compound WF14861 (Fig.Biological Activities of Fungal Metabolites 75 Figure 11 WF14861. 11). The compound.. Their participation in osteoclastic bone resorption in particular has been reported frequently. which was isolated from a Pseudomassaria sp. produced by Colletotrichum sp. CATHEPSIN INHIBITORS It has been suggested that cathepsins B. was shown to be selective for the insulin receptor versus other receptor tyrosine kinases. and cancer progression [197]. Screening for cathepsin natural inhibitors has resulted in the discovery of several fungal metabolites. 14. including rheumatoid arthritis. DMAQ-B1 (Fig. 13. phenopyrrozin and rumbrin. and K. [198]. the most representative being several transepoxysuccinyl type inhibitors. Compounds in this class inhibited cathepsins B and L at nanomolar concentrations in vitro and showed remarkable activity in a bone resorption mouse model [198–200]. but they also play significant roles in the process of muscle fiber destruction in inflammatory myopathy and several other disorders.

an antagonist of CCK receptor. the oral administration of the compound in two mouse models of diabetes resulted in significant lowering of glucose levels and suppression of the elevated plasma insulin levels associated with one of these mouse models. asperlicin. This discovery was celebrated as a breakthrough in the field for the development of orally active antidiabetic agents. paspalistrem A. assays. Moreover.76 ´ Pelaez Figure 12 DMAQ-B1. including stimulation of glucose uptake in whole cells. a potassium channel antagonist. The administration of DMAQ-B1 via central intracerebroventricular resulted in a dose-dependent reduc- . an activator of the insulin receptor tyrosine kinase. A synthetic derivative of DMAQB1 with improved characteristics subsequently has been reported [202.203]. The potential of these compounds as antiobesity agents also has been described recently.

Based on these observations. but the compound failed to show any efficacy [208].Biological Activities of Fungal Metabolites 77 tion of food intake and body weight in rats. resulting in neurotransmitter release and smooth muscle contraction. have been identified: CCK-A is the predominant peripheral CCK receptor subtype. Other derivatives that are CCK-B antagonists orally active in animal models recently have been studied for the treatment of gastroesophageal reflux disease [102]. Asperlicin (Fig. and it was used as a lead to develop other more potent antagonists [103]. and iberiotoxin. maxi-K channel agonists may be useful for treating neuronal and smooth muscle disorders. These are the indole diterpenes commonly referred to as tremorgenic mycotoxins. ION CHANNELS LIGANDS Potassium channels are involved in the modulation of a number of physiological processes. the high conductance calcium activated K (maxi-K or BKCa) channels. Claviceps. metabolites produced by a diversity of fungi. Therefore. Interestingly. aflatrem. such as margatoxin. 12) was discovered in 1984 by Merck scientists as a CCK antagonist produced by Aspergillus alliaceus [106]. asperlicin derivatives have been very useful as research tools for understanding the physiological role of CCK. Devazepide was tested as a potential antiulcer therapy. from the genera Penicillium. The discovery of asperlicin represented a major scientific breakthrough. Moreover. or paspalitrems (Fig. but safety issues precluded further progress [107]. with inconclusive results [209]. 12). CCK-A and CCK-B. This compound is a selective CCK-A antagonist. penitrem. Two receptor subtypes. Both MK-329 and asperlicin were shown to inhibit the growth of CCK receptor-positive human pancreatic cancer in athymic mice. the clinical candidate MK-329 was tested in patients with pancreatic cancer. Some fungal metabolites also have been described that are very potent ligands of maxi-K channels [210]. 15. and others [23]. Specifically. Some of these compounds have been tested in clinical trials. these compounds modulate the binding of other ligands such as charybdotoxin by positive or negative . this being one of the first nonpeptidic ligands described for a peptide receptor. such as the clinical candidate MK-329 (devazepide) and other compounds that have been tested in humans. charybdotoxin. Besides blocking the channels with high potency. oral administration of the synthetic derivative in a mouse model of high-fat. Aspergillus. and insulin resistance [104]. 16. diet-induced obesity reduced body weight gain. present in neurons and smooth muscle. have been useful tools to dissect the structure and physiological role of these channels. are opened by increases in the intracellular concentration of calcium or by membrane depolarization. other asperlicin derivatives selective for the CCK-B receptor subtype have been developed more recently and proposed as development candidates for the treatment of anxiety and panic disorders and pain [102]. adiposity. and CCK-B the predominant central CCK receptor [105]. CHOLECYSTOKININ ANTAGONISTS Cholecystokinins (CCKs) are peptide hormones and neurotransmitters widely distributed throughout the gastrointestinal tract and central nervous system that mediate a number of biological functions. including hypertension and asthma. such as paxilline. Peptidic channel blockers isolated from scorpion venoms. Despite the lack of success in clinical trials.

NEUROKININ ANTAGONISTS The tachykinin family of peptides. among other effects [211]. neurokinin A (NKA) and neurokinin B (NKB). NK2. and neuronal and immune system stimulation. 17. are involved in a wide variety of biological functions. and other neuropeptides. a neurokinin antagonist. Although the natural ligands bind to all Figure 13 WIN-64281. including smooth muscle contraction. . 8-methyl-pyridoxatin. and NK3. which includes substance P (SP). They mediate their effects through three G-protein coupled receptors termed NK1. secretion stimulation. an inducer of the EPO gene expression. a fibrinolytic enhancer.78 ´ Pelaez allosteric mechanisms [210] and stimulate the contractibility of smooth muscle in models of guinea pig and rat urinary bladder and rat duodenum. KOD.

which induced EPO gene expression by five-fold at a concentration of 0. It is known that high levels of the plasminogen activator inhibitor-1 (PAI-1) are a risk factor for thrombosis. at least partially. 13). originating in vascular endothelial cells. and an extensive search has been made of fungal metabolites with this potential by using a screening assay looking for inducers of the expression of the EPO gene fused to a luciferase reporter gene. new destruxins [218]. plasminogen is proteolytically activated by several factors. with Ki in the micromolar range. 20. tissue remodeling. Nonpeptidic antagonists of tachykinin receptors (mainly of NK1) have progressed to clinical trials for the treatment of diseases as diverse as depression/anxiety. by inactivation of the inhibitor PAI-1. The activation of plasminogen is enhanced on the surface of cells and fibrin. The most interesting compounds reported to date are WIN-64281 (Fig. which showed submicromolar Ki values against both NK1 and NK2 and was active in functional assays such as the rat vas deferens model [213]. respectively. 13) is mediated. emesis. however. isolated from Aspergillus sp. ERYTHROPOIETIN GENE EXPRESSION INDUCERS Erythropoietin (EPO) is the primary hormone regulating the proliferation and differentiation of immature erythroid cells. and several depsipeptides that are selective for NK2 with Ki values down to 27 nM [214. 18. cancer chemotherapy. In some cases. and others. and asthma [212].Biological Activities of Fungal Metabolites 79 three. 13).3 M [220]. inflammation. including fibrinolysis.. Recombinant EPO is in use as a treatment for anemia derived from chronic renal failure. An extensive search by several industrial laboratories has led to the discovery of several fungal metabolites with activity as antagonists of NK1 and/or NK2. 8-methyl-pyridoxatin (Fig. Several fungal metabolites have been described with this activity. to which it is able to bind. receptors and other biological processes. showed weak affinities. This effort has resulted in the discovery of at least three different families of compounds: namely. sesquiterpene tropolones [219]. to inhibitors (and activators) of enzymes. and other conditions. migraine. in the broadest sense. localizing fibrinolytic activity on cell surfaces and fibrin clots. An orally available alternative to recombinant EPO would present obvious advantages. In this system. The balance between activators and inhibitors regulates fibrinolysis in the blood vessels. each of which is regulated by specific inhibitors. CONCLUSION AND OUTLOOK As shown in this chapter. Agents able to promote the binding of plasminogen to fibrin and cells would be expected to be useful as fibrinolytic enhancers. such as staplabin and derivatives produced by Stachybotrys microspora [216]. and that PAI-1 expression is increased in atherosclerotic arteries. The most potent was the latter. therapeutically useful . and the novel fatty acid KOD (11-keto-9(E). Most of the compounds isolated. FIBRINOLYTIC ENHANCERS The plasminogen/plasmin system is involved in a variety of physiological and pathological conditions that require localized proteolysis. SP is more selective toward NK1. from antibiotics.215]. tumor metastasis. The effect of KOD (Fig. 19.12(E)-octadecadienoic acid) [217]. and a novel pyridone [220]. pain. whereas NKA and NKB preferentially bind to the NK2 and NK3 receptors. fungal metabolites have been described spanning a broad array of biological activities.

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and Tim Hunt together were awarded the 2001 Nobel Prize in Medicine. YE Cancer Research. it has long been known that production of secondary metabolites usually is associated with cell differentiation and development by organisms with filamentous growth and a complex morphology [1]. This book deals with a range of scientific and industrial issues concerning fungal secondary metabolism and bioactive secondary metabolites. Lee Hartwell.A. a link between biosynthesis of secondary metabolites and asexual development recently was established by analysis of a G-protein mutant in A. nidulans [3]. Indiana. It thus is not surprising to find that regulatory mechanisms of the cell cycle are conserved largely through evolution from fungi to humans. great insight into regulation of the cell cycle has come in the last three decades through a convergence of molecular genetic studies in model genetic systems of Saccharomyces cerevisiae. 1. Indianapolis. Although cell division is the cornerstone of biology. U. Schizosaccharomyces pombe. nidulans [2]. In recognition for their pioneering work on cell cycle research using model systems. Indeed. Reinhard Fischer’s laboratory has since provided further evidence supporting an impor93 . Eli Lilly and Company. However. INTRODUCTION Cell growth and division are the most fundamental cellular activities of eukaryotic organisms. Aspergillus nidulans. Paul Nurse. a role of cell cycle regulation in fungal secondary metabolism has not been investigated. It also was shown recently that an interaction between developmental regulators and cell cycle regulators is required for normal cell differentiation and morphogenesis during asexual development in A.4 Cell Cycle Regulation in Morphogenesis and Development of Fungi XIANG S. and biochemical studies in cell cycle extracts of activated frog and sea urchin eggs. Lilly Research Laboratories. Moreover.S.

I then more extensively discuss recent findings that help establish a key role of cell cycle regulation in fungal development and morphogenesis. apoptosis. A great deal of cell cycle research has been directed to study the regulation of cyclindependent kinases (CDKs). For instance. In fact. FCC1. The ensuring cytokinesis then separates each nuclei into two daughter cells. Readers are referred to recent reviews on fungal cell cycle regulation for more comprehensive information [7. 2. CELL CYCLE REGULATION IN ASPERGILLUS: A TALE OF TWO KINASES The cell cycle was originally divided into interphase and mitosis based on cytological observations. I hope this chapter helps provide a framework by which future studies can be designed to demonstrate the direct involvement of cell cycle regulation in fungal secondary metabolism. a C-type cyclin-like gene. DNA replication occurs once and once only per cell cycle. cell cycle progression in Aspergillus requires the function of a single cyclin-dependent kinase p34cdc2 encoded by nimX [15]. DNA is replicated during S phase and segregated equally into two daughter nuclei during mitosis. and when to enter into.5]. In addition. is shown to play an important regulatory role in both fumonisin B1 biosynthesis and asexual development in Fusarium verticillioides [6]. and G2 phases. and initiation of mitosis is coupled to completion of DNA replication [9]. S. especially when to start DNA synthesis. Similar to budding and fission yeasts.94 Ye tant role of cell cycle regulators in Aspergillus development [4. nidulans. and morphogenesis also is discussed. Most intriguingly. I first summarize recent progress in cell cycle regulation in the model fungal system A.8]. In this chapter. development. The p34cdc2 protein remains constant throughout the cell cycle and its kinase activity is regulated through its association with cyclins and by phosphorylation/dephosphorylation [10.11]. ranging from about 5 minutes in Aspergillus to about 30 minutes in mammalian systems. because they play key roles in regulating major cell cycle transitions [10]. initiation of mitosis in all eukaryotic cells requires activation of CDK1 [10. to ensure the faithful transmission of the genome. cells need to make a continuum of decisions about when to start and when to stop cell cyclespecific functions. as little cellular activities during interphase can be observed under a microscope [9]. at least through its role in fungal development and morphogenesis. The kinase activity of p34cdc2 requires both binding to cyclins and subsequent activating phosphorylation at T161 upon cyclin binding by CDK activating kinase (CAK) .11]. recent work from my laboratory demonstrating an important regulatory role of sphingolipid metabolism in Aspergillus cell cycle. which was first observed in sea urchin eggs to accumulate in abundance during interphase and is very rapidly degraded at each mitosis [14]. Mitosis occurs very rapidly. Therefore. CDK1 containing the catalytic kinase subunit p34cdc2 and the regulatory subunit cyclin B is a key regulator of mitosis and is universally conserved [11]. The function of p34cdc2 is required for both initiation of DNA replication and entry into mitosis [15]. The interphase occupies the majority of the cell cycle and is now functionally divided into G1. it is highly conceivable that cell cycle regulation plays an important role in fungal secondary metabolism. Indeed. mitosis. Thus. the conservation of cell cycle regulators was first realized when the human Cdc2 homolog was cloned by functional complementation of a Cdc2 mutation in fission yeast [12] and also the determination of frog mitosis-promoting factor (MPF) as the CDK1 complex with cyclin B as the oscillating protein [13]. and exit from.

proteolysis of NIMA also is required for exit from mitosis [32]. they must thus functionally coordinate with each other or may even regulate each other’s mitotic functions. Upon G2/M transition. tyrosine 15 phosphorylation of CDK1 also plays a key role in linking cell cycle progression to morphogenesis and differentiation (see section 3). CDK1 becomes abruptly activated by tyrosine 15 dephosphorylation to initiate mitosis. Analysis of temperature-sensitive.8]. originally isolated by Morris [28]. Studies in Aspergillus have established that activation of both CDK1 and NIMA kinases is required for entry into mitosis and that their subsequent inactivation by proteolysis is required for progression through mitosis into G1 [7.Cell Cycle Regulation in Morphogenesis and Development of Fungi 95 [16].19. This indicates that unlike other cell cycle regulators. As cells progress through mitosis.8. it is not surprising that tyrosine 15 phosphorylation of CDK1 plays key checkpoint roles in coupling initiation of mitosis to completion of S-phase and also in response to DNA damage to delay entry into mitosis until damaged DNA is repaired [10.11. the requirement of NIMA kinase for mitotic entry does not involve regulation of CDK1 kinase activity. NIMA becomes hyperphosphorylated and a MPM-2 antigen in a CDK1-dependent manner and its kinase activity is further activated . Interestingly. however. Activation of CDK1 is necessary but not sufficient for entry into mitosis as inactivation of NIMA kinase through the temperature-sensitive nimA5 mutation blocks cell cycle at G2 with fully activated CDK1 and no cytological signs of mitosis [7. known as NIMT in Aspergillus [17. very much like a mitotic cyclin [30]. Thus.30]. Tyrosine 15 dephosphorylation is catalyzed by the tyrosine phosphatase Cdc25. Thus. NIMA rapidly is proteolysed also in an APC-dependent manner [31]. However.26.20. Indeed. During G2/M transition.22–25]. NIMA. So far the mitotic cyclin B encoded by nimE is the only known partner of Aspergillus p34cdc2. cell cycle specific mutants in Aspergillus. the NIMA protein oscillates once each cell cycle.17. During G2 NIMA protein accumulates in a hypophosphorylated state but is active as a -caseine kinase. The p34cdc2 and cyclin B become associated with each other to form an inactive CDK1 complex known as pre-MPF during interphase as cyclin B protein accumulates from late S-phase through G2 and peaks at G2/M transition. like cyclin B.22]. proteolysis of cyclin B is required for exit from mitosis [26]. demonstrates that activation of another protein kinase. as cells transverse through mitosis. In fact. Then. a key regulatory step to initiate mitosis is tyrosine 15 phosphorylation/dephosphorylation of CDK1 [10. Whether there are also G1 cyclins as in budding yeast to regulate initiation of DNA replication in Aspergillus remains to be established.21]. For CDK1 and NIMA to promote initiation of mitosis in a timely manner during the cell cycle and in response to checkpoint controls. The level of NIMA protein increases during G2 and peaks at early mitosis.27].29. the NIMA protein does not contain a consensus destruction box motif required for APC-mediated ubiquitination and proteolysis by the proteosome pathway [7]. biochemical and cell biological studies show that CDK1 and NIMA kinases regulate each other’s mitotic functions at multiple levels. also is required to initiate mitosis [29. Whether APC-dependent proteolysis of NIMA is mediated through the ubiquitination-dependent proteolysis pathway like mitotic cyclinB remains to be determined. cyclin B becomes rapidly degraded by ubiquitin-mediated proteolysis promoted by the anaphase-promoting complex (APC) upon exit from mitosis into G1 [22.30]. The rapidity of tyrosine 15 dephosphorylation of CDK1 is promoted by positive feedback activation of Cdc25 by activated CDK1 [22]. CDK1 is kept in the inactive state through tyrosine 15 phosphorylation by the Wee1 kinase (known as ANKA in Aspergillus) during S to G2 phase [17–19]. Indeed. In addition.

Enigmatically. however.35]. Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena [36]. Many NIMA-related kinases (NEK) have . a suppressor of the nimA1 mutation (sonA) is a homologue of the nucleocytoplasmic transporter GLE2/RAE1 and suppresses nimA1 lethality at the restrictive temperature in an allele-specific manner [34]. the function of NIMA is unlikely to just phosphorylate histone H3 during mitosis. As mentioned earlier. NIMA kinase is required for initiation of mitosis at a point after activation of CDK1 by tyrosine 15 dephosphorylation. Third. whether histone H3 serine 10 phosphorylation is essential for DNA condensation in this system. Upon release from Cdc25 G2 block. Two lines of evidence indicate that NIMA kinase is required for nuclear localization of CDK1 in G2 [34]. Furthermore. As cells transverse through mitosis. an event closely associated with initiation of mitosis [33]. First. NIMA protein is mainly cytoplasmic. recent studies provide strong evidence at the molecular level for a mechanism by which CDK1 and NIMA kinases coordinate their mitosis-promoting activities through interdependent regulation of their mitotic functions. NIMA is able to promote this phosphorylation anywhere in the cell cycle. indicating the presence of a NIMA pathway of cell cycle regulation in other filamentous fungi [37]. In filamentous fungi. indirect immunofluorescence analysis shows that CDK1 is localized predominantly to the nucleus during G2/M and CDK1 nuclear localization and its association with the nuclear-associated organelles and spindle pole body requires NIMA functions. additionally. NIMA is an excellent substrate of CDK1 in vitro. Together. this suppression of nimA1 lethality is associated with the restoration of CDK1 nuclear localization. NIMA is rapidly transported into nuclei. suggesting that full mitotic function of NIMA kinase is regulated by CDK1. because inactivation of NIMA prevents all aspects of mitosis and. at least when it is overexpressed [30. NIMA can promote chromosome condensation in the absence of CDK1 function in many biological systems. Second. suggesting a role of NIMA at these sites during mitosis as well [33]. NIMA become associated with chromatins at initiation of mitosis. one of the mitotic functions of NIMA kinase is to regulate CDK1 cytoplasmic to nuclear transport during G2/M transition [34]. this phosphorylation is found to be dependent on NIMA function [33]. NIMA kinase is a founding member of an evolutionarily conserved family of protein kinases present from fungi to humans [7]. Then upon entry into the nucleus. Indeed. At Cdc25 G2 arrest point. NIMA nuclear localization during initiation of mitosis requires CDK1 activation by Cdc25 dephosphorylation [33]. Indeed. and CDK1 in vitro phosphorylation of NIMA also generates MPM-2 epitopes [30]. and all have a very high isoelectric point ( 10). It remains to be determined. as discussed previously. coincident with histone H3 serine 10 phosphorylation. Furthermore. However. Second. In Aspergillus. NIMA directly phosphorylate this residue on histone H3 in vitro [33]. The NIMA homologue (nim-1) isolated from Neurospora crassa can fully complement lack of NIMA function in Aspergillus.8]. indicating a key role of NIMA in promoting this mitotic event. First. A direct role of NIMA in this phosphorylation is supported by the following observations. part of NIMA functions is to phosphorylate histone H3 serine 10 to promote DNA condensation. NIMA kinases particularly are highly conserved and also very likely are functionally conserved. NIMA becomes transiently associated with the spindle microtubules and the spindle pole bodies. These kinases all contain an N-terminal catalytic domain and C-terminal regulatory domain with a coiled coil linker between the domains.96 Ye [30]. NIMA proteolysis is also required for progression through mitosis [7. when untimely overexpressed.

First. How developmental programs orchestrate these different levels of controls over the cell cycle is a fundamental biological question and thus attracts intense scientific interest because of the enormous importance in understanding human pathological conditions caused by the deregulation of cell cycle and growth controls such as cancer.Cell Cycle Regulation in Morphogenesis and Development of Fungi 97 been identified from humans and are implicated in human cell cycle regulation [7]. thus giving rise to multinucleate hyphal cells with up to 16 nuclei per cell [41]. Second. Metulae divide once to produce a second tier of cells. which then produce a chain of uninucleate spores by repeated asymmetric mitotic divisions. many rounds of nuclear division take place in the stalk in the absence of cytokinesis. or septation. However. Recent studies have indeed established a key role of developmental regulation of CDK1 activity in coordinating cell cycle control with morphogenesis and cell differentiation. Then during asexual development. cell division must also be coordinated with cell differentiation for the production of highly specialized cell types to perform specific physiological and cellular functions. Nuclear division and cytokinesis. The first event of conidiophore development is the production of aerial stalks crowned with vesicles from specialized anchoring foot cells. . A. nidulans has both a complex asexual and sexual lifecycle with highly defined morphogenic patterns [38]. called phialides. first cytokinesis normally occurs after the third round of nuclear division of germinating spores. Aspergillus is an attractive model to study the relationship between cell cycle regulation and developmental controls during morphogenesis and cell differentiation. An important role of CDK1 in Aspergillus asexual development was first noticed with a strain bearing a mutant CDK1 whose inhibitory phosphorylation residues threonine 14 and tyrosine 15 are mutated to nonphosphorylatable alanine and phenylalanine. Interestingly. This strain thus cannot negatively regulate CDK1 by Thre14 and Tyr15 phosphorylation and is shown to be deficient consequently in both the slowing S-phase and G2/M DNA damage checkpoint controls [19.23]. under laboratory conditions. just like the requirement for multiple CDKs. A series of cells. Perhaps. cell division must be coupled to growth and morphogenesis to allow organs and tissues to reach their appropriate size. respectively [19]. In fact. called metulae. extensive studies over the last several decades have established key regulators in both cell cycle control and development in this model experimental system [38–40]. Most important. Thus. Aspergillus elaborates several distinct cell types by both filamentous and budding growth to form the multicellular conidiophore structure from which the uninucleate conidiospores are derived by repeated asymmetric division. Remarkably. are formed from the vesicle by budding and one nucleus from the stalk migrates into each of them. none of the human NEKs so far studied show all the characteristics of NIMA mitosispromoting functions as observed in Aspergillus. NIMA mitosis-promoting functions observed in the lower eukaryotic system may have been delegated to multiple NEKs due to the increased complexity of human cell cycle regulation. 3. are not strictly coupled in vegetative hyphal growth. These dramatic changes in modes of cell division and patterns of growth during conidiophore morphogenesis thus necessitate intimate interactions between the developmental program and cell cycle control mechanisms [42]. DEVELOPMENTAL REGULATION OF THE CELL CYCLE IN MORPHOGENESIS AND CELL DIFFERENTIATION DURING CONIDIOPHORE DEVELOPMENT Cell division must be carefully controlled during the development of multicellular organisms.

Thus. stuA. Depletion of maternal string leads to a G2 cell cycle delay at cycle 14 to allow cellularization. other mutant strains deficient in tyrosine phosphorylation of CDK1 also display similar morphogenic defects during asexual development. As the mode of cell cycle changes from multinucleate to uninucleate cell division during conidiophore development. whereas the mutant conidiophores display grotesque deformities and consequently lack symmetry.40]. and wetA. with brlA functioning as the focal point for integration of developmental cues [38. nuclear division in the stalk normally occurs in the absence of septation. However. Subsequent light and scanning electron microscopic examinations show multiple gross defects in conidiophore morphogenesis [3].98 Ye this mutant strain is viable and is able to grow vegetatively without significant cell cycle defects. Ectopic expression of brlA or abaA under normally conidiation-suppressing conditions also markedly induces CDK1 activity [3]. which coordinates mitosis and morphogenesis during fly ventral furrow formation by regulating string/CDC25 proteolysis. this mutant strain conidiates very poorly [3]. This increase in CDK1 activity is closely correlated with the increase of both nimX/Cdc2 mRNA and protein. which encodes a master transcriptional regulator in conidiophore development [3]. Two recent studies identified a new cell cycle regulator. Furthermore. Following fertilization. CDK1 activity is highly induced during initiation of conidiophore development and this induction is dependent on brlA. tribbles. a morphogenic checkpoint is mediated by tyrosine phosphorylation of CDC28. perhaps increased CDK1 activity is needed to couple each nuclear division to cytokinesis/septation. Perhaps this mechanism coordinating the cell cycle with morphogenesis is conserved through evolution. Even in the unicellular budding yeast. Together. normal conidiophores are highly symmetrical. In addition. a key morphogenic event [43]. The central genetic pathway controlling asexual conidiophore development consists of five regulatory genes: brlA. Interestingly. the homologue of CDC25 tyrosine phosphatase [43]. Third. Additionally. This is the first observation showing developmental regulation of CDK1 activity through regulated Cdc2 transcription. these results clearly demonstrate that tyrosine phosphorylation of CDK1 plays a key role in coordinating cell cycle regulation with cell fate determination during conidiophore morphogenesis. The upstream regulatory domains of nimX/Cdc2 contains multiple brlA regulatory elements (BRE). part of the functions of brlA is to regulate positively the expression of nimX/ Cdc2 and consequently increased activity of CDK1. a homologue of CDK1. First. abaA. Second.45]. through the Swe1/Wee1 protein kinase activity to coordinate cell cycle with bud emergence [46. the fly embryo undergoes 13 rapid syncytial blastoderm divisions driven by maternally provided proteins and RNAs. This is consistent with the observation that a slight de- . coordination between cell division and morphogenesis in Drosophila also is mediated through tyrosine phosphorylation of mitotic CDK regulated by string. but the mutant strains still fail to develop the correct cell types of the conidiophore [20]. the lethality caused by a mutation of nimT/CDC25 can be suppressed by multiple copies of nimE/cyclin B for vegetative growth. As mentioned above. brlA is necessary and sufficient for induction of CDK1 activity during asexual conidiophore development. thus providing further evidence supporting an important role for the regulation of CDK tyrosine phosphorylation in linking cell cycle regulation to morphogenesis in the fly [44. the mutant conidiophore stalks all have multiple septa. Reinitiating mitosis is then elaborately controlled by regulated expression patterns of string/CDC25 in time and space by developmental programs [43]. These defects are apparently caused by inability of the mutant strain to develop the correct cell types sequentially [3].47]. medA. many mutant conidiophores form multiple vesicles as a result of reiteration of the same cell type.

to five-fold due to slower mitotic division in phialides. pclA [4].Cell Cycle Regulation in Morphogenesis and Development of Fungi 99 crease in CDK1 activity using the nimT23/Cdc25 mutation at a semirestrictive temperature suppresses septation while nuclear division continues normally [41]. whereas growth factors stimulate rapid. nidulans and has uncovered novel biological functions for sphingolipids. and metabolism of sphingolipids are largely conserved from fungi to humans. These results again point to a key role of CDK1 regulation through tyrosine phosphorylation both in cell cycle regulation and development. transient generation of S-1-P catalyzed by sphingosine kinase [48–50]. the level of cellular ceramide remains very low. Recently. It is now well documented in many biological systems that stress signals rapidly and transiently elevate the level of cellular ceramide. Perhaps part of the increased NIMX/ Cdc2 during conidiophore morphogenesis is used to form complex with the Pcl-like cyclin to carry out yet unknown cell cycle functions required for spore production. APOPTOSIS. sphingoid bases (sphingosine. It has been suggested that ceramide and S-1-P together act as a cellular rheostat. Our research in Aspergillus shows that sphingolipids play a central role both in the establishment and in the maintenance of cell polarity via polarized organization of the actin cytoskeleton. We further find that inositol phosphorylceramide (IPC) synthase. and highly polarized hyphal growth is the most salient feature of fungi. The pclA expression also is developmentally regulated in a brlA dependent manner. 4. Subsequent biochemical analysis shows that the PCLA protein physically interacts with NIMX/Cdc2 kinase as demonstrated by coimmunoprecipitation experiment. dihydrosphingosine. and sphingoid base phosphate are highly bioactive molecules implicated as second messengers mediating a wide array of cellular activities. Establishment and maintenance of cell polarity is essential to the growth and development of multicellular organisms. such as ceramide. Among these. including stress response. cell cycle. Many metabolites derived from complex sphingolipids or from de novo synthesis. Deletion of pclA markedly reduces spore production by three. and phytosphingosine). the level of cellular ceramide is rapidly and transiently elevated and the increase in cellular ceramide accompanies closely a transient inhibition of cell cycle progression and cell growth. REGULATION OF CELL CYCLE PROGRESSION. apoptosis. biosynthesis. AND DEVELOPMENT BY SPHINGOLIPIDS Sphingolipids are major components of eukaryotic cell membranes and are emerging as an important signaling mechanism [48]. perhaps mediated through functions of lipid rafts [51]. consequently with higher kinase activity. S-1-P is a high-affinity ligand of the edg family of G-protein coupled receptors. Because the basic chemical structure. and its ligation with the receptors is shown to promote cell survival and proliferation by antagonizing ceramide-mediated apoptosis [50]. our laboratory has employed both genetics and pharmacological inhibitors to manipulate the sphingolipid biosynthesis pathway to gain new insight into cellular functions of sphingolipids in genetically tractable A. determining whether cells undergo apoptosis or continue to proliferate [50]. a genetic screen using restriction enzyme mediated integration (REMI) mutagenesis with SmaI for Aspergillus mutant defective in conidiophore morphogenesis identified a homologue of budding yeast Pcl cyclins. Conversely. inflammation. CDK1 mutants deficient in inhibitory phosphorylation. which catalyzes the synthesis of . and cancer development [48–50]. and that the immunocomplex contains H1 kinase activity [5]. Upon stress treatment such as heat shock. promote septation [41]. In normal growing cells. ceramide and sphingosine-1-phosphate (S-1-P) are most extensively studied.

DHS and PHS induce large-scale DNA fragmentation. organelle degradation. but not the typical oligonucleosomal ladders associated with caspase-mediated apoptosis [53].100 Ye complex sphingolipids. Further analysis reveals that DHS and PHS inhibit fungal growth via rapid induction of apoptosis [52]. Dihydrosphingosine (DHS) and phytosphingosine (PHS) are the predominant sphingoid bases in fungi. In fact.and PHS-mediated apoptosis are not understood. . Therefore. Cell death associated with heterokaryon incompatibility shows microscopic and ultrastructural features—vacuolization of the cytoplasm. the central regulator of asexual development of Aspergillus. we suggest that IPC synthase activity plays an important role in coupling cell cycle to cell growth during stress response via its function in sphingolipid synthesis and regulation of the level of cellular ceramide. heterokaryon incompatibility results in cell death after anastomosis. forced expression of brlA. which is found to be accompanied by extensive hyphal death in the central portion of the colony. and DNA fragmentation but not DNA condensation requires protein synthesis [52]. exposure of phosphatidylserine. DNA condensation can occur in the absence of DNA fragmentation. At present. cell cycle progression is tightly coupled to cell growth. First. unpublished data). Perhaps sphingolipid metabolism plays a role in the cell death associated with heterokaryon incompatibility and conidiation. and plasmolysis—consistent with some of the apoptotic features in animals [58. Remarkably. how cellular levels of endogenous DHS and PHS are normally regulated and under what biological conditions A. nidulans undergo DHS. such as nutrient limitation promote asexual sporulation. Two lines of evidence demonstrate that apoptosis induced by DHS and PHS is very similar to caspaseindependent apoptosis recently described in mammalian systems and in Dictyostelium discoideum [52. although they undergo vegetative growth normally as the wild type. only the naturally occurring DHS and PHS are biologically active in our assays. Fungal apoptosis induced by DHS and PHS exhibits all major features characteristic of apoptosis originally described in animals. whose cellular contents are recycled for spore production [40]. because sphingolipid biosynthesis is essential for cell growth and increases in cellular ceramide cause G1 arrest. Indeed.59]. during vegetative growth promotes conidiophore formation and also causes rapid cell death [60]. In support of this assumption. accumulation of ceramide upon inhibition of IPC synthase activity causes cell cycle arrest at G1 [51].54–57]. and generation of reactive oxygen species (ROS) [53]. which shows exquisite structural and stereo-chemical specificity [52].. because several different chemical classes of potent antifungals all targeting fungal membrane functions do not induce apoptosis [52]. in A. In addition. indicating an essential role of sphingolipid metabolism in conidiation (Cheng et al. High structural and stereo-chemical specificity in induction of apoptosis by DHS and PHS suggests that their activities are physiologically relevant and must act on very specific molecular targets to bring about apoptosis. such as rapid DNA condensation independent of mitosis. plays a key role in mediating the level of cellular ceramide. This apoptotic response is shown to be specific to the action of DHS and PHS. We further find that DNA condensation and DNA fragmentation in fungal apoptosis are in fact separate cellular events. Normally. DNA fragmentation. we found that Aspergillus mutant strains with compromised SPT activity are unable to conidiate. apoptosis induced by DHS and PHS is independent of the highly conserved fungal metacaspase [54]. In filamentous fungi. presumably as a self-defense mechanism to prevent transmission of infectious elements [58]. DHS and PHS have potent antifungal activity against Aspergillus. Second. nidulans environmental stresses.

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ARLENE M.2]. are less labor intensive and inherently faster.5 Agrobacterium-Mediated Transformation of Filamentous Fungi JAN S. and they have been used successfully with certain fungi. tumefaciens that allows the delivery of genetic information across the boundaries of biological kingdoms. enabling the application of today’s powerful gene manipulation strategies for purposes ranging from the elucidation of fundamental biological phenomena to the improvement of commercial biological processes. a bacterium widely employed in the transformation of plant cells. NAGEATTE IBRAHIM. DAHL-ROSHAK. PARESS Merck Research Laboratories.A. It makes use of Agrobacterium tumefaciens. tumefaciens is capable of introducing DNA into fungal cells. 1. U. and PHILIP S. AGROBACTERIUM BACKGROUND A mechanism for the mobilization of DNA has evolved in A.S. For two decades. INTRODUCTION Procedures to introduce exogenous DNA into a cell in a form that can be inherited and expressed for phenotypic change constitute a key technology. the most commonly used transformation method for filamentous fungi has been the calcium ion–polyethylene glycol (PEG) procedure that requires first the conversion of the target organism to spheroplasts for the uptake of the DNA and subsequently the regeneration of cell walls for the resumption of hyphal growth. this chapter is devoted exclusively to a new method that offers the advantages of simplicity and general applicability. Other methods. In as much as comprehensive reviews detailing the use of these procedures with filamentous fungi have appeared recently [1. TKACZ. New Jersey. including electroporation and particle bombardment. Rahway. [3] that A. The 105 . 2. and it stems from the 1998 report by de Groot et al.

Following introduction of the donor plasmid into an Agrobacterium strain that carries a resident disarmed Ti plasmid. which manifests itself as a crown gall tumor. The T-DNA is delimited by imperfect 25-base pair repeats called the right and left border sequences (RB and LB. and agrocinopine [9]. known as the T-DNA. Another portion of the Ti plasmid comprises a cluster of about 35 virulence (vir) genes that encode the proteins required to mobilize the T-DNA. as a single-stranded molecule. The first feature is that the vir genes and the T-DNA need not reside on the same plasmid [11. RB first. whereas pTiA6 and pTiAch5 have been placed in the octopine group. Examples of opines include octopine. The T-DNA region of a Ti plasmid encodes genes for the synthesis of both plant growth hormones and unusual amino acid derivatives known as opines. Although the hormone and opine genes play an integral part in crown gall tumor formation. tumefaciens and Escherichia coli carries the RB and LB sequences between which one or more genes of interest may be cloned. respectively). As mentioned. Any DNA element that is placed between the RB and LB can be mobilized by the vir gene products. A donor vector able to replicate in both A. Adjacent to the RB but outside of the T-DNA region there is an element (known as overdrive) that binds the virC1 gene product and enhances T-strand production. tumefaciens cell carrying a disarmed Ti plasmid when the Ti plasmid’s T-DNA is introduced into the cell as part of a separate replicon [11]. 1). Virulence is restored in an A. nopaline. mannopine. In the older literature the Ti plasmids were classified according to the predominant opine associated with the crown gall tumor they cause. so that today the bacterium provides the preferred method for transforming cells of both dicotyledonous and monocotyledonous plants. they are not necessary for mobilization of the T-DNA. histopine. The second significant characteristic concerns the nature of the T-DNA itself.106 Tkacz et al. However. tumefaciens but are incapable of producing crown gall tumors. Ti plasmids that are disarmed by removal of the T-DNA segment propagate in A. Despite the distinctions rooted in the opine genes. The majority of the vir genes are quiescent until induced by phenolic compounds typically released by wounded plant cells. tumefaciens cells and constitute the benefit the bacteria derive from the expression of the opine genes within the plant. has its origin in large extrachromosomal elements. Many phenolics may act as elicitors. Most often activation is achieved by the exogenous addition of acetosyringone.12]. Further mechanistic details on the establishment of this intriguing host-pathogen relationship can be found in a number of worthwhile reviews [4–10]. Over- . These two properties have permitted the adaptation of the Ti plasmid into a binary plasmid system for DNA delivery (Fig. known as tumor-inducing or Ti plasmids. pTiT37 and pTiC58 have been considered nopaline plasmids. Ti plasmids themselves are not readily manipulated. These derivatives serve as nutrients for the A. the T-DNA portion of Ti plasmids encodes genes for opine production. Establishment of a crown gall tumor requires the transfer of a specific segment of DNA. the genes that are flanked by the border sequences are subject to mobilization upon activation of the vir genes. Because of their large size ( 100 kb). With the characterization of increasing numbers of Ti plasmids. there is very little difference in the RB and LB sequences in the various Ti plasmids. it is now clear that this classification system is imperfect [6]. two characteristics of the DNA mobilization machinery have made it possible to harness Agrobacterium as a means of gene delivery. Transport of the T-DNA into the plant cell is a polar process: the T-DNA is introduced. acetosyringone and coniferyl alcohol are two examples. tumefaciens. pathogenic association of this Gram-negative prokaryote with certain plants. Thus. found in natural populations of A. from the Ti plasmid into the plant cell by a process that resembles bacterial conjugation [4].

Hooykaas reported the transformation of a wide variety of filamentous fungi (both ascomycetous and basidiomycetous) to hygromycin B resistance resulting from cocultivation of the fungi with A. J. Replication of the donor plasmid in E. 3. tumefaciens (Table 1). FUNGI AS TARGETS FOR AGROBACTERIUM-MEDIATED DNA TRANSFER Although the Ti plasmid system has evolved as a mechanism of plant pathogenesis. J. the transcriptional regulator of the vir cluster. Earlier reports from the same laboratory [14. The donor plasmid has an overdrive (OD) sequence proximal to the RB to enhance T-strand formation. T-DNA (T. coli allows it to be manipulated by standard procedures before it is introduced into the Agrobacterium cell. and this leads to activation of the virG protein. Nester’s group [16] had demonstrated the transfer of wildtype yeast URA3 and TRP1 . tumefaciens has the capability to deliver T-DNA to fungi under conditions that simulate those within a plant wound. a drug resistance marker is provided for selection in both bacteria.Agrobacterium-Mediated Transformation of Filamentous Fungi 107 Figure 1 Binary plasmid system for Agrobacterium tumefaciens. respectively) is mobilized and transferred to the target cell where it enters the nucleus and integrates into genomic DNA. Acetosyringone activates a cell surface receptor that is the product of virA. A disarmed Ti plasmid resident in the bacterium carries a cluster of virulence genes (vir) encoding proteins that mobilize the TDNA. A.15] and from E. but there appears to be functional equivalence among them [13]. W. The Dutch group led by P. Cocultivation was performed in the presence of acetosyringone to mobilize a T-DNA element containing the hygromycin B phosphotransferase gene driven by a fungal promoter [3]. drive sequences differ in the various types of Ti plasmids. shown as a black segment of the donor plasmid) flanked by right and left border sequences (RB and LB.

single copy integration frequent Bacteria that had not been exposed to acetosyringone prior to cocultivation yielded a higher proportion of fungal transformants with single site insertions.18 20 31 Organism Donor Plasmid Aspergillus awamori pBin19. single-copy ectopic insertions predominate Inducer-dependent transformation. cocultivation parameters explored 26 . frequency decreases as size of T-DNA increases Transformation of yeast and germinating conidia.108 Table 1 Fungi That Have Been Transformed by the Agrobacterium Procedure Selectable Genea Features hph (gpdAb) pyrG hph (gpdAb) Hc URA5 hph (gpdAb) hph (cpc1) hph (gpdAb) hph (gpdAb) hph (trpC) Ref. higher efficiency with nutritional selection.18 23 25 Coccidioides immitis pAD1310 Colletotricum gloeosporioides Fusarium circinatum Fusarium oxysporum pBin19 Inducer-dependent transformation of conidia or protoplasts. single site insertions predominate Inducer-dependent transformation of yeast cells. Magnaporthe grisea pBHt2 First transformation of this organism. inclusion of ColE1 ori and gene for chloramphenicol resistance in the T-DNA facilitated recovery of the integrated DNA by thermal asymmetric interlaced PCR Inducer-dependent transformation of conidia and rehydrated freeze-dried material Inducer-dependent transformation of vegetative mycelia. TRP1 gene replacement more efficient for ssDNA delivered as T-DNA than for dsDNA introduced by electroporation Inducer-dependent transformation of conidia. single-site insertions predominate Inducer-dependent transformation of conidia. pSDM14 pSDM14 Blastomyces dermatitidis pBIN19 Calonectria morganii pTAS5 24 21 3. efficient gene replacement Transformation of yeast form was more efficient with nutritional selection. cocultivation parameters explored. 3. cocultivation parameters explored Inducer-dependent transformation of conidia pPZP201 pCAMBIA1300 Fusarium venenatum pBin19 hph (gpdAb) 3 33 31 19 Glarea lozoyensis pAg1 Histoplasma capsulatum Kluyveromyces lactis pBin19 hph (trpC) Flanked by pks1 hph (gpdAb) Hc URA5 URA3 Flanked by TRP1 hph (trpC) Tkacz et al. first transformation of this organism Use of vector with N54D variant of virG for inducer-independent transformation of arthroconidia.

ori Very high transformation rates with T-DNA containing an ars and telomere sequences (mini-chromosome).pBIN19 pCGN1589 pBin19 pBin19 27 3. 1 Au: “germlings” correct or do you mean “germlines”? 109 . promoters and disarmed Ti plasmids compared Transformation procedure optimized.17 3.18 25 3 28 22 32 Hebloma cylindrosporum pCAMBIA1300 Paxillus involutus Pleurotus ostreatus Inducer-dependent transformation of cells or protoplasts at equivalent frequencies. in vivo circularization of T-DNA containing the 2. c Glyceraldehyde-3-phosphate dehydrogenase promoter of Agaricus bisporus. d Glyceraldehyde-3-phosphate dehydrogenase promoter of Schizophyllum commune. 60% of transformants resulted from single-site integration Transformation of an ectomycorrhizal basidiomycete Transformation of an ectomycorrhizal basidiomycete Inducer-dependent transformation of conidia and germlings1 Transformation of an ectomycorrhizal basidiomycete 30 30 18 30 29 Suillus bovinus pBin19 pBin19 pBin19 pBin19 pCAMBIA1300 Sh ble (gpdd) Sh ble (gpdd) hph (gpdAb) Sh ble (gpdd) hph (gpdc) a Agrobacterium-Mediated Transformation of Filamentous Fungi b Heterologous promoters are shown in parentheses. Inducer. disarmed Ti plasmids compared Characterization of randomness and fine structure of ectopic integrants Inducer-dependent transformation of conidia Cited as unpublished data Transformation of germinated basidiospores Transformation of germinated basidiospores and vegetative mycelia Transformation of gill tissue and fleshy tissue of the fruiting body. Glyceraldehyde-3-phosphate dehydrogenase promoter of Aspergillus nidulans.and vir gene-dependent transformation of yeast cells.18 14 hph (gpdAb) flanked by MgAtr2 hph (gpdAb) URA3 Mycosphaerella graminicola Neurospora crassa Saccharomyces cerevisiae “ “ TRP1 URA3 hph (gpdAb) hph (gpdAb) hph (gpdAb) hph (gpdb) hph (gpdc) 16 pBin19 Trichoderma reesei Verticillium dahliae Agaricus bisporus pBin19 pBin19 pBin19 pBin19 pCAMBIA1300 15. efficient gene replacement Inducer-dependent transformation of conidia. homologous integration of T-DNA into the genome.

Significantly.0-kb segments from opposite ends of pks1. provided that yeast replication origins or telomeric sequences were absent from the T-DNA [14. stable transformation is established by ectopic insertion at random genomic loci.and 4.16]. lactis.1-kb segments from the MgAtr2 gene provided null mutations at the MgAtr2 locus of Mycosphaerella graminicola in 44% of the hygromycin B resistant transformants [27]. replacement of the TRP1 gene with the URA3 gene of S. when the URA3 gene in the T-DNA was flanked with 0. whether the recipient is a yeast [15. a filamentous fungus [3]. with the possible exception of electroporation. germinated conidia. The organisms transformed by de Groot et al. Preparations of lytic enzymes and osmotically fortified solutions for protoplasting as well as knowledge of appropriate conditions for cell wall regeneration are not required. In K. In fact. Successful use of the technique with other fungal species subsequently has been reported by several laboratories [18–33]. genes from A. for example.110 Tkacz et al. the single-stranded nature of the transforming DNA may favor homologous recombination. lactis. This is primarily because the T-DNA is delivered to the fungal cell and integrates into the genome predominantly as a single copy [3. a melanin-deficient phenotype was produced in 59% of hygromycin B–resistant transformants of Glarea lozoyensis when the resistance cassette in the T-DNA was flanked by 3. the method does not require the target fungus to be converted to protoplasts. When incoming T-DNA is devoid of homologous sequences. the gene may be reiterated between the RB and LB in the donor vector before the plasmid is used to transform the fungus [20]. or a plant cell. Refinement of the currently available donor vectors and selection strategies may be expected. The Agrobacterium method is far less complicated in execution than any of the earlier methods. .and 1. When an increase in gene dosage is the desired goal. roughly equivalent transformation frequencies were obtained with either conidia or protoplasts in the case of Aspergillus awamori [3] and of Mycosphaerella graminicola [27]. Similarly.31.17]. cerevisiae was observed in 71% of the Ura transformants. even fruiting tissue have been successfully transformed by means of Agrobacterium-mediated DNA transfer. Stable yeast prototrophs were obtained predominantly by homologous recombination. it would appear that the technique will play an increasingly prominent role in the study of fungal biology and in the manipulation of commercially important fungal strains. ssDNA delivered by Agrobacterium was remarkably more efficient in producing gene replacements than a comparable DNA segment delivered as a double-stranded molecule via electroporation [27]. neither is specialized equipment for the ballistic delivery of DNA coated microparticles. Targeted gene replacement is also readily achieved if the T-DNA contains sufficiently large stretches of homologous DNA. [3] included a broad range of fungi (Table 1).and 4.4-kb segments homologous to the ends of the target TRP1 gene [19]. The opportunity to use intact fungal cells as recipients is perhaps the most appealing aspect of the technique. in the case of basidiomycetes. a rehydrated lyophilized fungal preparation and. From these initial experiences with Agrobacterium-mediated transformation. Another advantage of the Agrobacterium method is that the outcome of DNA integration is more predictable than it is with other transformation procedures. Larger regions of homology are required to give high rates of gene replacement in filamentous fungi.6. Use of T-DNA comprised of a hygromycin B resistance cassette flanked by 2. a gene whose product catalyzes the formation of a polyketide precursor required for pigment synthesis [33]. conidia.32].9. vegetative mycelia. tumefaciens to Saccharomyces cerevisiae auxotrophs. In K.8. In some cases.21–27.

the T-DNA flanked by the LB and RB. the Pseudomonas plasmid pVS1 [40. tumefaciens having this donor plasmid is no longer strictly dependent on acetosyringone for DNA mobilization. coli). To remedy this.Agrobacterium-Mediated Transformation of Filamentous Fungi 111 4.g. and the RK2 replicon [36].8-kb) plasmid with relatively few unique restriction sites located between the LB and RB. 1). A derivative of pAg1 bearing the N54D form of virG has been constructed in our laboratory. nptIII. Transcriptional activation of these genes is under the control of a two-component regulatory system composed of constitutively expressed vir proteins (Fig. repA in the case of SA and trfA of RK2) have been omitted from certain donor vectors. Incorporation of this N54D mutant form of virG into a donor vector provides inducer-independent expression of the vir genes. The new plasmid. We have made a streamlined version of pBin19 [33] by a PCR strategy similar to that employed by Xiang et al. and a second ori for propagation in A. personal communication) within the T-DNA region. tumefaciens and E. a transmembrane protein that autophosphorylates in response to environmental stimuli (plant phenolics. and temperature) [8].41]. It is important to note that in the interest of minimizing the size of these dual-origin donor vectors and providing for biocontainment. the cytoplasmic virG protein. limiting their use to specific Agrobacterium host strains in which a copy of the replication gene is either incorporated into the genome or carried on a compatible companion plasmid. the donor plasmid pBIN19 (Table 1) is a large (11. pAg1 (Fig. Seeing no practical advantage over pAg1. for high copy number in E. or the wide-host range plasmid pSA [42]. A practical drawback of donor vectors based on the RK2 origin of replication is their propagation at low copy number in E.. even though A. One component is the virA product. acidic pH. namely. A mutation within virG that converts a neighboring asparagine residue (N54) to aspartic acid allows the gene product to behave as a transcriptional activator without being phosphorylated [43–45]. we do not routinely make use of the pAg1 N54DvirG derivative. coli. The activated form of the virA protein phosphorylates an aspartic acid residue (D52) in the second constitutively expressed component. DONOR VECTORS A number of donor plasmids tailored for plant cell transformation have been described [34]. and such a plasmid has been used in the transformation of Coccidioides immitis [21]. Donor plasmids also may contain a mutant form of virG to alter the expression of the inducible vir genes. but many of these contain segments that are inconsequential for fungal transformation. It was derived from the nopaline plasmid pTiT37 [12] and incorporates the Gram-negative broad host range RK2 origin of replication. [37]. and we found that. Modeling of the 3D structure of the virG protein based upon its similarity with .. such as the ori from the Agrobacterium rhizogenes Ri plasmid [39]. which consists of the 700-bp oriV sequence and a trans-acting gene designated trfA. as well as the overdrive element characteristic of octopine Ti plasmids [38]. genes for obligatory trans-acting replication functions (e. whose products then mobilize the T-DNA. DeSanti. Less than 4 kb of pBin19 is required to encode its essential functional features. 2). The extent of this stimulation. the ColE1 ori of pBR322. several vectors have been developed that have two replication origins. sugars. has a size of only 4 kb and incorporates the generous multiple cloning site of pANT846 (C. a streptococcal kanamycin resistance gene that provides dominant selection in A. tumefaciens. For example. The trfA gene encodes two proteins required for initiation of DNA replication [35]. a drug-resistance marker (e.g. is only equivalent to that which could have been achieved with the inducer acting in concert with the wildtype virG gene of the resident disarmed Ti plasmid. enabling it to become a transcriptional activator for the expression of the remaining vir genes. transformation frequency remains subject to further stimulation by addition of inducer. however. coli.

Restriction sites of the MCS that remain unique in pAg-H4 are marked by asterisks. overdrive element. Because transfer of TDNA is polar beginning with the RB.35-kb hygromycin B resistance cassette at the KasI and SacI sites of pAg1. . selection for a marker placed near the LB ensures that each transformant will have received the entire T-DNA element. a transacting gene of the RK2 replicon required for DNA replication. The hph gene in this cassette is under the control of tandemly arrayed promoters: the trpC promoter for expression in fungi and the EM7 promoter for expression in E.112 Tkacz et al. OD. Figure 2 Map of donor plasmid pAg1. nptIII. a 4-kb streamlined version of pBIN19 with a generous multiple cloning site (MCS) in the T-DNA segment delineated by the left and right border sequences (LB and RB. coli and A. kanamycin resistance gene. tumefaciens. trfA. respectively). pAg-H4 was made by inserting a 2.

and to phosphinothricin by an acetyltransferase from Streptomyces hygroscopicus (bar) [55. 5. Resistance is provided to hygromycin B by an E. cerevisiae than the same donor in combination with the disarmed version of pTiA6 [16]. resistance is given by an acetyltransferase encoded by nat1 [57]. coli hygromycin B phosphotransferase (hph) [51–53].5 to 15. DOMINANT SELECTION Complementation of an auxotrophic mutation in a fungal recipient is a means of selecting transformants that is available only in the well-studied filamentous fungi [2]. the gpd promoters of Schizophyllum commune.5 kb has been found to decrease the efficiency of transformation [20]. selection based on dominant drug resistance provides a more generally applicable approach because it requires only that the target organism is sensitive to an agent for which a resistance gene is available. Both the loxP site for the P1 bacteriophage Cre protein [49] and the site for the lambda phage att system (Gateway . Three drugs that have proven useful in this context are hygromycin B. and Ustilago maydis are available [61. found that a donor plasmid used along with the disarmed form of pTiBo542 was more efficient in transforming S. In certain areas of fungal biotechnology. therefore.62]. Phanerochaete chrysosporium. The efficiency of this process is improved by increasing the gene dosage for virG and providing for extra copies of the virE2 protein [48]. For most strains of agricultural and industrial importance. and changes in two amino acids (residues 7 and 106) near this pocket apparently account in large measure for the remarkably strong activity of the virG protein encoded by the hypervirulent plasmid pTiBo542. Invitrogen) [50] have been used.Agrobacterium-Mediated Transformation of Filamentous Fungi 113 the cheY protein suggests that D52 is located within an ‘‘acidic pocket’’ [46]. increasing the size of T-DNA from 3. A vector with the ColE1 ori and a gene for chloramphenicol resistance in the T-DNA region has been used to allow recovery of fungal genomic DNA flanking the integration site by plasmid rescue [25]. maydis. Transfer of very large segments of DNA into filamentous fungi has not yet been reported. Expression of these bacterial genes in fungi under various growth conditions and at different stages of the life cycle requires that the genes be placed under the control of strong fungal promoters. whereas disarmed pTiC58 did not [22]. Piers et al. Two from Aspergillus nidulans (trpC [58] and gpdA [59]) and one from Neurospora crassa (cpc1 [60]) have been widely used in ascomycetous fungi. to phleomycin by a drug binding protein from Streptoalloteichus hindustanus (ble) [54]. The actin promoter from Cryptococcus neoformans has been used to express the genes for hygromycin B and nourseothricin resistance in this basidiomycetous human pathogen [64. although without making provisions for augmented levels of virG and virE2. the ability to transfer the genes for an entire biochemical pathway would offer tantalizing prospects. phleomycin (Zeocin). Nourseothricin also may be useful in certain cases. It is noteworthy. disarmed pTiBo542 supported transformation. Agaricus bisporus. that the Agrobacterium-mediated transfer of 150 kb of DNA into plant cells has been achieved [47]. .56]. Similarly for fruiting tissue of Agaricus bisporus. For basidiomycetes. and phosphinothricin (glufosinate ammonium). hygromycin B phosphotransferase has been expressed under the control of the organism’s heat shock hsp70 promoter [63]. in U. Agrobacterium donor vectors have also been refined by inserting site-specific recombinase sites within the T-DNA to facilitate the subcloning of DNA fragments from other plasmids.65].

coli cells containing the hph gene downstream of the fungal hsp70 promoter do not express resistance to this drug [63]. They can be used with a variety of donor plasmids. General methods for handling and preserving Agrobacterium strains may be found elsewhere [68. pAg1 derivatives containing the transposon can be recovered overnight in E. These cells carry plasmid pAL4404. ATTC51350) and AGL1 (with a disarmed hypervirulent succinamopoine plasmid derived from pTiBo542 [46]. The dual-origin of replication vector. 2). The resulting plasmid. because cells of this organism carrying pAg-H4 are resistant to hygromycin B at 50 g/mL. ATCC BAA-101). Glarea lozoyensis. Prompted by this example. despite its low copy number.67]. Similar tandem promoter constructs could be prepared for other drug resistance genes to extend the utility of this gene knockout strategy for elucidating gene function in fungi [66. coli. It is derived from the nopaline plasmid pTiT37 [12].2. New England BioLabs) has prompted interest in drug resistance markers that may be selected in both bacterial and eukaryotic cells [66. pAg-H4 allows robust growth and overnight colony formation in the presence of hygromycin B at 100 g/mL. In plasmid pTEF1/Zeo (Invitrogen). The new cassette was inserted into the T-DNA of pAg1 proximal to the LB to produce plasmid pAg-H4 (Fig. Those with insertions in the gene (or region) of interest are identified by restriction analysis or sequencing and moved into A.g. Transfer of the T-DNA bearing the insertionally inactivated gene into the fungus from which the gene was originally obtained results in replacement of the homologous gene by the inactive allele in a high proportion of the hygromycin B resistant transformants. Donor Vectors pBIN19 can be obtained from the American Type Culture Collection (ATCC37327). tumefaciens LBA4404 ready for electroporation may be purchased from Life Technologies (catalog number 18313–015). coli. tumefaciens [33] to allow growth of these bacteria in the presence of hygromycin B at 25 to 50 g/mL.69]. 2). followed by the EM7 promoter for expression in E. The strain also is available from the American Type Culture Collection (ATTC23341). and it has been installed in pGPS3 (New England Biolabs) in place of the kanamycin marker (Fig. tumefaciens. we have constructed a tandem trpC/EM7 promoter and placed hph under its control (unpublished). 6. which is a disarmed derivative of the octopine plasmid pTiAch5 [11]. 6. The recent commercial introduction of transposon systems that can be used to modify target DNA in vitro (e. the ble gene for Zeocin resistance is under the control of two tandem-arrayed promoters. In E. or a Nodulisporium isolate results in hygromycin B resistant fungal transformants. Following the random insertions of the transposon into the target DNA in vitro and destruction of the residual pTaH6.1. As expected. This activity profile makes the new tandem-promoter cassette an ideal transposon marker.. as are strains A136 (carrying no Ti plasmid. but E. the Tn7-based GPS. the TEF1 promotor for expression in yeast. tumefaciens. GUIDELINES FOR AGROBACTERIUM-MEDIATED TRANSFORMATION 6. It also appears that the EM7 promoter is active in A. Fortuitous expression of the hph gene in a trpC promoter-hph construct [53] is apparently sufficient in E.67]. provides a transposon that is very convenient for generating insertional mutants of fungal genes cloned within the T-DNA region of pAg1. coli [67] and A. pTaH6.114 Tkacz et al. Agrobacterium-mediated transfer of the T-DNA from pAg-H4 to either Penicillium paxilli. . coli on the basis of hygromycin B resistance. Agrobacterium tumefaciens Preparations of A.

70. pH 7. Vectors are propagated and manipulated in E. Electroporation Donor plasmids may be introduced into A. Sources for other plasmids have been summarized in the vector guide compiled by Hellens et al. Except where noted. Agrobacterium broth (AB) and glycerol induction (GI) media are defined media used for A. after autoclaving and cooling to 55 C. colonies appear after 2 to 3 days. Immediately following the electrical pulse. coli by standard methods making the accommodations necessary for those replicating at low copy number.0) Bacto yeast extract 0.3. tumefaciens by electroporation [68. pH 7.5 g MgSO4 7H2O 0. Although they share common components. 6. is also in the ATCC collection (ATCC37493). and ´ Belanger et al. tumefaciens just before and during the cocultivation of the bacterium with the fungal recipient. Transformants are subsequently recovered by plating on YM agar containing the required selective agent. Culture Media for A. The electrical pulse parameters appropriate for Agrobacterium in a specific electroporation apparatus can often be obtained from the equipment manufacturer.2 g NaCl 0. the stock solutions are sterilized by autoclaving.5) Bacto tryptone 10 g Bacto yeast extract 5 g NaCl 10 g YM broth (per liter. the cells are diluted with 1 mL of YM broth and incubated for 3 hr at 28 to 30 C with gentle agitation for expression of the plasmid’s resistance. may be fortified with appropriate antibiotics. their differences are necessitated by the fact that induction of the vir genes is favored at pH values and phosphate concentrations lower than those optimal for the growth of the bacterium [72]. 6.5% to 2% (w/v) and. LB broth (per liter. [68]. [71]. These media may be conveniently prepared from concentrated sterile stock solutions (Table 2). [34].4 g Mannitol 10. At 28 to 30 C. tumefaciens The media specified here are those described by Cangelosi et al. . Lin [70].Agrobacterium-Mediated Transformation of Filamentous Fungi 115 pC22.0 g K2HPO4 0.4.1 g LBYM broth LB broth 1 volume YM broth 1 volume These broths may be converted to plating media by the inclusion of agar at 1.73–79]. GI agar is cooled to 55 C before acetosyringone is added.

Mullins et al. The new culture is incubated under the same conditions. Table 2 Formulation of defined media. A dilution of this suspension is prepared in phosphate-free GI broth to achieve the desired cell density as measured by light scattering at 660 nm (discussed further in a subsequent section).0 mg 30.81]. tumefaciens for Cocultivation Two days before the fungal recipient is ready for cocultivation.18.0 g 50. The resulting culture is diluted 1:100 into 50 mL of AB with the selective agent added.0 g 97. at the low pH of GI broth.0 g 3. reported that single site integration was more frequently found when bacteria were not exposed to acetosyringone prior to the cocultivation step [25]. c Sterilized by filtration. and after 20 to 24 h the cells are collected by centrifugation and suspended in one-tenth the volume of phosphate-free GI broth. Agrobacterium cells bearing the donor plasmid are grown in LBYM broth containing the appropriate selective agent. . 6.0 g 50. because the bacterium is an obligate aerobe.19.0 g 1. certain strains have a propensity to clump upon incubation.27].116 Tkacz et al. from a fresh 103× stock solution prepared in ethanol or dimethylsulfoxide. To induce the vir genes during the cocultivation period. If an electroporator is unavailable.2) Sterile H2O Sterile 2× agar a b 1 vol — — — 6 vol — — — 1 vol 1 vol 6 vol — — 1 vol 1 vol 1 vol — 5 vol 3% agar Phosphate-free. d 2-Morpholinoethanesulfonic acid monohydrate.5′-dimethoxy-4-hydroxyacetophenone. acetosyringone (3′.9 g 18.0 mg 25. In our experience as well as that of Malonek and Meinhardt [24]. Aldrich catalog number D-13440–6) is added to the GI agar at a final concentration of 200 M. Beyond these practical considerations.0 g 10. Solution AB salts 1 Components NH4Cl MgSO4· 7H2O KCl CaCl2·2H2O FeSO4·7H2O K2HPO4 NaH2PO4·H2O Glucose NaH2PO4·H2O Glucose Glycerol MES/NaOH 10×Stock (per L) 10. indeed.5 g GI AB 1 vol 1 vol 1 vol GI Brotha 1 vol 1 vol — Agarb 1 vol 1 vol — AB salts 2c AB phosphate (adjust pH to 7. The culture is incubated at 28 to 30 C for 24 h with shaking at 220 rpm in a vessel that allows free transfer of oxygen.16.0) AB glucose GI phosphate GI carbon GI MESc.d (adjust to pH 5. the older freeze-thaw method may be used for transformation [80. Exposure to acetosyringone during the several days of cocultivation is sufficient to induce the T-DNA mobilization apparatus. Preparation of A.5.0 g 6.22.5 g 100. it is unnecessary at this point to incubate the bacterial cells with inducer as described in other reports [14.

Detergents. The organism is grown in a clear (nonparticulate) liquid medium with agitation. Preparation of the Fungus for Cocultivation Cocultivation is conveniently conducted on membrane filters that allow the cell mixtures to be kept for a period on GI agar and then moved to a selective agar medium. For other fungi. For other fungi. Often short lengths of mycelia may be separated from masses of tangled mycelia by mixing the culture with several volumes of sterile water in a conical tube. The fungal recipient and the Agrobacterium cells carrying the donor vector are applied to the filters and cocultivated at 20 to 25 C.82]. and the conidia are harvested. cefotaxime at 100 g/mL may be required. Millipore Corp.Agrobacterium-Mediated Transformation of Filamentous Fungi 117 6. lifecycle. and behavior of each fungus in culture will largely define the forms of a particular organism that could be used for cocultivation. appear to interfere with the transformation process. potato dextrose agar) containing two selective agents. allowing larger clumps to settle to the bottom of the tube. one to kill the Agrobacterium cells and the other to provide selection of the fungal transformants.45 m. Cocultivation in the absence of acetosyringone is an important control to confirm that resistant fungal colonies arise only as a consequence of the induction of the T-DNA mobilization apparatus in the bacterium.05% (w/v) Tween 80 and stored at 4 C..7. they are purified by a second plating on the same type of selective medium.). notably ALG1. TF series mixed cellulose ester filters or Durapore PVDF membrane filters are satisfactory (0. . As discussed earlier. the filters are moved to plates of a fungal growth medium (e. Their concentration should be determined.1% (w/v) Tween 80 to allow their harvest as a suspension.6. If the organism has a tendency to grow as pellets or balls in submerged culture. it may be necessary to place the spores on filters and allow germination on a different medium prior to transferring the filters to GI medium and applying the Agrobacterium cells. Agrobacterium-mediated transformation has been successfully employed with fungal recipients in various cellular forms. and the cells are suspended in ten times that volume of GI broth (without phosphate). carbenicillin at 500 g/mL can be used. they can be washed free of detergent with phosphate-free GI broth and mixed directly with the Agrobacterium cells for placement on the filters. Because the morphology. the volume of the pellet is estimated. The fungus is allowed to conidiate. If the yield from this procedure is low. the best form of recipient cells may be vegetative mycelia. and the protocol should include measures to remove or minimize them. and drawing off the suspended mycelia for collection by low-speed centrifugation. For many Agrobacterium strains. The plates are incubated at a temperature that is appropriate for the fungus. Following the last centrifugation. formulation of the growth medium with 0. 6. Cocultivation and Selection of Fungal Transformants Detergent-free membrane filters are placed on GI agar containing acetosyringone at 200 M. Often spores need to be wetted with 0. After the appropriate period. and once putative fungal transformants appear.4% agar may discourage aggregation of the hyphae. they can then be washed in 0. the culture might first be mildly homogenized to generate mycelial fragments (an Omni PCR Prep Homogenizer with disposable probes is convenient for this process). but for others. In this case the objective is to obtain a suspension of dispersed mycelia. T-DNA transfer is optimal at temperatures lower than those for the growth of the organism [32. even in trace amounts. Perhaps the simplest situation is presented by a fungus capable of readily producing conidia that behave as propagules. only general protocol guidance can be provided here. If the spores can germinate rapidly on GI agar.g. The mycelia are washed twice by suspension in sterile water and centrifugation.

and slow. respectively. and G. 7. tumefaciens offers a promising means of surmounting what has often been a significant obstacle in the study of many fungi. key variables such as the choice of promoter and resistance gene and the appropriate concentration of the selective agent are already known. clumps that are not removed during the preparation of the suspension may eventually appear as small fungal colonies during selection. a Nodulisporium isolate. lozoyensis). Troubleshooting When transformation of the organism of interest has already been achieved by another method. To illustrate. and Glarea lozoyensis to hygromycin B resistance using pAg-H4. The filters were incubated on GI agar containing acetosyringone for 1. When fungal mycelia are used as recipient. Because T-DNA is transferred and integrated primarily as a single copy. or 3 d for P. It can be helpful in setting these parameters to be guided by the radial growth rate of the fungus on an agar medium. 100. . 2. If transformants are obtained in the initial experiment. respectively.26. Hygromycin B resistant transformants were obtained under selective conditions after 4 to 5 days (P. It has been successful with organisms throughout the fungal kingdom. Two significant variables that are worth exploring in the first transformation experiment are the ratio of bacterial and fungal cells and the length of the cocultivation period [21.118 Tkacz et al. For Nodulisporium and G. 6. These are recognized by their failure to grow during the second plating on selective media. Agrobacterium-mediated transformation is likely to become an indispensable experimental technique in many fields of mycology.0 or 4. For P.32]. The radial growth rates of these fungi may be characterized as fast. Bacterial cells carrying disarmed Ti plasmids are readily available. Nodulisporium.25. its sensitivity to selective agents should be determined as a basis for choosing the most appropriate resistance gene. and donor vectors may be adapted or constructed to meet specific needs. mycelia were employed as recipients and suspensions were mixed with bacterial preparations that had been adjusted to an OD660 of 1. in our laboratory we have transformed Penicillium paxilli. but excessive growth of either organism at this stage may confound subsequent efforts to select transformants. lozoyensis. 47-mm membrane filters. In each case.31. paxilli). The method is simple and relatively rapid in execution and requires no lytic enzymes or specialized equipment. lozoyensis.0. and careful consideration is needed in deciding which promoter is likely to drive expression of the resistance gene as discussed earlier in this chapter. 6 to 7 days (Nodulisporium).0. Lengthening the cocultivation period may provide improved transformation rates. moderate. paxilli. a small number of subsequent trials exploring the variables identified in the previous section should optimize the procedure for routine laboratory use. For an organism that has never been transformed. or 9 to 10 days (G. before being transferred to selective agar. a spore suspension (2 108/ mL) was mixed with an equal volume of an Agrobacterium cell suspension with an OD660 of either 2.L portions of the mixtures were spread evenly on sterile. paxilli. Overgrowth of either organism might be addressed by lowering the glucose in the GI agar from 10 mM to below 5 mM. CONCLUSION The delivery of DNA through a binary plasmid approach mediated by A. Experience with Aspergillus nidulans [18] demonstrates that a stringent selection procedure is a prerequisite for a successful outcome with this method. analysis of the transformants tends to be uncomplicated.8.

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We start by reminding users of biodiversity of how the Convention on Biological Diversity (CBD) has altered the use of biological resources.. tools. Many newcomers to microbial screening are as unacquainted with wild species of fungi as they are unknowing of what can be done with fungi in the context of the technology of pharmaceutical/industrial screening. which are the most genetically apt to produce secondary metabolites. and then follow with an analysis of recent organizational and technical trends of natural products discovery programs. INTRODUCTION The vision for this chapter stems from our experiences working in and managing industrial microbial screening programs and as professional mycologists. Madrid.A. Kyowa Hakko Kogyo Co. Finally. we have attempted to set up a framework of the basic methods. Ltd. especially filamentous ascomycetes and basidiomycetes. or enzyme discovery laboratories. Rather than exhaustively list methods for obtaining fungi from nature. Spain 1. We provide some stepby-step approaches for what we have found to be some of the most fruitful techniques for coaxing wild species into cultivation.6 Fungal Germplasm for Drug Discovery and Industrial Applications TORU OKUDA Tamagawa University Research Institute. 123 .. Tokyo. Japan KATSUHIKO ANDO Tokyo Research Laboratories. and organizational principles that we have found most useful for channeling fungal germplasm into the academic.. ˜ S. Tokyo. Merck Sharp & Dohme de Espana. pharmaceutical. Japan GERALD BILLS ´ ´ Centro de Investigacion Basica. especially in Japan [1].

our ability to exploit patterns of fungal diversity and the mechanisms that shape them is influenced by the approaches we use to evaluate the ecological and evolutionary structure of fungal communities.g. or acquire fresh strains with in-house personnel. http://tolweb. Therefore. and how they regulate species densities and species occurrences. What is the nature of products sought? Are they enzymes or small molecular weight molecules? What might be their potential targets? Where do we look for leads in realm of natural products and natural products pathways? In the past 10 to 15 years.html). the types and intensities of interspecific interactions. Consequently. lysergic acid. and even unseen. at the outset. Other important considerations are whether to use preserved organisms from public service collections or from the company’s in-house historical collections. The parameters that characterize a community and that are central for assessing aspects of its biodiversity include species composition. The organisms that serve as the basis of screening are very much an essential infrastructure for the discovery program. organisms. like -amanitin. Consequently. several questions need to be carefully considered. collaborate with a third party to deliver strains of interest. disturbance and fragmentation of the habitat. the means and facilities for strain preservation need to be factored in at the planning stages. and product expression is likely to be very sensitive to how the organism is grown. If the starting point is a known product and it is associated with a known gene family or gene cluster..124 Okuda et al. and psilocybin. . ergotomine. muscarine. However.g. it is likely that only a small portion of the microbial universe is genetically equipped for producing the natural product metabolite or enzymes of interest. questions of whether the source has been exhausted of useful chemical structures or what types of natural products are likely to interfere with discovery assays need to be considered [4]. therefore. In fact. much has been discovered about the magnitude and extent of bacterial and fungal diversity. to their position in the phylogenetic universe (Tree of Life Web Project.. Elaborating from a known point with a phylogenetically or ecologically directed search may be a valid strategy. along with its functional and genetic correlates. starting with known organisms from culture collections may be sufficient. Although much of the newly found bacterial diversity has been claimed to be inaccessible because of its unculturability. we call attention to the need to be curious and critical and to continuously strive to evaluate and improve the contribution of the microorganism to the screening process. Linking strain selection to growth and assay is critical because the growth forms and rates among the fungi are extremely heterogeneous. Historical. The fact that a wide cross-section of the usable microbial universe has already been mined heavily for their antimicrobials and other pharmaceutical agents during the past 50 years further clouds the discovery landscape [2–4]. historically important fungal metabolites.org/tree/phylogeny. and strategic considerations are thus essential in determining what kind of microbes should be examined as the materia prima. Consistently reproducing the product and its activity are equally critical. after considering what would be the necessary genetic makeup of an organism that will produce the desired profiles of products. Glomales and Uredinales). were discovered and elucidated before their source organisms were cultivated in vitro. Reliable microbial phylogenies and easy access to marker gene sequences now provide a practical and facile means to map unknown. All such plans should be closely integrated with decisions on how the organisms will be preserved and grown and how their cells and culture media will be treated to extract their products. theoretical. and the dynamics of those attributes over time and space or as they change as a consequence of past or present human intervention (e. fungal biologists have long been aware of the inability to culture several major groups of fungi (e.

the authority to determine access to genetic resources . one must address all aspects of community organization and dynamics.S. equipment and automation. plus the artifacts the nature-to-laboratory transition introduces into the system. A renaissance in biological diversity research has been spurred in part by the U. Fortunately for those interested in using biological diversity. after 30 countries ratified it. and space are apt to create bottlenecks in the discovery process and limit which kinds of microorganisms can be used. Perhaps the most outstanding feature of the program from an industry viewpoint is that it has provided a training ground for future scientists in natural products management and discovery and has been instrumental in putting scientific infrastructure at the sources of biological diversity. the regulatory environment governing their use and transport. planned. during the Rio Earth Summit (the United Nations Conference on Environment and Development) and entered into force in December 1993. The industrial microbiologist seeking to optimize the discovery opportunities from wild microbial species faces the same challenges of integrating limited resources as these landscapescale inventories. Article 15. have ratified the CBD.S. the playing field for designing a screening program has broadened considerably in the past 10 years. and financial resources required to observe and enumerate all the organisms in a complex ecosystem. The U. These projects have contemplated the human.all-species. and Fogarty International Center have sponsored multidisciplinary. Agency for International Development. Article 15 is probably the most relevant provision of the CBD with respect to use of microbial resources. technical. the concept has evolved along a totally different course during the subsequent 10 years. or initiated in Costa Rica [7]. CONVENTION ON BIOLOGICAL DIVERSITY AND FUNGI As late as November 1983. Biological Complexity. the members of the Food and Agriculture Organization (FAO) adopted the International Undertaking (IU) on Plant Genetic Resources. 2. The goal of the ICBG program is to integrate and link the needs for improved human health products.Fungal Germplasm for Drug Discovery and Industrial Applications 125 transport.1 referred to the ‘‘Recognizing the sovereign rights of States over their natural resources. The CBD was opened for signature in June 1992. Furthermore. and the development of new modes for sustainable economic activity [5].S. and storage). thereby providing at best a superficial assessment of fungal biodiversity and its chemical complexity. including the fungal and bacterial kingdoms. These latter considerations constrain the focus of screening programs to only a few aspects of microbial communities.org). the logistics of moving microorganisms. drug discovery.org). National Science Foundation has established programs for Biotic Surveys and Inventories. National Institutes of Health. the Great Smokey Mountains National Park (www.discoverlife. incentives for conservation of biodiversity. multi-institutional awards dubbed the International Cooperative Biodiversity Groups (ICBG). all-organism inventory projects [6] have been proposed. including the European Union. Since 1993. As of October 2002. Article 5 of the IU referred to the ‘‘universally accepted principle that plant genetic resources are the common heritage of mankind and consequently should be available without restriction. Several large-scale. and The Tree of Life that have supported basic studies in phylogenetics and ecology. Microbial Observatories.S. and the Hawaiian Islands (www. the U.’’ However. government’s investment in biological sciences. the U. although on a smaller multiple dimension. personnel with their expertise and biases. 186 countries. To truly take advantage of patterns of biodiversity and mechanisms contributing to those patterns.

the national government of the original country has the final decision about the material transfer. Article 15. Practical guidelines on the access and benefit-sharing process is described in ‘‘Bonn Guideline on Access to Genetic Resources and Fair and Equitable Sharing of the Benefits Arising Out of their Utilization.126 Okuda et al. rests with the national governments and is subject to national legislation.biodiv. shall be on mutually agreed terms and subject to the provisions of this Article. in a case of using biological materials of another country. as illustrated in Fig.’’ and is interpreted to mean that in the transfer of biological resources from the country of origin to another country. a user who wants to use biological resources of another country. the user and the provider would negotiate a contract that includes a material transfer agreement and the benefit-sharing procedure under Mutually Agreed Terms (MAT). Therefore.4 Access. 1. The Article 15 of the CBD referred to access to genetic resources and states: Article 15. a user has to obtain appropriate permission for the use from the relevant government authority. Therefore. where granted.7 Each Contracting Party shall take legislative. as the first step. .’’ which was adopted at the Sixth Meeting of the Conference of the Parties (COP-6) held in the Hague. or policy measures. Article 15.org/). And if the possibility exists. administrative. Such sharing shall be upon mutually agreed terms. the Netherlands in April 2002 (http://www.5 Access to genetic resources shall be subject to prior informed consent of the Contracting Party providing such resources. unless otherwise determined by that Party. as appropriate … with the aim of sharing in a fair and equitable way the results of research and development and the benefits arising from the commercial and other utilization of genetic resources with the Contracting Party providing such resources. inquires at an appropriate national authority the possibility of the utilization with Prior Informed Consent (PIC). Figure 1 Simplified structure of an access and benefit sharing agreement for biological materials.

and Fisheries. Fungal Acquisition and the Convention on Biological Diversity Japan has maintained 23 collections of microorganisms and other biological resources that could function as national biological resource centers. the goal of the BRC is to operate as a mediator between providing countries and the private sector for access to biological materials. and 70% of the organizations thought that high-throughput screening (HTS) was an advantageous complement to the program. 3%. Sports. The purpose of the survey was to investigate how these organizations run their current natural product-based drug discovery (NPDD) programs. Department of Biotechnology. 2–5–8 Kazusakamatari. the Japanese government. but several multinational companies were also polled. We considered the replies to reflect industry trends because the private sector comprised 92% of the respondents.Fungal Germplasm for Drug Discovery and Industrial Applications 127 2. industry. Forestry. In addition to these existing culture collections. unique chemical structure was not the only important issue for screening natural products. and what were their objectives. It was created to implement a basic plan. Although discovery of truly new chem- . Chiba. the Ministry of Health and Welfare. Japanese research organizations have bought into the concept and have adopted the view that a good relationship among providing countries. In general. Although industry research groups have had concerns that government involvement would introduce more bureaucratic hurdles and thereby impede the use of biological resources. intended to meet the requirements of the life sciences and biotechnology in the 21st century. Opinions varied widely on different operational facets. and biomedical industries in Japan and a few multinational companies [1]. universities or colleges. and (3) effects of the CBD and the new BRC established in Japan in 2002. the Ministry of Education. Japan established a new national Biological Resource Center [BRC (NITE Biological Resource Center. General Aspects of Natural Product Screening Programs Responding organizations fell into the following categories: pharmaceutical industry. 3. other private companies. and Culture. Obtaining leads based on a single. However. 5%. the Ministry of Agriculture. Poor understanding by government agencies of the difficulties and risks involved in biological innovation may exacerbate unrealistic expectations and interfere with exchange of biological resources on the open market. Most were Japanese. the government may have an important role if it clarifies procedures and facilitates access. agreed upon by five ministries: the Ministry of International Trade and Industry. chemical.1. mainly pharmaceutical. The NPDD organizations consistently considered that such programs were capable of delivering lead structures beyond the imagination of the synthetic chemists. Sustainable use of biological diversity and equitable sharing of derived benefits is desirable in the long run. 292-0818 Japan)] in April 2002. (2) various technical aspects. Twenty-seven of 37 responding organizations (73%) ran NPDD screening programs. and other research organizations or nongovernmental organizations. Science. Kisarazu-shi. The major issues addressed were (1) the general state of NPDD. 73%.1%) of the 48 industrial organizations contacted responded. and the Science and Technology Agency. Thirty-seven (77. 19%. TRENDS IN ACQUISITION OF MICROBES IN INDUSTRY IN JAPAN The Kanto Branch of the Mycological Society of Japan surveyed 48 organizations.1. and academia is advantageous. National Institute of Technology and Evaluation. Positive opinions are summarized here. 3. because minor structural differences often revealed important structure–activity relationships.

plants. Consequently. not unusual chemical structures. the objective of NPDD is to find drug leads. mitosporic fungi and ascomycetes were most widely used. medicinal plants. Oomycetes and Zygomycetes were men- . can be counterproductive because the structural complexity of some natural products may be technically intractable for a medicinal chemistry program. Fusarium. Their regular use was no doubt a function of the convenience in obtaining high numbers of strains. a number of organizations have reduced or abandoned the use of plants. whereas insects were rarely exploited. Company-sponsored OJT is complemented through interaction with academic societies. Screening Sources Almost all organizations with NPDD programs used actinomycetes and fungi as screening sources. bacteria. none so far have reached advanced clinical development [8. even for scientists involved in NPDD. Furthermore. The latter situation recently seems to have dominated in Japan. Soil fungi (74%) and litter fungi (52%) were the ecological groups most frequently mentioned. In Japan. respondents made several negative statements.’’ although always a possibility in NPDD. The same series of screenings protocols and various assay formats cannot be adopted for all targets with heterogeneous and complex natural products. taxonomic input is not necessary. and medicinal herbs were the next most frequently used organisms. The suggestion of ‘‘beyond synthetic chemists’ imagination. in recent years. Unicellular bacteria. the Kanto Branch of the Mycological Society sponsored a series of workshops for academic and industrial scientists on taxonomy of Acremonium. ical entities from natural sources was difficult. 3. However.9]. microalgae. The Role of Systematics More than 90% of organizations routinely isolated a broad range of microbes without targeting a specific taxon. Other organizations simply reported they attempted to obtain as wide a range of taxa as possible. Finally. When specific taxa were mentioned. Some respondents said that they targeted specific ecological or taxonomic groups of fungi. If a company does not operate NPDD. About 40% have developed their own methods for optimizing microbial isolations when existing methods did not meet their needs [10–15]. because Japanese universities have de-emphasized microbial systematics and students are less interested in the subject. many HTS formats are often poorly suited for NPDD. whereas 33% of organizations have increased the proportion of fungi among their screening sources. Some sought microbes from unusual environments. Statements such as ‘‘beyond synthetic chemists’ imagination’’ and ‘‘diverse fungi provide diverse compounds’’ have been so overused during the past 10 years that they are no longer a driving force. and aquatic fungi. paradigms for screening natural products differ among targets based on new genomic-derived and other newly developed targets. Another negative opinion was that probably the majority of basic structural types and biosynthetic pathways have been found. Interestingly. For example.3. Japanese industries conducting NPDD generally have skilled taxonomists on their staff. it was also doubtful whether innovative compounds have been synthesized and identified using combinatorial chemistry. Although some companies may screen natural products without any systematic guidance. taxonomists are either university educated or learn within the company through on-thejob training (OJT). However.2. 3. although many hits have been generated from combinatorial chemical libraries.128 Okuda et al. In fact. Invertebrates and microalgae were screened by some. or actinomycetes.

. 26%. However. strains and their corresponding biological activities. 3. or variations in genetic markers). maintaining that useful microbes can still be discovered by carefully examining the Japanese microbial flora that spans from boreal to near tropical environments.g. or lack thereof. But most organizations (67%) preserved and reused strains. Cosmopolitan species often were excluded when it was judged the species had been adequately represented in the screening collection.5. In fact. Impact of the Convention on Biological Diversity Fifty-six percent of the industrial groups collected at least part of their original samples themselves. microbial strains. different geographical origin. Most laboratories (67% to 70%) did not necessarily discard duplicate strains of the same species. 56% expected government organizations to help them gain access to foreign biological materials. Unreasonable expectations for financial investment will prevent many countries from participating in the drug discovery process. About 41% obtained their materials within Japan. inactive isolates are not preserved and are discarded after being screened (30%). Based on anecdotal evidence. General Considerations for Collecting and Transporting of Fungi The process of collection of and culturing of fungi generally can be delimited into three stages: obtaining and transporting the isolation source. Construction of microbial extract collections for HTS therefore was thought to be worthwhile for certain applications. 3. 22%. However. probably as a result of their limited history of secondary metabolite production [16. Almost all pharmaceutical companies (96%) were conscious of the CBD and agreed with its principles. Examination of infraspecific and population-level variations in metabolite production generally was thought to be valuable. Seventy-four percent of organizations stated that complaining about the CBD was useless. some believed that the activity of fresh isolates is superior to that of strains preserved in culture collections. 48%. cultural differences.4. 37% organizations removed duplications based on complimentary genetic or chemical analyses [18–20]. 4. cell separation or trapping tech- . are duplicated in the screening results.1. 41% of the organizations expressed concern that the CBD has hindered natural product screening activities or necessitated realignment their operation. and duplicate species were submitted to screening assays when there was some reason to suspect they could be genetically distinct (e. Strain Management When thousands of isolates are collected. at least to some degree. To ensure extract diversity for HTS. and extracts. The strain duplication is either ignored or various strategies are invoked to overcome it. some companies have abandoned use of biological materials from overseas. whereas some acquired raw materials from overseas with the following frequencies: microbial substrata.Fungal Germplasm for Drug Discovery and Industrial Applications 129 tioned infrequently. Such recycled strains either served as the basis of a screening sample library to meet the demands of HTS or were used to pilot new assays. BRINGING FUNGI INTO THE LABORATORY 4. Many have proactively contracted collaborative research agreements with a foreign partner to provide biological resources. As a result.17].

transport. The ensuing challenge is how to put what arrives at the laboratory into a usable form and then make the most of it. conspicuous fruiting bodies of mushrooms. invertebrates. and manipulations of growth in isolation media. Almost anything imaginable harbors fungi: materials range from desert rocks or high mountain snow with no apparent fungal growth to large. Although finding natural substrata devoid of fungi is difficult. their relative proportions of ubiquitous to substratum-specialized species. and storage that cause attrition and artifacts in the perception of fungal diversity. The most often used materials are those that bear large numbers of culturable fungi and include soils. and their durability and survivability during handling and trans- Figure 2 Effects of human intervention. animal dung. The three stages are interdependent and determine the success of the isolation. and stored and processed food. certain substrata are clearly more useful than others with respect to their numbers of species. other fungi. The factors that influence which components of fungal kingdom can be captured and brought in live into a screening program are illustrated in Fig. . living and decomposing plants. 2. niques for establishing in vitro growth. thalli of lichenized fungi.130 Okuda et al.

However. Aureobasidium pullulans. fungal fruiting bodies with high water contents die. infested with nematodes and mites. Cladosporium cladosporiodes. Arnold [23] estimated that approximately 350 genetically distinct species were recoverable from 100 leaves of a single tropical forest tree. Phomopsis species in woody plants.. . Fungi and fungi-containing materials should be processed soon after collection. Before going to the field. they are kept at room temperature until processed or are stored at cool temperatures. can be carefully air-dried and packed for long-distance transport and later rehydrated. and Epiccocum nigrum. Particle filtration applied to leaf litter may yield 80 to 400 species for 600 to 1000 isolates [22]. then the habitat relationships become especially crucial (see Chapter 10).Fungal Germplasm for Drug Discovery and Industrial Applications 131 port. the number of substratum samples and the number of isolates taken for examination need to be considered carefully. and some Aphyllophorales. can yield hundreds of taxa. or apothecial ascomycetes.. Freezing usually is avoided. examined intensively. but the organisms can be identified readily and often are obtained in no other way. especially many perithecial. therefore. and inundated with yeast and bacterial films. After handling many isolates and becoming familiar with the mycota of a particular substratum. each harbors its characteristic core group of species. we think reviving a dried sample is preferable to working with a wet and poorly handled sample that was stored in an anaerobic state. certain cosmopolitan fungi. and desiccate. Obviously. or litter samples. A skilled mycologist may be able make up to two dozen isolates from single spore or tissue isolations in a day. rot. Phoma species in arid soils. e. asymptotes for species-isolate curves range from about 60 to 150 species for 600 to 1000 isolates [21]. plant. some fungi. Deciding what to culture and how much material is necessary to provide sufficient isolates needs planning.g. it becomes obvious that each type conveys its own set of omnipresent genera and species (e. building a screening program based on soil isolates or endophytes ignores the vast majority of culturable fungi. In other words.g. Abundant anecdotal evidence indicates that complex fungal communities rapidly deteriorate when separated from their habitat. Especially wet or humid material needs to be air-dried to prevent mold contamination in storage. Even small numbers of soil. It cannot be emphasized enough that all fungi are not in one substratum. Although some fungi may be lost during air-drying. Therefore. Time constraints and costs of materials must be weighed against the priority of experimental questions or screening goals. but some fungi appear to be in all substrata. In cases where certain enzymatic properties are targeted. although some conidial fungi from temperate soils can survive freezer storage [24]. Some fungi can quickly lose their forcible spore discharge. seemingly can be recovered from any substratum anywhere. isolations from field-collected fruiting bodies yield fewer taxa. When materials arrive in the laboratory. the maximum number of isolates that can be adequately managed by two full-time experienced researchers using conventional microbiological techniques at a single time in a well-equipped laboratory is about 2000 to 3000. If suspension plating or particle filtration is used to isolate fungi in small composite samples of temperate or tropical soils. stromatic. badly decomposed. consider the following questions: (1) What kind of materials are to be collected for which targeted fungi? (2) Where do you need to go for those materials and do you have or need permission to take them? (3) How will the materials be packed and transferred to the laboratory? When planning a fungal inventory. In contrast to mass-isolation techniques. transportation is a major obstacle when the laboratory is distant from the collection sites. Penicillium chrysogenum. or Trichoderma species in temperate forest litter). Among the fungi of these materials. As a practical guide.

a refrigerator to store media and reagents. a semipermanent air-conditioned laboratory outfitted with incubator. Avoid its inhalation and contact with skin. the U. especially soils and plants. Costa Rica).. and a clean room or incubator for growing cultures. when mites have infested cultures and threaten to destroy them. Consult with your local state. and mite infestation is a chronic problem in laboratories where large numbers of fungi are cultivated.2. Penicillium. For example. The approach guarantees entry of sets of fungi that in no other way would ever be tested. Fusarium.26]. Soils Soils have been one of the most effective and popular materials for isolating large numbers of fungi. Collaboration with a microbiology or phytopathology group near the site of interest is one way to gain access to these fungi. an autoclave (or large pressure cooker). physical separation of living cultures and natural materials. For natural products discovery. preferably an air-conditioned room with a sink. Useful Substrata for Fungi 4. a laminar flow hood or biosafety cabinet. A similar medium treatment can be made using lindane at a concentration of 75 g/mL [25]. when working with materials from outside your country. and E.g. reduction of humidity. When importing fungi or other living materials from a foreign country. and a routine fumigation schedule can help prevent infestations.S. Santo Domingo. during the Merck Research Laboratories’ collaborative project with InBio (Instituto Nacional de Biodiversidad.g. Animal and Health Inspection Service in the U. especially fungi with fleshy or watery fruiting bodies that will not survive transport. their substrata may contain harmful insects or invertebrates. that these materials be contained under the appropriate biosafety levels (P2 in the U. many of the most historically important and metabolite-rich genera (e. whereas media preparation and staging for shipment were carried out at InBio’s main facilities in Santo Domingo. or national agricultural authorities (e. is highly regulated because a very small percentage of fungi potentially are plant pests or human pathogens.A. Mites and fungi coexist in nature. It is imperative that. Dieldrin is extremely toxic to animals. media can be made lethal to mites by adding dieldrin at a concentration of 20 g/mL. and laminar flow hood was set up in an existing field laboratory at Santa Rosa National Park. Add 1 mL of an acetone stock solution (200 mg dieldrin in 10 mL acetone) to a liter medium before or after autoclaving. your collaborator likely will require phytosanitary export certificates and other commercial export licenses. Aspergillus. but seems not to affect fungi. microscopy equipment.).2.U. Isolations and specimens were prepared at the remote laboratory. Laboratory hygiene. The minimal requisites are a clean work area.132 Okuda et al..1. investment in setting up a field laboratory in collaboration with local investigators or working with a university or government laboratory to upgrade its facilities may be worthwhile. some . 4. Glassware and used media need to be handled as toxic waste material.S.S. In fact.) to learn what permits and certifications are needed to import and work with foreign biological materials. If all else fails. Guides on culture collection operation and maintenance recommend many mite control practices [25. An alternative to basing the entire screening program only on fungi that survive long-distance transport is putting the laboratory near the habitat. If no facility exists. and Trichoderma) are abundant in soils.D. Movement of fungi and their associated substrata. Furthermore. provincial. There seems to be a misguided notion among natural products scientists that soil fungi have been exhausted as source of secondary metabolites. refrigerators.

the Glomales and many ectomycorrhizal basidiomycetes) have consistently defied attempts to isolate them into pure culture. and Barron [29] included 202 genera of soil-dwelling mitosporic fungi. and even within the known universe of soil fungi. Soil usually is collected with some kind of digging tool and packed in plastic. Stratification is minimal and the litter–soil interface can be very abrupt. and Aureobasidium pulullans are dominant fungi in L-layer.. are in F1-layer. Domsch et al.2. . and human intervention are therefore useful in devising collecting and isolation strategies [21. and soil fungi are isolated dominantly from the H-layer [34. informed selection of isolates. plus their mycoparasites. Nonetheless. 4. Diversity is usually highest in the uppermost horizons. In humid tropical forests. An understanding of the ecology of soil fungi and how soil fungal communities respond to geography. or after application selective or baiting techniques. In temperate forests. some major groups of soil fungi. yields a wealth of taxa obtained from no other habitat. In arid or semiarid regions. litter decomposes very rapidly.2. For example. species-rich genera frequently are isolated. the numbers of taxa are formidable. The number of soilinhabiting fungi is unknown. and specialized taxonomic references combined with ribosomal DNA sequence data is essential to cope with their identification. whereas their conidia reside in the soil’s spore bank. Thysanophora and Chaetopsina spp. Even a set of exhaustive isolation of soil fungi on a small scale of a few hundred isolates may yield dozens of genera and upward of 50 to 100 species. In temperate forests. usually persisting only a few months. Some Aspergillus and Penicillium spp. therefore. Plant Litter Litter is an accumulated composite of plant parts in various stages of decay plus the organisms associated with those decay processes. including some contributing the greatest biomass (e.2). In most forests. the litter is often highly stratified. collecting near the surface is more effective. or heavy paper bags. It is probably true that many species from temperate soils have been comprehensively surveyed. and that many soil fungi produce troublesome mycotoxins that interfere with assays. Species in taxonomically complex. For example. and the species of fungi recovered varies with the depth of the litter layer. especially from the tropical and remote regions. and invertebrate associated fungi. digging deeper may be necessary to reveal horizons with moisture or accumulated organic matter.Fungal Germplasm for Drug Discovery and Industrial Applications 133 companies actually promote their collections as being superior because they avoided soil fungi. The spatial-temporal heterogeneity and complexity of litter is the main cause of its extreme richness in fungal inhabitants. the main fungi that colonize litter are saprobic basidiomycetes.38–41].2. Fungi that start their lifecycles in living plant leaves and stems can remain viable in the litter.30–33]. However. the fungal communities also exhibit succession downward through the litter and to a lesser extent through soil horizons [34–37]. conifer litter exhibits a classical vertical succession. probably are specifically adapted to colonize seeds and other litter components. [28] listed 153 common genera in their compendium. The litter and humus layers that overlie mineral soil harbor distinct mycotas. and sporulate there to complete their lifecycles. cloth. Almost any soil or sediment is potentially useful. a spectacular diversity of ascomycetous and anamorphic fungi that seem specifically adapted to decompose litter. Trichoderma and Mortierella species are in the F2-layers.g. and they should be examined separately (see section 4. vegetation. Representatives of many orders of fungi can be found in soil. Cladosporium sp.

fog.2. Xylariaceae. or sometimes the sample can be consumed by a single basidiomycete mycelium.48]. An extensive literature has accumulated on all aspects of the biology of the endophytes. Many of the endophytic fungi belong to orders with a rich history of secondary metabolite production and. the physical location of the wood (standing. because such data may aid in identification of the resultant fungi. and other discrete components may be separated for direct examination or for preparation of moist chambers. dry place. the fungi are collected by sampling rainwater and water deposited from fog and mist on living leaves and barks and those draining from living leaves and barks [50. including methods on how to optimize their isolations from nature [47. The involvement and interactions of fungi in mediating the wood-decay processes have been extensively reviewed [42. have become a mainstay of natural products screening programs.51]. and mist. consequently. Air-dried samples that cannot be treated immediately should be kept in a dark. they should be air-dried. often loosely termed as endophytes [46]. they are rich in biosynthetic gene clusters [49]. their isolation from living leaves and bark by usual isolation methods is very difficult. especially in plastic. Hypocreaceae. The terrestrial aquatic fungi grow slowly and their sporulation is very transient compared to other phylloplane fungi. Individual leaves. 43 species of wood-decay basidiomycetes were observed along five 500-m transects [45]. to a lesser extent. A total of 157 species in 87 genera and 42 families (30% in the families Lasiosphaeriaceae. If possible. Litter can be collected as a composite vertical section or segregated into its strata. they also may be rapidly extracted by indirectly plating colonized wood fragments onto an isolation medium (see section 4. in one of the few studies ever to estimate ascomycete diversity on dead wood.2. pyrenomycetes were collected over a 1-year period on dead wood and bark of 31 woody plants along a 500-m transect in Puerto Rico [44]. Their conidia are concentrated by centrifugation and either separated manually or with a micromanipulator or plated directly. In a lowland rainforest in Panama. as now is being revealed. Fungi associated with wood are generally assessed by visually inspecting the surfaces and collecting fruiting structures. 4.1).43]. basidiomycetes. small-diameter woody debris. and Tubeufiaceae) were found.3. Dead Wood Dead wood supports complex fungal communities. nematodes. encourages contamination with bacteria. A particularly elusive group of plant-associated fungi are the terrestrial aquatic fungi [50. . The fungi of living plants are an extremely diverse and heterogeneous assemblage of ascomycetes and. Therefore. identify the plants and other components and make note of the degree of decomposition. Some of these fungi are phytopathogens and their secondary metabolites have been selected as plant virulence factors. abundant conidial fungi.134 Okuda et al. Living Plants Fungi living internally in live plants. Litter samples should be collected into paper bags or envelopes.4. and the structure and the community composition varies with the stage of succession in the decay process. and saprophytic fungi. in service). Extended storage of wet litter.4. For example. seeds. and if wet and cannot be processed immediately. and species of the tree or shrub. 4. Although wood-colonizing basidiomyceteous fungi usually are obtained by isolations from their fruiting bodies. Sporulation of terrestrial aquatic fungi responds to water stimulus such as rain.51]. fallen. plant pathogenic fungi. Evidence from their study in tropical forests ranks them among the most species-rich assemblages of terrestrial organisms [23]. Short conidiophores develop rapidly and release conidia adapted for dispersion in water films.

Therefore. Coprophilous ascomycetous fungi have proven to be a surprisingly productive source of new bioactive metabolites.. Conventional plating of water and sediments are inadequate to observe these fungi. Specific groups among the zygomycetes. ascomycetes. the spores of aquatic hyphomycetes [64.2. the conidial stage of the coleopteran parasite Cordyceps subsessilis [56] that is used for commercial production of cyclosporine A [57]. including domesticated and wild species. are resistant to axenic culture. More than 300 genera of ascomycetes. Paecilomyces lilacinus. and basidiomycetes have adapted to colonize dung. e. and some basidiomycetes are known among these fungi. . Relatively few of these fungi have been examined for secondary metabolites [66. Some species of the Clavicipitaceae. parasites of microinvertebrates generally are more accessible because they can be baited from soils and organic debris.2. such as river foam. Only a few nematodeassociated fungi have been thoroughly examined for secondary metabolites [60]. and Basidiomycetes infest insects [55]. produce abundant conidia that are readily cultured from soil and plant litter. For example.53]. P.g. Tolypocladium inflatum. they would appear to be a valid source for secondary metabolites. occur in remote regions.5. except for one important exception. 4.7.2. based on the fact that many belong to or are closely related to orders of ascomycetes with secondary metabolite biosynthetic pathways. and are time consuming to collect in the field. Dung Coprophilous fungi are those fungi that are adapted to live and reproduce on animal dung [52. Unlike most insect-associated fungi. carneus. these methods will recover fungi associated with the partially digested plant material and sometimes soil fungi that have colonized the dung after its deposition on the soil surface.Fungal Germplasm for Drug Discovery and Industrial Applications 135 4. They most commonly are observed by examining submerged plant debris or wood baits after incubation in humid chambers. especially antifungal metabolites [54]. certain species of the Clavicipitaceae with high saprobic capacity. Ascomycetes. Furthermore. The dung most commonly used is that of herbivorous mammals.65] can be collected from natural traps.59]. 300 anamorph genera. Metarhizium anisopliae. about 3000 species of fungi in five orders spanning the Zygomycetes. Invertebrates Invertebrate-associated fungi are considered to be some of the most diverse and biologically attractive of all fungi. Other important groups of invertebrate pathogens are the fungi that trap and infect nematodes.6. and Chaunopycnis alba. insect-associated fungi have not figured largely in the results of industrial screening. By and large. The most historically important soil-borne entomopathogenic fungus is Tolypocladium inflatum.67]. Beauveria bassiana. or sporulation of aquatic hyphomycetes can be induced in the laboratory by aerating aquatic debris in water at cool temperatures. and therefore have received widespread attention from natural products researchers [2]. rotifers. The fungi are usually obtained by incubating field-collected dung in humid chambers and observing the development of fruiting structures over time. However. Less elegant and precise methods of quickly extracting large numbers of strains from dung are based on solvent pasteurization [10] or particle filtration techniques. 4. In addition to coprophilous fungi. Freshwater Habitats Several different assemblages of fungi and fungal-like protozoans complete part or all their lifecycles in freshwater habitats [61–63]. insect-associated fungi have been underused because they are often rare. are difficult to transport alive. and other microscopic invertebrates [58.

terrestrial fungal propagules are thought to accumulate because of the mycostatic effect of seawater and to form an inactive spore bank [68]. mycelia. In marine sediments. However.). the importance of knowledge of fungal biology. Such methods are simple and usually do not require complex equipment or protocols. sands. Because marine fungi. Such reports rarely have critically evaluated the nature of the organism and its relationship to its substratum. seashore and estuary plants. In recent years. 4. Spartina and Juncus spp. 4. to provide access to fungi that are not cultivable by other means. salina). Methods for collecting and culturing this challenging assemblage of fungi were reviewed by Kohlmeyer and Kohlmeyer [68] and Vrijemoed [77]. the actual contribution made by true marine fungi is unclear. Asteromyces cruciatus. mangroves.3. and tissues from fruiting bodies or substrata can be used to initiate cultures. and some oligotrophic yeasts from open oceans appear to be terrestrial soil fungi [69]. Almost all taxa isolated from these habitats. and manipulations of single spores and dissection of hyphae. especially the filamentous ascomycetes. Many of these reports of new metabolites are as likely to reflect the novelty that the fungus was fermented in a seawater-based medium as they are a reflection of a metabolic adaptation to a marine habitat..76]. Dendryphiella arenaria. Tools for Dissection and Micromanipulation Effective removal. and marine and fresh water habitats [68.136 Okuda et al. Marine Habitats Marine fungi are fungi that have evolved to complete their lifecycles in the oceans and their estuaries [68. and tissues require practice and adequate tools. there is little doubt the organisms being cultured are distinct from each other and. rather than lack of ability to produce secondary metabolites. microscopic colonies. to make the most of fungal materials. manual dexterity. A sewing needle or . and D. seaweeds. many articles have appeared in natural products journals describing metabolites from ‘‘marine-derived’’ fungi that often belong to typical terrestrial genera or are not identified at all [72–74]. Although fungi isolated from marine habitats have been screened intensively in industrial programs. for the most part. belong to classes and orders that either are rich in secondary metabolite encoding genes or are sister groups. with the exception of a few facultative marine mitosporic fungi (e. The most speciesrich materials seem to be the senescent and dead remains of mangrove trees and shrubs and intertidal monocots (e. True marine fungi almost exclusively are associated with dead wood. and sediments of estuaries. The main advantages of these methods for a screening program are that the organisms often can be identified with certainty. Direct Isolations Spores. separation.1. experience.2. the suggestion that such fungi are somehow marine organisms may be misleading [69].3.75. Seashore foam concentrates the appendaged conidia and ascospores of marine fungi.g. animal exoskeletons. the rarity of reports may be due to the relative difficulty in collecting and culturing them and their often slow growth in agar and liquid culture. and adequate tools are worth considering. Reports of metabolites from true marine fungi are relatively rare [70. and sea sand [68.g. some halophilic black yeasts.. and therefore can provide material for starting cultures from single spores. Various investigators have isolated filamentous fungi from soils.77]. Aspergillus insulicola.8.71]. Isolation Strategies 4.69].

The fine pins are glued into a glass or Teflon capillary. Extra fine insect pin ( 000). which in turn is fixed into a handle or pin vise. E). Two pieces of tungsten wire are connected to a variable transformer (e.. The pieces should be moved vertically into and out of the solution so that a finely sharpened needle is obtained. Insect pin ( 2).Fungal Germplasm for Drug Discovery and Industrial Applications 137 a hypodermic needle mounted into a wooden or plastic handle often is sufficient and inexpensive (Fig. A new needle also can be further sharpened or the tip can be modified into knifelike blade or a flat spatula. D. minuten pins) need to be ethanolsterilized and the ethanol removed by immersion in clean agar because they deteriorate when repeatedly sterilized by flame.2. Poor quality needle from a student’s dissecting kit. C. A. 3D). The tips of the two pieces are dipped in the solution and electrolyzed in an alternating current at 5 to 10 V for several seconds. Very fine steel needles (e. 1 mm. Very fine Elgiloy needle. Scale bar uals. Typical sewing needle. .g. Too-high voltage and too-long electrolysis will shorten Figure 3 Assorted dissecting needles.g. Tungsten needles [41. E. Insect pins are very good for microscopic manipulations and come in a variety of sizes (Figs.78] can be made by cutting 0. 3B).5-mm diameter tungsten wire into pieces in about 5 to 10 cm long. F.. Larger needles may obliterate fine structures and are clumsy under higher magnifications. a Slidacs variable transformer or a 6-V microscope transformer). normally used for mounting specimens of microscopic insects. Sharpened sewing needle. The cut ends are sharpened to a point by electrolysis in a solution of 50 g NaOH and 150 g NaNO3 dissolved in 900 mL distilled water. Deteriorated needles can be sharpened with a very fine whetstone (Fig. B.to 0. 3C. An especially useful pin for fine work is the minuten pin.

We have found this device indispensable for isolation of many kinds of fungi and for decontamination of fungal cultures. If the micromanipulator is used extensively. Elgiloy orthodontic wire. B). We believe that it is worthwhile to describe in detail its basic operation.). 3F) should not be flame-sterilized. The L-ring is fastened to the objective lens(10 ) of the microscope. 4A. for about $1000 U. Elgiloy needles (Fig. Its use has become very popular among Japanese mycologists. Skerman’s Micromanipulator Micromanipulators are convenient tools for isolating single spores or separating individual cells and hyphae from natural substrata under a microscope. vibration-free stage is needed. but rather simply dipped in ethanol and washed in the agar medium. Ltd. 0.138 Okuda et al. and glass capillary tubes (about. we recommend purchase of a dedicated microscope to avoid repeated assembly and disassembly. After electrolysis. Set up of the Skerman’s micromanipulator kit. Two needles are obtained because alternating current electrolyses both ends simultaneously.S. unpublished). 2.and V-rings perfectly. The kit’s 10 objective lens is made to fit the L. the microforge (Fig. 3. The angle of the capillary can be adjusted by rotating the pin.2 to 0. a scalpel or microcutter can be improvised by cutting a razor blade into small pieces and shaping them with a hammer and a file [79].. Okada. Fine disposable scalpels are indispensable for dissecting fruiting bodies and substrata. 4 cm long) is inserted through the tube fixed on the base of the magnet and is fixed with melted paraffin between the capillary and the tube. if at all. 4A). Skerman’s micromanipulator was first developed for separation of bacterial cells and later applied to fungal isolation. Preparing Microtools with the Microforge: 1. The operation of the microforge requires use of the transformer (Fig. is also excellent for formation of microneedles because of its tensile strength and resiliency and ease of bending (G. or curve the wire. Tungsten needles are hardened when sterilized in the flame of an alcohol lamp. and a kit that includes a 10 objective lens designed to fit the manipulator is sold in Japan (Toyo Rikoki Co. . If scalpels are unavailable. 4B). which often bends or curls when electrolyzed. a cobalt-chromium-nickel-molybdenum-iron alloy. the operation of the microforge to make fine glass needles. especially because the manufacturer’s operating instructions are in Japanese. The magnet with the capillary is set on the grooved steel slide of the L-ring.83]. 4A. Wire 0. and the capillary fixed to the point of the pin with paraffin. 4B).81].8 mm in diameter). the L-ring (Fig. a fine insect pin can be inserted through the tube. and the manipulation of spores are done with the same microscope. Cells are manipulated with a microtool mounted on the magnet attached to the V-ring (Fig. Elgiloy wire is more easily straightened into needles than tungsten wire. the Skerman’s micromanipulator may be one of the least known yet most useful and attractive devices for mycologists in terms of its reasonable price and simple handling [82. C). Other objectives may not fit as well. Additional techniques and tools for singlespore isolations are have been described elsewhere [80. the magnet (Fig. Although various kinds of micromanipulators are available on the market. A compound microscope with a movable. Alternatively.3 mm in diameter is cut into pieces about 5 to 10 cm long and electrolyzed in 50% H2SO4 as described above. the alkaline solution is washed away in water. A glass capillary tube (about.

8.Fungal Germplasm for Drug Discovery and Industrial Applications 139 Figure 4 Skerman’s manipulator kit and its set up. 100 m. The transformer’s rheostat is lowered and the heated filament is moved carefully until it just meets and melts the tapered tip. 5. retracting the tip of the tapered glass capillary until it is once again in the centered in the field of view. The transformer is turned on.. 4. Scale bar uals. C. or microloop). The mechanical stage and filament temperature are manipulated to forge the tip of tapered capillary into the desired shape (e. The magnet is moved forward in the groove slide until the glass capillary is focused in the center of the visual field. 6. The filament is then retracted in a quick and smooth action that stretches the bead into fine tapered point. 4C). sliding magnet. Hooked microtool of a Skerman’s manipulator with fungal conidia. B. The filament is then raised or lowered into focus with the fine adjustment of the microscope stage (the capillary is already in focus). 10. V-ring. and L-ring set up on the microscope stage. and microtool mounted on microscope. The microforge connected to the transformer is placed in the slide caliper of the microscope stage (Fig. the magnet is slowly pulled out of the L-ring and set aside. 7. pushing. The filament and glass capillary are then aligned along the same axis and moved together until they nearly touch each other (Fig.g. The magnet is pulled back along the groove. Once the tip is forged into the desired tool. . An L-shaped microhook is especially useful for dragging. Microforge. U-shaped tip and fastened into the vise clamp of the microforge. sliding magnet. and the power increased so that the heating filament reddens and expands toward the glass. because the wire filament expands when heated. 4B). The tungsten heating filament is bent into a tight. Note rheostat for microforge in the lower right. or lifting fungal spores across the agar (Fig. The filament is pushed into the glass tip with the mechanical stage forming a bead of melted glass. A. 9. where the microtool will not be broken. a microknob. microhook. 4B). The tip of the heating filament of the microforge is moved to the center of the field of view with the microscope stage. The tip of the heating filament should be near but not touching the capillary.

7. 4C). such as filter paper or Perlite. Using the marked circles as guides. are removed for culturing and identification. 12. The magnet is lowereded by hand while observing through the microscope until the microtool is visible and centered in field of view. Sticky. or inconspicuous forms. The stage is slowly moved. spores dissected from ascomata. 5. once developed. or conidiophores are placed above the marked circles. 9. either dragging or pushing the spores to a new clean surface (Fig. The microscope stage is lowered and the Petri dish is set on the stage. or can break the dormancy of resting spores and sclerotia. The container should be transparent or translucent so that the contents are visible and illuminated from outside. Use of moist chambers increases the likelihood of observing nonfruiting. A substratum is maintained moist in a chamber where ambient conditions can be manipulated so that fungal development and sporulation can be observed directly. Moist chamber incubation also rehydrates desiccated specimens. The level of confidence it provides in accurate establishment of strains and the ability to rescue contaminated cultures more than compensates for the initial expense and effort to learn its use. or filamentous spores or cells may be gently lifted and moved to a new location. 10. Moist Chambers Moist chambers are a mycological enrichment technique used to observe elusive fungi that may fruit sporadically or only under very specific conditions. Micromanipulating Single Spores: 1. 11. 4A). the procedure is very simple and. Alternatively. Petri dishes. . 3. At this point. The substrata are placed inside a container lined with a clean absorbent material. about 15 mm from the edge of the isolation plate (Fig. it is a very rapid and accurate. After the manipulator’s initial setup. or the separated spores can be moved to fresh agar when they start to germinate (8 to 48 hours. the tip of the microtool also should be in focus and centered in the field of view.to 300-mL volume polystyrene cups with polyethylene snap lids. The V-ring is secured to an objective lens (10 ) of a microscope.plates can be used. permits maturation of immature specimens.140 Okuda et al. Either 60. The microtool can be pushed into the agar by raising the stage to mark the new location of the cleaned and separated spores. The separated spores can be removed immediately with a needle. 2. 6. pycnidia. viscous.or 100. The fungi. 8. with practice. Mark one or more circles on the bottom of Petri dish of agar isolation medium to facilitate location of the spores on the dish. but 100-mm plates provide more area for movement. The magnet with a microtool is slowly inserted into the grooved steel channel of the V-ring. spores targeted for separation are located and centered in the field of view. A small amount of spore suspension is pipetted on the agar surface above one or more of the marked circles. The stage is lowered again and the Petri dish is removed from the stage. Examples of useful moist chambers include 200. Dragging and pushing spores scrubs bacteria and smaller spores from the target spores and therefore eliminates contamination from the spore surface. depending on the species). small. 4.

hyphae.g.84. Spores. possibly the fungus is immature or spore discharge was arrested by transport of refrigeration.77] and freshwater habitats [60]. Humidity adjustment requires some experience. the fruiting bodies fastened to the cover should be removed to avoid contamination. Substrata should be examined every few days for newly emerged fungi for at least up to 1 month. The containers are incubated at room temperature. and rotifers for their respective parasitic fungi [58. Best known among baits for recovery of specialized saprobic or parasitic fungi from soils are cellophane and other cellulosic materials for isolation of cellulolytic fungi [86. but materials are easily dried up in a few days. airtight plastic container may better retain moisture while restricting movement of invertebrates associated with the substrata. basidiospores or ascospores are ejected and land on the agar.. basidiomycetes that form ectomycorrhizal symbioses or species of the Geoglossaceae). and to best capitalize on the technique. sclerotia. soil or river water) believed to contain the targeted fungi in the hope that they will preferentially colonize and reproduce on the substratum or host organism. Spores are observed periodically until they germinate. although spore discharge can be adapted to trap almost any forcibly ejected spore from microscopic fungi. nematodes. If no spores are observed on the agar. hair or feathers for keratinolytic fungi [87–89]. because containers are opened frequently to examine the substrata. . As soon as possible after germination. As with selective media.59. too much moisture encourages growth of fast-growing mucoraceous fungi and nematodes and inundation with actinomycetes. and insects [90]. such as Tupperware. Petri dishes give good aeration conditions. or other fruiting or vegetative structures are dissected from the substratum and planted onto fresh isolation media or further separated with a micromanipulator. When Petri dishes are used. Forcible Spore Discharge Directly discharging spores onto an agar medium is an essential technique for culturing basidiomycetes or ascomycetes with large fruiting bodies [92.g. and its low profile may interfere with development of tall fungi from large substrata. water should be added every few days. although slightly cooler temperatures may help prevent overgrowth by aggressive fungi.Fungal Germplasm for Drug Discovery and Industrial Applications 141 or lidded plastic food containers. it is necessary to know which types of fungi are unlikely to germinate on agar (e. Not all spores of all fungi germinate on agar media. In a few minutes to several hours. the need to assay for difficultto-isolate destructive plant pathogens has motivated the development of many baiting techniques [32. Aeration usually is not a problem. such as agarics from dung. fragile fruiting bodies can dry out or never develop. The best known form of the technique is to fix the hymenium (spore-bearing surface) of a basidiomycete or apothecial ascomycete onto the inside of a Petri dish lid with petroleum jelly. If it is too dry. Sterilized wood blocks and mesh bags containing sterilized leaves have been the preferred baits for trapping fungi from marine [68. and wool into the soil or invasion of plant seedlings by damping-off fungi. Baiting Baiting is a nutritional enrichment technique whereby an organic substratum or living organism is placed in contact or mixed with the habitat sample (e. hide. germ tubes emerge in a few hours to several days.91].85]. such as. at least several individual spores or small groups of spores should be transferred to new plates or tubes of media.93].87].. A lidded. Once spores have been deposited on the agar. depending on the fungus. the decay and incorporation of a dead sheep’s bones. however. The bait-substratum mixture is designed to mimic the natural differential colonization or infection process. Apothecial or stromatal ascomycetes can be placed on the inside of the lid with agar surface inverted above to trap ascospores that are forcibly discharged upward.

the pre-cut lid is replaced with a new one. conidia attached to the leaf surfaces float to the water’s surface. Sometimes media and a convenient place to set up an isolation are unavailable. Another variation is to place decayed vegetation or aquatic debris in a shallow container or Petri dish and partially cover it with sterile water. tissue transfer tends to be more successful with saprobic or decomposer fungi than with obligately symbiotic or parasitic fungi. where they can be collected for separation or pipetted and spread onto an agar medium. A precut lid is disinfected with 70% ethanol and placed over the isolation medium. cut to an appropriate size. The tissue should be fresh and free from other organisms. The foam forming on the surface is collected and pipetted onto a Petri dish of agar medium. the airflow stimulates conidia production and release of conidia from the leaf and debris surfaces. and Hypal Isolations Basidiospores or ascospores often do not discharge or germinate on agar. Leaves and stems infested with stromatic pyrenomycetes or loculoascomycetes can be used similarly to trap discharged spores [94]. After spores are collected. The containers are placed onto a rocking platform and incubated at cool temperatures (15 to 18 C) for several days to a week.. The lid is rotated gradually.g. Discharged spores of larger fungi can be collected in a clean. or stipe. Cordyceps. empty Petri dish or on a clean paper surface (spore prints) for transport or mailing back to the lab. .142 Okuda et al. dispersing and separating the spores on the medium. Tissue. The conidia accumulate on the meniscus layer of the water and are trapped with a fine screen or glass cover. Single conidia are separated with Skerman’s manipulator. the relative positions of spores are used to locate the ascomata of origin so that the fungus can be identified. but observing them sometimes is difficult. After the surface conidia are dislodged. glebum. because germination characteristics vary among fungi and viability can be negatively influenced by handling. Square or rectangular holes of varying sizes (5 5 mm up to 10 30 mm) are cut into the lids of 100-mm plastic Petri dishes with a hot knife. A handful of litter is soaked in 500 mL of sterile water in a beaker and gently mixed. e.95–97]. Epichloe. and fixed on the lid of the dishes. or psuedothecia are soaked in water. fragile. A small piece of the clean inner tissue is excised with a sterile scalpel or forceps and transferred to an agar medium. and Balansia species). stromata. the water is replaced. Dead leaves or stems with black perithecia. Freshly emerged hyphal strands from decaying wood. Professor Robert Bandoni developed a method to stimulate sporulation and release of those spores of these fungi by incubation of the litter in water [50. After several minutes.g. is broken open to expose clean inner tissue. Spore prints should be kept cool until they can be cultured by dilution or streaking onto fresh isolation medium. ¨ or when the fruiting body is very large and does not fit well inside the Petri dish. Success with preserved spore deposits may be erratic. The beaker is aerated for 1 week with a submerged aquarium pump. the pileus. Bandoni’s Method Specialized aquatic or terrestrial aquatic fungi inhabit moist surfaces of leaf litter and woody debris. The thickest part of the fungus. leaves. stems. This variation avoids damage to scant.. Once again. Rhizomorph. If ascopores are discharged on to the agar. The fertile part of fruiting body or substratum with a fruiting body is positioned over the hole so that the spores shoot onto surface. or wood can be dissected and propagated onto agar as well. and conidia are separated with Skerman’s manipulator. Conidia released from plant debris and adapted for water dispersal will float to the surface. or small fruiting bodies (e. and tissue isolations may be useful for such species [93]. A variation on this theme is to use Petri dish lids with precut holes for spore discharge [79].

if present on the stems. Remove about 8 to 15 fragments along the inner edges of each stem section. Occasionally. The benomyl is omitted if Xylariales. especially where insect activity is apparent. Tunneling insects introduce contaminating fungi and render the wood useless for this technique. cream-colored. can be selectively separated from wood by taking advantage of their relative resistance to the fungicide benomyl and their vigorous ability to decay sound wood of standing or recently fallen trees. Diatrypales. some will produce chlamydospores. usually the wood is discolored and loses strength and hardness. Select recently dead standing stems. Identifications of basidiomycetes from cultural features are difficult. verifying that it is a basidiomycete. The most troublesome contaminating fungi in wood are likely to be species of Trichoderma. Indirect Isolation Methods 4. Differentiated conidiophores are rare. heavily fragmented wood. actively decaying wood is largely free of other fungi except for the fungus that is colonizing the wood.100. have isolation media prepared and ready in the laminar flow hood.101]. or even a . black or dark brown zone lines will be apparent where different fungal individuals meet inside the wood. In this case. but basidioma.4. Several standard methods and protocols are used to describe and classify basidiomycetes based on their morphology in culture [93. Small fragments of decayed wood can be dissected and placed on an agar medium to yield pure cultures of basidiomycetes. At each site. Cladosporium. and the grain will be less apparent. and Penicillium.99]. Splitting stems into quarters or eighths will yield many triangular edges from which it is easy to cut and remove wood fragments. Basidiomycete mycelia tend to be colorless.4. Split stem sections longitudinally with a quick blow with a hatchet or by prying the stem apart with a heavy knife. or light brown. Insert 5 to 10 fragments on a plate of malt extract-benomyl media [98. Basidiomycetes will emerge slowly from fragments. Fresh.1. Isolation of Basidiomycetes from Decaying Wood Wood-decay basidiomycetes. pale orange. Inspect the wood for signs of decay. cut a series of small fragments (less than 0. Diaporthales. Cut stems into manageable lengths (5 to 10 cm) that can be split easily with a hatchet or large knife. or recently fallen wood. Basidiomata or their primordial may form occasionally in older cultures.5 cm) along the triangular edge of the split stems. select only the most recently dead. yellow. white or lightcolored mycelium of the basidiomycete will be visible on the wood surface underneath the bark or in cracks and fissures. more rarely by holoblastic. Avoid highly porous. Stems should be small enough to be cut and split with hand tools (3 to 15 cm diameter). Zone lines near the surface are a good indication of colonization by fungi of the Xylariales or other sapwood-colonizing ascomycetes. Many species will form clamp connections at the septa. conidia. Label plates with collection data or collection code and incubate them at 15 to 25 C for 2 to 6 weeks. close familiar and generic approximations. However. Using a flamed.Fungal Germplasm for Drug Discovery and Industrial Applications 143 4. if not impossible. Do not touch the inner surfaces of the wood with your fingers. insect-free stems and make isolations from the sapwood. primarily Aphyllophorales and Agaricales. Occasionally. ensuring that a wide variety of basidiomycetes will be encountered. steel-handled scalpel or a sharp knife. hanging branches. Often. arthrosporic or. white. choose a variety of dead stems and branches from different tree species. or other ascomycetes characteristic of dead sapwood are sought. On return to the laboratory. may provide clues. appearing as light-colored mycelium.

85% saline. Soil samples often are sieved through a 2. Soils are sometimes dried at room temperature.005% to 0. Soil Swabbing and Stamping Very small quantities of soil can be directly applied to isolation plates. Tween 80. Heat and Chemical Pasteurization Partial killing of a fungal population with dry heat. Cotton balls. Soil is pulverized or sifted into a sterile Petri plate lid or plastic weighing pan. but soil samples can be used immediately without drying.2% agar or carboxymethyl cellulose so that soil particles remain suspended. as in desert soils. The suspension is mixed or vortexed vigorously and between 50 and 200 L 102 to 104 diluted suspensions are pipetted onto isolation medium. swabbing or stamping can be applied to any soil and can even be inoculated in the field. may be added to aid dispersal of soil aggregates. With care and a light touch. numerous genera of coprophilous fungi. touch the soil surface and streak particles. onto the surface of the agar. sponge stoppers. species match. Ethanol pasteurization is a very effective method for extending the number of kinds of fungi recoverable from soils while lowering the total number of isolates of yeast species. following a bacteriological purification pattern. Usually the first two or three plates are overwhelmed by colonies. A typical dilution scheme suspends 200 to 500 mg of soil in 5 mL sterile water or 0. may be possible from ITS ribosomal DNA or near large subunit ribosomal DNA sequences [102. Serial Dilutions Serial dilutions are the classic method for isolation of soil fungi and fungi from other materials [31]. The method works well with soils where propagule density is low. With a sterile cotton swab.. has effectively increased the frequency of isolations of ascomycetes from soils. starting at the first plate. A detergent. Chaetomium species. or felt replicate pads also can be used to disperse soil particles across agar surfaces [104]. Xylaria species. transferring emerging hyphal tips to agar slants. Incubate plates at 15 to 20 C and review daily. Stamp a cotton ball or a felt pad lightly onto the soil. or solvents has been employed for preferential isolation of species that have resistant propagules. or sclerotia. steam. Other researchers have researched isolates from ethanol. stamp the entire surface of the agar in each plate in succession in a clockwise pattern.g. The suspension is evenly spread with a sterile glass rod. but the later plates yield a manageable number of colonies.103].to 5-mm mesh to remove large debris and to homogenize the sample. Then. Such soil treatments shift the population of isolates toward ascomycetes and basidiomycetes that have ascospores. thus increasing isolation of ascosporic Eurotiales. as well as many species with thin-walled conidia. a variation of ethanol pasteurization. An alternative method is to mix the suspension with a pre-cooled agar medium. Formalin pasteurization [112]. e. Anecdotal evidence indicates the isolation results can differ depending on the soil pretreatment. Some investigators have combined ethanol treatment with mild heat treatment. as is the case for actinomycetes isolations. Place a row of 6 to 10 plates with the desired isolation medium to one side. Place finely divided soil from each sample in a Petri dish lid.144 Okuda et al. One mL of 103 to 105 diluted suspensions is poured into an empty plastic dish and about 19 mL of pre-cooled agar medium (45 to 55 C) is mixed with the suspension.or phenol-treated soils for taxonomically novel fungi [106–111]. and possibly some ascosporic yeasts. We often increase the viscosity of the suspension by adding or 0. which is thought to stimulate ascospore germination [105]. thick-walled chlamydospores. Chemical pasteurization is useful .

the profile of isolates differs from that of suspension plating [31.Fungal Germplasm for Drug Discovery and Industrial Applications 145 for recovery of large numbers of fungi from herbivore dung and reduces interference from contaminating yeasts [10]. The wash solution is decanted and replaced for up to 10 times. bryophyte leaves. the washed fragments are plated on an agar medium and incubated for up to 1 month. 1. A similar method using a sucrose density gradient was used to separate. or very thin materials in which disinfectants will kill cells.005% aerosol OT solution (di-iso-octyl sodium sulfosuccinate).2 to 5. but less damaging than. such as fine roots. therefore. or other surfactant. The precipitate is suspended in 0. Germany. Sieved soil samples (1 g) are added to 10 mL Percoll. and a thin layer of agar facilitates dissection of colonies embedded in the agar.5 mL of sterile water or with a few milligrams of sterile sand grains. Overlay the soil particles with 8 to 10 mL of medium while gently agitating the plates to disperse particles. Density Gradient Methods Differences in the specific gravity and size permit separation of mixtures of organic debris and inorganic soil particles containing spores and mycelia [13. The solution is prepared in a tube and centrifuged at 60. The method is fast and easy for culturing soil fungi.15M NaCl is applied prior to the addition of soil suspension.’’ were developed as an alternative to suspension plating for characteriza- .41]. small invertebrates. Differential centrifugation also can separate large-spored predatory nematophagous fungi and soil microfauna infected with small-spored endoparasites [58]. Molten isolation medium is prepared and maintained at about 45 C in a hot water bath. surface sterilization. The supernatant (5 mL) is mixed with 45 mL distilled water and centrifuged at 2150 g at 20 C for 10 minutes. also known as ‘‘soil washing. The quantity of soil will vary widely with propagule density and soil texture. submerged colonies need to be cut from beneath agar.0 mL Percoll (Pharmacia). and 2. The Percoll solution contained 7. Fractionation of the gradient more thoroughly extracts fungal propagules from soil than a typical serial dilution. and vortexed. The soil can be dispersed by agitation in 0. and Sweden. Warcup Soil Plates This method differs from other soil isolations because the particle structure of the soil is relatively undisturbed. Particle Filtration In laboratories in Canada. which is then gently applied on the top of the Percoll pregradient. Each fraction is diluted and spread on an agar medium.113]. and invertebrate parts [114]. Serial Washing Repeated washing of small substratum fragments removes surface spores. Add 0. The effect is similar to. Tween 80. Young.0 mg of finely pulverized soil to the bottom of a sterile Petri dish. deteriorated. After air drying on a sterile filter paper overnight to reduce bacterial contamination. particle-filtration techniques. concentrate.0 mL 1. and directly enumerate basidiospores and sclerotia from soil in Pinus contorta forests [116]. although obtaining optimal colony densities can be tricky.115]. The Percoll pregradient with 0. plant litter fragments. The suspension is sonicated for 2 minutes and left to settle for 2 minutes while heavy soil particles sink. The material is added to a large volume of sterile water with 0. The solution is centrifuged at 240 g at 20 C for 3 minutes and fractionated from the bottom up. and generally is used with porous. Samples are cut into small pieces and put into tubes or vials.000 g at 20 C for 15 minutes to form the pregradient.5 mL distilled water.5 M NaCl.0 mL distilled water. enough times to remove surface propagules [40.

and it has been shown to be effective in controlling foliar fungi in greenhouses [128]. tion of soil fungal communities [30.122.120. Theoretically.124]. Recently. the method often reveals numbers and kinds of fungi rarely seen by those acquired by conventional techniques. and plated the washed particles on a medium made selective for basidiomycetes by the incorporation of benomyl. filtered syringes. cost. Bills and Polishook [11. has been used for surface disinfection because of its flexibility and less corrosive and damaging effects on disinfected surfaces.122]. fungicolous. wood-decay.to 210. The medium also contained alkali lignin to encourage growth of ligninolytic fungi. Isolation of species with chlamydospores embedded in organic particles may be favored. lichenicolous.m particles to study fungi in forest litter.122]. nested sieves. whereas conidia of undesired anamorphic fungi (e. anything that can be broken into small particles can be used.g.127].1% hypochlorite solution. which acts as a colorimetric indicator of the lignin-modifying enzymes laccase and peroxidase.123] used 105. hypochlorous acid. However. the technique was substituted for the traditional ethanolhypochlorite disinfection for isolations of endophytic fungi [129]. The agar adjacent to a few of the 5 to 25 colonies that developed on each plate turned dark brownish red as guaiacol was oxidized to a quinone. and complex self-flushing/refilling devices have been used as washing devices [30. Particle-filtration techniques favor isolation of fungi from mycelial fragments immersed in the substratum while reducing recovery of fungal colonies initiated from spores. and availability of materials. Despite the need for more preparation. Acidic electrolyzed water often is used for nonthermal disinfection of hospital instruments. food processing. and guaiacol. Choice of device seems to be a matter of personal preference. Simple vials. Good results usually are achieved only after considerable practice and careful preparation of materials. Particle filtration is readily adaptable to many kinds of organic substrata. [125] used the particle filtration technique to remove spores of anamorphic fungi and Zygomycetes from soil. presumably due to nonselective reducing .117–120]. A total of 67 basidiomycete isolates were recovered from 64 soil samples from 35 sample plots. and the method has been applied successfully to isolate terrestrial and aquatic litter. because their propagules probably are not entirely removed by washing [121]. and coprophilous fungi..146 Okuda et al. Prevention of intraparticle competition is the key to its success and is achieved by reducing particle size so that a colony:particle ratio of less than 1 results [117. the activity of electrolyzed water decreases rapidly with increased contact with organic material. Effectiveness of these devices depends on the ability to obtain well-washed particles. and chlorine. 5) [126. and water purification. clearly separated into descending size categories. Thorn et al. The acidic electrolyzed water containing 40 mg/L sodium chloride showed similar antimicrobial activity to that of 0. Alternaria and Trichoderma species) are avoided. Recently. Surface Disinfection with Acidic Electrolyzed Water Immersion in hypochlorite and ethanol solutions is probably the most widely used disinfection method for removal of superficial fungi from plants and other materials (Fig. Baath [117] recommended particles in the range of 50 to 80 m in diameter for conifer soils.114. prepared by the electrolysis of an aqueous sodium chloride solution and resulting in a dilute mixture of hypochlorite. acidic electrolyzed water. These colonies are scanned under low magnification (40 to 100 ) and putative basidiomycetes selected for transfer. Choice of ˚˚ particle size depends on substratum texture [117.

Additional recipes and procedures can be found among previous compilations of mycological methods [26. typical leaf-surface fungi. 5.. In one experiment employing leaves of Pieris japonica soaked in electrolyzed solution for less than 10 minutes... amino acids. followed by air-drying on a sterile filter paper. Therefore. The treated leaves were washed thoroughly in sterile water with 0. Phomopsis sp. and characterization. Pestalotiopsis versicolor.5 g/L Tween 80. peptides. Intact leaves were soaked in 1 L of the fresh electrolyzed solution at intervals of 10 minutes up to 1 hour. melanogenum. Yokohama.85. A disinfection method was developed for fungal endophytes that used 2 L of 1% NaCl solution electrolyzed for about 20 minutes using electrolyzing apparatus (Amano Co. Tests of the antifungal activity of the electrolyzed solution indicated that both conidia and mycelia of all these fungi were about equally susceptible to the water. Most culture media contain at least water.Fungal Germplasm for Drug Discovery and Industrial Applications 147 Figure 5 Effective chloride concentration as a function of pH in hypochlorite solution generated by acidic electrolysis. SPORULATION. lipids. Aureobasidium pullulans. Longer treatments increased the ratios of Phyllosticta sp.. bacteria and fungal propagules on plant surfaces will be killed with a brief exposure.131].32. Japan. and Colletotrichum gloeosporioides. Fig. but endophytic fungi within the leaf are expected to survive.130. a simple or complex source of carbon (carbohydrates. AND MAINTENANCE Appendix 1 provides formulae for some media that we have found useful in the laboratory for isolation of fungi into pure culture and for their cultivation. CULTURE MEDIA FOR ISOLATION. amino . They were then cut into small pieces (ca. organic acids) and nitrogen (peptides.93. and the differential isolation was not due to the differential susceptibility.. pullulans var. true endophytic fungi. 3 mm2) and plated onto a fungal isolation medium. it is believed that isolations of fungi from plant tissues can be more precisely controlled by varying treatment times with acidic electrolyzed water than by immersion of tissues in with hypochlorite-ethanol solutions.27. agents. 6). A. and Tripospermum sp. Cladosporium spp. predominanted. As a result. microscopical examination.

Yokohama. . These factors are deliberately varied to influence the growth form. Figure 6 Electrolyzing apparatus. humidity).or disaccharides tend to favor vegetative growth. Nutrients can be supplied as pure reagents. and sometimes vitamins and essential amino acids. hay infusion. dilute. essential metal cations. and to the physical environment (light. peptone.148 Okuda et al. Fungal growth and development are extremely sensitive to the composition of the medium. acids. Media with high concentrations of mono. as complex plantor animal-derived additives (oatmeal. sulfur. phosphate. as is often the case. when all media components need to be defined and reproducible or. Super Oxseed Labo (Amano Co. to laboratory conditions. Japan). For example. carrots).. ammonium. lifecycle. nitrate). low nutrient media with only polysaccharide carbon sources or plant-derived infusions often favor sporulation and suppress mycelial growth. temperature. and metabolic products of fungi in culture.

is possible. chlortetracycline. AutoGen Inc. The first phase of isolations. An exception is chloramphenicol. Addition of antibacterial antibiotics to fungal isolation media often is essential to prevent bacterial growth. which can be autoclaved and usually is used at a concentration of 100 mg/L medium. Most antibiotics are heat labile and are added to cooled media (45 to 50 C) just prior to pouring plates. Kuhner AG. and certain actinomycetes. Selectively toxicity toward certain fungi. with as few as steps as possible. particularly oomycetes and basidiomycetes. and we have never observed them to adversely affect establishment of basidiomycete or ascomycete cultures from direct plating of spores.. The concentrate is added to media to yield a final concentration of 30 to 100 mg/L of medium. The precise challenge is to increase the numbers of samples. chloramphenicol. Many of the necessary manipulations have been incorporated into automated devices developed by Olympus. cherry-picking. Automated strain manipulation started with ac- . and macroarraying [134]. if selective toxicity is suspected. A good combination of antibiotics for isolation of most filamentous fungi is chlortetracycline (50. ampicillin.136].. so that they are numerically competitive with synthetic chemical collections.133] for isolating homogenous colonies from clonal libraries ¨ of bacteria. However. Hitachi. Automation of Strain Isolation and Management The evolution of HTS laboratories and their demands for increasingly larger chemical libraries and numbers of natural product samples for testing have challenged microbiologists to streamline and improve the manual processes of making microbial extracts. The technology has been further elaborated for preparation and management of clone libraries through colony replication.Fungal Germplasm for Drug Discovery and Industrial Applications 149 whereas extremely high carbohydrate or salt concentrations may inhibit growth by reducing water activity. The concept is based on building a set of interacting automated devices that carry out parallel handling of strains in a format compatible with current methods for automated liquid handling systems.0 mg/L) and streptomycin sulfate (50. One of the only high throughput cultivation systems that was adapted for use with wild fungal and actinomycetes colonies was the CT4000 developed by Hitachi Electronics Engineering Company [135.0 mg/L). Acidification of media to pH 4.1. the preparation and application of soil dilutions to isolation plates. and Bio-Rad Laboratories. the pin-transfer tools employed in conventional colony pickers designed for transfer and array of bacterial or yeast genomic libraries are often incompatible with many kinds of filamentous fungal growth. while maintaining high levels of chemical complexity and uniqueness among the samples. GeneMachines. Tomtec. Discussions of strategies for improving the productivity of microbial screening invariably lead to the suggestion that automated devices can supplement the traditional microbial processing techniques. oxytetracycline. Streptomycin sulfate. These antibiotics specifically interfere with bacterial ribosome function. was still done manually. Such genomics tools are available from Genetix Ltd.5 with lactic acid can help to suppress bacterial growth. substitute other antibiotics 5. Most antibiotics are prepared in sterile water or filter-sterilized and stored as concentrated stock solutions. and vancomycin frequently are used. Rose bengal in isolation media may inhibit growth of many bacteria and reduce radial extension of fungal colonies. yeasts. Tens of hundreds of colonies can be repetitively transferred from mother plates to daughter plates and arrayed as desired. and others [132. among others. used in the microbial screening program at Nippon Roche. Toyo-Sokki. rather that sequential handling of individual strains for flask or tube fermentations.

whereas others can inundate the plate in a few days. for example soil. Fungal colonies from nature are very heterogeneous in terms of size. The CT4000 had an automated colony recognition and picking system that replaced the largely manual and repetitive task of selecting and transferring microbial colonies from soil isolation plates to clean growth media. the underlying assumptions disregard differential growth rates. design of an isolation system for wild fungal colonies needs to overcome obstacles inherent in the varied ways fungi grow and reproduce: 1. The CT4000 usually worked very accurately without excessive errors from the time it was completed and installed. therefore. mainly because of reasons explained below and because manual selection of colonies for picking was still needed. 2. Implementation of such an automated system potentially could be counterproductive in terms of producing the diversity of metabolites. Rarely were they operated for selection of wild strains for screening. and some may not even appear on selective media for days. and hardness. texture. . tively growing soil isolation plates. The range of motions that the human eye and hand match to each type of fungal colony in order to dissect and lift it is unlikely to be replicated by a robot. its use for wild colony selection was abandoned. and that organisms are behaviorally and morphologically equivalent and therefore can be delivered to the system in a standardized format. 3. That standardized format generally is assumed to be a Petri plate bearing wild colonies from soils. undifferentiated or immature colonies of different species may appear to be the same. can be prepared in a homogeneous format. it is doubtful whether it saved human resources. The system was used in bacterial screening for a while and. so that automating the recognition and discrimination of all colonies is difficult. Dry spores that interact with static electricity may scatter during the transferring process and cause contamination. color. in the long run. The inverse situation can limit the system. The computer was programmed to recognize and select colonies and send instructions to a robot. and the biased and narrow spectrum of the microbial flora that develop on soil isolation plates (Fig. in the case of fungi. out of the range of a tool that operates at or above the surface. which would pick them. Colonies of fungi may grow at very different rates. and transfer cells. Proposals for automated microbial isolation usually are based on the often-erroneous assumptions that the source of the organisms. however. and it was used for manipulations in the strain improvement programs. The robot’s picking needles accurately touched the selected colonies and transferred them onto an agar medium. A single universal picking tool employing a single mechanical action is inadequate to dissect. The Hitachi CT4000 also inoculated liquid media with the isolated strains. lift. Images of colonies grown on agar were captured with CCD camera and the images were binarized so that the positions of the numerous randomly distributed colonies are recognized by a computer. and those that produce spores do so in a myriad of ways. 2). We are aware that several other Japanese pharmaceutical laboratories operated robotic colony picking systems. but rather were employed in mutation and strain improvement screening in fermentation facilities. Some fungi may grow appressed at the agar surface or submerged within the agar and. the CT4000 was never well accepted by microbiologists. because reliance solely on large numbers of soil-derived strains compatible with surface growth on agar limits screening to a relatively restricted diversity of fungi and ignores the majority of the fungal kingdom. Ultimately. Furthermore. Despite its sophisticated features. However.150 Okuda et al. variable disseminated propagules.

An alternative route for parallel separation. Bacterial cells from soils.g. Therefore. and by differential application of enrichment cultures on the encapsulated population. FACS-separated microdroplets contain single-cell–derived microbial colonies that range from one to many cells. and the high viscosity of large mycelial pellets and submerged growth can obstruct or complicate pipeting. The encapsulation-FACS technique has several obvious advantages over the use of colony pickers on soil plates. engineering and programming of custom-built robotics are not required. or other materials are concentrated and then added to a nonpolymerized gelling agent (e. Encapsulated cells can be cultivated in a continuously fed open culture system that more likely simulates natural environments and thereby enables cultivation of microbes not normally seen on conventional agar plates. especially in industrial programs that may be providing natural products samples to multiple therapeutic target areas simultaneously? . how does one know that the goals established by the program are met. replication plating. isolation. 6. Cell throughput of FACS is potentially orders of magnitude higher than could be achieved with mechanical colony separation. a much higher percentage of the microbial flora would be cultivated and assayed. All cell types from the environment are equally likely to be encapsulated. As a result. isolation of most fungi is likely to still rely on manual techniques and cognitive abilities of trained scientists. Cell separation by FACS is achieved with commercially available (albeit costly) flow cytometry equipment that is often accessible at major research centers. then the probability of entrapment of single cells inside individual microdroplets is high. and cultivation of microbes recently has been developed and relies on microencapsulation of microbial cells followed by cultivation under selective nutrient or environmental conditions and separation of encapsulated cells by fluorescence activated cell separation (FACS) [15.. aquatic environments. separation. including cell types generally thought to be unculturable [15]. different components of the population can be favored for growth. The gelling agent is then dispersed in an emulsifying agent with either a shearing device or by filtration through a microporous filter to form a homogeneous emulsion of microdroplets. addition of shearing devices.137]. and those that initiate cell growth are separated from those that are empty or dead by FACS. Not only do growth forms vary among organisms. There is no doubt that success ultimately rests in validity of the target and implementation of screening assay and on how they correlate with in vivo activity. Parameters of the FACS are set to channel a subpopulation of living microdroplets to a collection vessel.Fungal Germplasm for Drug Discovery and Industrial Applications 151 4. the encapsulation-FACS technique has only been applied to bacteria. CODA: EVALUATING AND GUIDING THE OUTPUT Success in microbial screening. cell-containing microdroplets can be distributed into wells of microculture plates for subsequent cultivation steps. as defined by immediate recognition of a drug lead. but they are profoundly influenced by the vessel shape. but it is easily imaginable that such a system could be adapted to entrapment. has always been an elusive event. The microdroplets are harvested and cultured briefly. alginate or agarose). therefore. and downstream analyses. Liquid inocula of fungi are similarly heterogeneous. and parallel cultivation of environmental fungi. rotation speed. If the volume of gelling agent is sufficiently high relative to the cell density. If the FACS is equipped with an automated cell sorter. So far. Then in absence of discovery of a blockbuster active.

Judgments based on one-parameter measurements. Several different parameters and outcomes that contribute to the overall goal of providing microbial chemical diversity can be evaluated. Attention should be paid to types of metabolites with promiscuous activities and the qualitative differences between the discoveries of a totally new bioactive scaffold versus the discovery of new analogues within known scaffold series. or whether it is excessively repetitive. Chemometric analyses and identification of extract components are detailed in other chapters of this volume. Furthermore. Phylogenetic or taxonomic diversity of the strains is not directly translatable into chemical diversity and alone is an imperfect surrogate for measuring the chemical complexity of the output. and especially of strains that have yielded specific activities and compounds. Maintenance of historical records of molecules discovered and which organisms produced them discovered will reveal repetitive patterns and potential needs to shift focus in strain selection. and whether is it targeted toward chemically relevant taxa. rather than attempting to maximize hypothetical probabilities from dimensionless measurements. and certain kinds of organisms are more genetically disposed to producing metabolites of different biosynthetic pathways than others [12]. as well as the unique physiological and biochemical characteristics of the organisms responsible for their biosynthetic capabilities. The challenge in screening large cross sections of microorganisms and fungi for metabolites is to focus on physiological and genetic components common to all fermentations while anticipating the recognition of a yet-defined product. The role of the microbiologist is the selection of organisms that have the best characteristics for the biosynthesis of the required products in quantities sufficient for detection. The assumed equation between biological and chemical diversity becomes even more tenuous when one considers that strain selection must be tightly coupled with fermentation and extraction techniques to give a chemically meaningful result. or simply genera of terrestrial fungi whose propagules germinated on the isolation plates? Such confirmations need not be exhaustive. just as certain synthetic chemical families are more useful for particular types of targets than others. confirmation. and a natural tendency exists to focus on those unique features that lead to specific product formation. and hit rates. do isolations from freshwater or marine habitats really obtain taxa adapted to those habits. Finally. each fermentation is unavoidably unique. unknown taxa. can be evaluated to some degree. numbers of extracts produced. We believe that parameters with a genetic or physiological underpinning or those that lead to product formation should be emphasized. phylogenetic breadth of the screening population. Bioactivity and chemical complexity of culture broths and extracts are more direct measurements of the utility of different fungi. like number of strains isolated. Maintaining records of diagnostic analyses of screening populations. can provide a valuable historical map of where the program has traveled and where it has not.152 Okuda et al. and scale-up of primary actives. For example. Direct probing and analyses of genes of biosynthetic pathways can confirm the genetic potential of target . However. An analysis of isolates at the generic and species level can confirm or reject whether isolation methods are achieving the claimed results. Scrutiny and analysis of such numbers out of context can lead to bizarre predictions and conclusions. it cannot be emphasized enough that quality control by spot-checking the identity of active strains in our screening programs has greatly improved reproducibility. However. The reasons are simple: unrelated organisms often produce the same kinds of metabolites. are essentially meaningless because the units of measurement are undefined. certain microbial products will be more or less useful. Environmental and nutritional conditions need to be identified that favor product formation. but can be carried out on subsets of screening populations by means of morphological and rDNA-sequence analyses.

. but they can help identify ineffective organism-medium-environment combinations. For example. Performance. A high proportion of the basidiomycetes (45%) produced some type of antifungal or antibacterial activity. . fermentations.g. but generally less than some of the better-known ascomycete orders (e. Eighteen percent of the strains produced activity against at least one of the strains. synthetic chemical libraries. the Eurotiales and Hypocreales). The levels of activity were comparable to those of some ascomycete orders. although comparing strains at the family level. A more rational strategy for the discovery of fungal metabolites probably lies near the proposals of Dreyfuss and Chapela [12]. was assay dependent.g. Researchers at Sandoz submitted a set of endophytes from woody plants (N 400) to a series of 14 varied assays. Staphylococcus aureus) and one of two yeasts (Saccharomyces cerevisiae or Candida albicans). while refining and redirecting the search as new leads are discovered. whereas others are yet to become apparent. measured by detection of actives between the endophytes and the control set. although overall a greater proportion of the actives were detected among the endophytes. have only been derived from metabolites and enzymes of about two handfuls of different fungi. detection of antibiotic activity is a test of the combination of the genetic capacity of an organism and its response to its cultural conditions. relegates discovery to an accident and would be analogous to screening unlabeled. Mapping those few organisms onto the vast evolutionary tree of the fungi raises expectations that many new products await discovery. The exploratory stage gradually progresses to a larger scale where parameters such as sample and organism selection. Results were contrasted to those of an undefined control group (N 764). and phylogenetic criteria. undifferentiated colonies from isolation plates. In reality. arbitrarily selected. e. Some of these groups of fungi are well known. and pathway data to identify areas and correlate them with relevant chemical activities. Programs need to be maneuvered toward biosynthetically creative pockets of organisms using logistical. more than 30% of the strains the Dermataceae and Hyaloscyphaceae were active. and production of antibiosis can be used to compare efficacy of production conditions for a given sets of strains. Simple antibiotic readouts may not be able to guide the program to an optimal state of productivity. Evaluation of microbial extracts from their antimicrobial activity is another common method and is based the assumption that antibiosis is a primary or secondary effect of the production of one or more complex secondary metabolites [138]. worse. Therefore. to date. The selection of broad cross sections of different fungal taxa or the search for novel chemical structures [54] among poorly studied species by themselves are not enough ensure discovery success. The relative antibiotic activity of 317 isolates of basidiomycetes from different orders were surveyed with a single fermentation medium and compared to those of major groups of ascomycetes [140]. Conversely.. the Pezizales and Xylariales. The first step in setting the path is to carry out enough preliminary screening to be able to integrate ecological. blind and arbitrary selection of strains or.Fungal Germplasm for Drug Discovery and Industrial Applications 153 organisms and likewise are described in other chapters of this volume. phylogenetic. negative results can mean either biosynthetic capacity is lacking or the fermentation parameters are inadequate. and extractions are incrementally improved. Commercial products. four of which were antimicrobial assays [12]. Hosoya [139] gathered comparative antimicrobial data on 350 strains of Helotiales tested against a panel of three bacteria (Bacillus subtilis. Escherichia coli. especially in the antibiotic assays. ecological.

and rhizomorphs. Blakeslee’s Malt Extract Agar (BMEA) [142. tissues of basidiomata. With the ability to identify loci of secondary metabolism independent of expression. Growth varies slightly depending on manufacturer of the malt extract and peptone. 20 g Peptone. 15 g Agar. 1. like Neurospora crassa [141]. 15–20 g Benomyl. along with knowledge of the biochemistry of the targets. .154 Okuda et al. Other antibacterial antibiotics may be substituted for isolations from natural substrata. This new front in natural products research suggests that typical fermentation methods underestimate the production of antibiotics and other secondary metabolites. 0. Refrigerate the stock solution. before or after autoclaving. Recommended for identification of Penicillium and Aspergillus spp. natural-product gene clusters and the genetic and regulatory basis of metabolic creativity and versatility. dilute the ethanol solution to 100 mL in warm water. Use: Isolation of basidiomycetes from decaying wood. 1000 mL Antibacterial antibiotics of choice are added after autoclaving. Cloning and sequencing of specific pathways and comparative genomics of fungi and actinomycetes are uncovering cryptic. and microfungi from leaf litter. improved phylogenetic understanding. 2 mg Water. add 40 mg of benomyl to 50 mL of 95% ethanol. Use: Isolation and sporulation of freshwater hyphomycetes and ascomycetes. but Difco.143] Malt extract.0 g Yeast extract. it should be possible to expand and better define paths to the most relevant creative pockets of the fungal kingdom. To prepare a stock solution of the antifungal components.5 g Distilled water. 20 g Distilled water. or BDH products often are used. 4. 100 mg. and add 20 mg of dichloran. 1000 mL Use: Many fungi grow robustly on this medium. 1000 mL After autoclaving. 1 g Glucose. Basidiomycete Isolation Medium (BDS) [99] Malt extract. APPENDIX. 2 mg Dichloran. FORMULAE FOR MEDIA USED TO ISOLATE AND CULTIVATE FUNGI Bandoni’s Sorbose–Yeast Extract–Tetracycline Medium [96] L( ) sorbose. cool to 50 C and add tetracycline (in 95% ethanol). hyphal strands. Oxoid. 20 g Agar. and add 10 mL to the malt extract medium. even by common model organisms.

0 g KH2PO4.01 g Agar.01 g CuSO4. 0.0 g MgSO4.0 mL Boil water and add cornmeal. Slowly decant all but the bottom 200 mL. 1000. 0.05 g FeSO4. including animal pathogenic fungi.0 mL For convenience.0 g Distilled water. 0.001 g Agar. 0. Medium will probably be lumpy. Czapek-Dox Agar (CZA) Sucrose. 15.7H2O. Cornmeal Agar (CMA) Yellow cornmeal. Allow this infusion to rest 15 minutes.5 g FeSO4. Sodium carboxymethyl cellulose forms a highly insoluble gel.005 g ZnSO4. 0. Filter through three or four layers of gauze. Use: Medium appropriate for isolation and cultivation of a wide variety of fungi. Add antibacterial antibiotics after autoclaving. 2. 0.Fungal Germplasm for Drug Discovery and Industrial Applications 155 Carboxymethyl Cellulose Agar (CMC) [35.5 g CaCl2.30. 1.5 g KCl.145] Sodium carboxymethyl cellulose. 1000.0 mL Antibacterial antibiotics should be added when used for isolations from natural substrata. 15.0–20.0 g K2HPO4.5 g KCl.0 g NaNO3.7H2O.0 g NaNO3.7H2O.0 g Distilled water.7H2O. Minute hyphal growth is easily seen with a dissecting scope in this nearly transparent medium. Dehydrated cornmeal agar is available . the last five salts can be combined as a stock solution.0 g Yeast extract. 1. stirring constantly. Use: A low-nutrient medium for isolation of soil and litter fungi that supports only limited colony development. 2. 1000. 40. 10. 1.01 g MnSO4. 0.7H2O. 0. 0. 15. It is also commercially available as a dehydrated liquid broth or agar medium.144. Bring volume back up to 1000 mL and add agar. 0. Combine all dry ingredients in a flask.0 g Agar. 0. Slowly add hot water. Allow mixture to stir for 15 to 30 minutes. Simmer for 15 minutes. and some brands are easier to dissolve than others.0 g MgSO4. most lumps disperse after autoclaving.4H2O.0 g Water.5 g (NH4)2SO4. and fungi from extreme environments.

0 g Peptone. 10.1 g Agar.0 mL Glycerol (analytical reagent grade).0 g KH2PO4. 0. 1 mL is added to each liter of medium to attain a concentration of 2 mg/L. After heating to dissolve agar. which limits mycelial growth. 1. 15.955. Use: Isolation of osmotolerant. 1.025 g Chloramphenicol. 1. Use: A good medium for isolation of soil fungi. Final pH is 5. 5.7H2O. Dichloran-Glycerol-18 Medium (DG18) [143] Glucose.0 g MgSO4.156 Okuda et al. .0 mL Dichloran has low water solubility.0 g KH2PO4. 0.1% aqueous solution of chlortetracycline to medium before pouring to give a concentration of 5 g/mL. It is an excellent medium for making single-spore isolations because of its transparency and because the main nutrient is starch. giving final concentrations of 18% (wt/wt) glycerol and 2 g of dichloran/mL.1%. add glycerol and dichloran solution.6. food-associated fungi.0 g Distilled water.7H2O. Dichloran can also be dissolved in a few milliliters of acetone and added to medium prior to autoclaving.002 g Rose Bengal.5 g Dichloran (0. and xerophilic fungi. but other combinations of antibacterial antibiotics can be added after autoclaving. 5.0 g MgSO4. 220. Antibacterial antibiotics should be added after autoclaving if the medium is used for isolations from natural substrata.0 g Bacteriological peptone.0 mL Chlortetracycline (0. 0. in 95% ethanol). 15. Dichloran-Rose Bengal-Chloramphenicol Agar (DRBC) [145] Glucose. The manufacturer of DRBC recommends use of chloramphenicol.2%. Use: Often a good medium for induction of sporulation. 0. aqueous) to give 5 g/mL (after autolaving) Add all ingredients except glycerol. Dichloran is highly toxic and should be used with extreme care and under proper ventilation. dichloran. A convenient stock solution can be made by dissolving 0. xerotolerant. 10. 1000. from various distributors. and fungi from dried seeds and plant materials. Add filter-sterilized 0.0 g Distilled water.0 g Agar. 1000. and chlortetracycline to 800 mL distilled water.2 g of dichloran in 100 mL ethanol. 0. Chloramphenicol can be purchased as a supplement in ampoules or can be weighed and added directly to the medium. Adjust final volume with distilled water to 1 L. aw is 0.5 g Dichloran.

Malt Extract Agar with Cyclosporin [11.0 g MgSO4. 0. 0. 1000.0 g Yeast extract.2 g Agar.0 g MgSO4. 1.7H2O. 20.0 g Water.147] Malt extract. Add antibiotics and antifungal agents after autoclaving. 2. 0.0 mL Use: The 2% version of malt extract agar is one of the widely of all media for cultivation and characterization of fungi. depending on fungi to be isolated. 0. produces malt-yeast agar (MYA).0 g Water. 2. 10. Use: Isolation of soil fungi.0 g KH2PO4. 1000.0 g Water. 1.0 g KH2PO4.7H2O.2 g KCl.0–10. Use: A very versatile isolation medium. Miru Agar (LCA) [148] Glucose. and adjust pH if desired.5 g Rose Bengal. 1000.2–2. Antibacterial antibiotics and cyclosporin A (if control of colony expansion is desired.0 g Yeast extract (optional).0 g NH4NO3.03 g Agar. Malt Extract Agar Malt extract.0 g Distilled water.0–20. 2. 20.0 g Agar.2 g NaNO3. 1. Substitution of 2–10 mg cyclosporin A for the rose bengal is a useful variation of GAN.Fungal Germplasm for Drug Discovery and Industrial Applications 157 Glucose Ammonium Nitrate Agar (GAN) [146] Glucose.0 mL Adjust pH to between 6. malt-cyclosporin A agar) added after autoclaving. 20. Use: Isolation and sporulation of freshwater hyphomycetes and ascomycetes.0 g Agar.5 and 7.0 mL Add antibacterial antibiotics after autoclaving. but especially for isolation of soil and leaf litter fungi. 0. 0. 20.0 mg Quantity of malt extract is often varied. 15. 1000. Yeast extract optional. 1.0.0 mL Cyclosporin A. .

squeezing the potatoes to extract all the liquid. 30.0 mL Agar. 1000. filter the mixture through a layer of gauze. Commercially prepared versions of OA also are available. 200. Strain autoclaved potatoes and liquid through several layers of gauze. to make 1 L Boil oats for 15 to 30 minutes. 20. because the medium usually foams excessively when autoclaved. 500. .0 g Tap water. PDA from Commercial Preparation Potato dextrose agar. grind uncooked rolled oats in a blender. Prepare this medium in an oversized flask.0 g Use: This medium is useful for stimulation of sporulation in culture. 40. Potato Carrot Agar (PCA) [27] Potato-Carrot Extract Peeled carrots.0 g Tap water.0 g Glucose. Potato Dextrose Agar (PDA) [26] PDA from Fresh Potatoes Potatoes. 1000. and autoclave for 45 minutes.0 g Peeled potatoes. 40.0 g Agar. add to water and boil for 15 minutes.0 g Distilled water. 20. Add enough water to bring the potato extract to 1000 mL. It can be used as isolation medium for soil and litter fungi by adding antibacterial antibiotics after autoclaving.0 g Agar. 500. Alternatively.0 mL Tap water. Add glucose and agar and autoclave 15 to 20 minutes at 15 psi. Oatmeal Agar (OA) Rolled oats. 20. 39. 1000. Potato-Carrot Agar Potato-carrot extract.158 Okuda et al. Use: Very good medium for studying development of fungal morphology and for induction of sporulation in nonsporulating isolates.0 mL Shred carrots and chop potatoes into small pieces.0 mL Antibiotics can be added to medium after autoclaving. Autoclave for 20 minutes at 15 psi. Autoclave 30–40 minutes at 15 psi. Filter through cheesecloth or gauze. combine with agar and water. Bring filtrate volume to 1 L.0 mL Chop potatoes and place in water. Add agar. 20. The extract maybe autoclaved for future use.0 g Distilled water.

35. World Federation for Culture Collections Newsletter 2002. 1000.0 mL Glucose. 900.7H2O.0 mL Soil extract: steam 1 kg soil (preferably sandy loam) in 1 L distilled water at 100 C for 1 hour.5 g Glucose. Several formulations are in the literature.0 mL Use: Isolation of endophytes.5 g Agar. Headfast/CD Software.0 g Distilled water. Use: Widely used for isolation. Water Agar (WA) Agar. 15. 2. one is provided here.2 g Sucrose. 0.0 g MgSO4. Filter first through cheesecloth. Okuda. 0. . then centrifuge to clear.2 g Agar. Head Software International. 20.Fungal Germplasm for Drug Discovery and Industrial Applications 159 Use: PDA is one of the most commonly used culture media for fungi because of its simple formulation and its ability to support mycelial growth of a wide range of fungi. 20 g Distilled water. The recipe for commercially prepared PDA is provided as well. 0.149] Soil extract. Soil Extract Agar (SEA) [27. and roles of biotech ventures and biological resource centers.0–20. 0. Autoclave extract for 1 hour on each of 2 consecutive days before preparing medium.7H2O.0 g Distilled water. induction of conidia and identification of Fusarium and Clonostachys species and other hyphomycetes with slimy conidia. 0. 1. 1000. Use: Widely used for isolation of soil fungi and fungi associated with plant roots. 100. 1. 0. 21–26.5 g KCl. Spezieller Nahrstoffarmer Agar. REFERENCES 1. Chapman & Hall Dictionary of Natural Products: 1999. T.0 g KNO3. Trends in natural product-based drug discovery. plant pathogens and soil fungi.0 mL Add antibacterial antibiotics after autoclaving if used as an isolation medium. 0.5 g MgSO4. Anonymous.5 g K2HPO4. Various combinations of antibiotics can be added to medium after autoclaving. or Synthetic Low-Nutrient Agar (SNA) ¨ [27] KH2PO4.

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and an animal growth promoter (zearalenone). ROBERT GIACOBBE. Spain INTRODUCTION Filamentous fungi produce a vast array of bioactive secondary metabolites [1]. S. HARRIS. cephalosporins.7 Expression of Cosmid-Size DNA of Slow-Growing Fungi in Aspergillus Nidulans for Secondary Metabolite Screening ZHIQIANG AN. the host 167 . most efforts have been focused on fungi that can be easily isolated. PING LU. JERROLD LIESCH. DEBORAH ZINK. GERALD BILLS Merck Sharp & Dohme de Espana. and strobilurins). migraine and obstetrics pharmacologics (ergot alkaloids).A. New Jersey. ˜ Madrid. but expansion of fungal natural products sources to unculturable and slow-growing fungi may lead to even more new structural classes. and WILLIAM STROHL Merck Research Laboratories. and maintained on laboratory media. plant growth hormones (gibberellins).. Rahway. ANDY LIAW. but some fungi may never be cultivated. GUY H. Despite these successes. In this approach. U.A.S. RAJINDER SANGARI. including antibacterials (penicillin. One way to capture the genetic diversity of unculturable or slow-growing organisms for secondary metabolite discovery is to introduce large segments of genomic DNA from these organisms into faster growing. griseofulvin. cultured. antihypercholesterolemia agents (lovastatin and pravastatin). genetically amenable hosts. antifungals (pneumocandins. immunosuppressants (cyclosporin A and mycophenolic acid). We expect to continue to discover new secondary metabolites from easily culturable fungi. PRAKASH S. MASUREKAR. BERT GUNTER. VLADIMIR SVETNIK. STEVEN GOULD. Improved cultivation techniques can help to take advantage of less manageable fungi. and fusidic acid).

MI). Pequannock. A. 4 g. 20 g). 2. nidulans strain MF5999 was grown in appropriately supplemented A. Total genomic DNA from fungal biomass ground to a powder in liquid nitrogen was isolated by a phenol-chloroform extraction procedure [10. Inc. This tendency for genes to cluster allows us to gather most. 2. HPLC peak-guided isolation procedures resulted in the isolation of two compounds from extracts of two of the outlier transgenic strains that were not produced by either the expression host or the bulk of transgenic strains..168 An et al. 10 g. Louis.4. malt extract. 4 g. Furthermore.2. and reagents from other sources are identified in the text. and sometimes even in distantly related organisms [2–7]. nidulans transformations were carried out as previously described [9]. Filtering out the bulk of samples using these tools yielded a manageable number of samples that were individually examined and subjected to further chemical separation and biological testing. MA). we developed a reproducible procedure using 24-well microplates for high-throughput small-scale fermentation and a semi-automated mass spectroscopy protocol that quickly and sensitively characterized the sample strains. if not all. NJ) on YME agar (per liter: yeast extract. nidulans for secondary metabolite screening. and Promega (Madison. of the genes for a particular secondary metabolite within one large fragment of DNA.11]. KS) and . is asked to express genes from the donor organism and to produce secondary metabolites that it does not naturally produce. Inc. Kansas City. PA). Construction of Cosmid Cloning Vector pANARGB2 and Cosmid Libraries pANARGB2 was constructed as follows: the 1. nidulans was isolated from plasmid pDC1 (FGSC.8 kb XhoI/BamHI fragment containing the argB gene of A. Sigma (St. Ardamine PH was obtained from Champlain Industries. New England Biolabs (Beverly. agar. Bel-Art. which can then be introduced into a recipient host for potential production of recombinant metabolites. we describe the heterologous expression of cosmid-size genomic DNA fragments from five strains of slow-growing cranberry endophytic fungi in A. Genomic DNA Isolation Fungal isolates from cranberry plants were grown on a layer of cellophane membrane (dialysis membrane.3. In this chapter. 2. Media components and chemicals were from Difco Laboratories (Detroit. Chemicals and Reagents Restriction endonucleases and DNA modifying enzymes were from Gibco BRL (Rockville. EXPERIMENTAL METHODS 2. MO).1. Soybean meal was from Centra Soy Co. Transformants of A.. During the course of this study. glucose. This is possible because transcription and translation control sequences from one organism often function in closely related organisms. and Fisher Scientific (Pittsburgh. 2. WI). nidulans minimal medium [8]. genes involved in the biosynthesis of major microbial secondary metabolites are often clustered. nidulans strain MF5999 containing cosmid genomic DNA clones were selected for L-arginine prototrophy. tomato paste was from Beatrice/HuntWesson. A. MA). nidulans Transformations A. we also have developed data analysis/pattern recognition procedures to assist in the prescreening. Because visual examination of the thousands of spectra to find those few that look different is both laborious and prone to error.

Hybridization to 32P-labeled random-primed DNA fragments (Multiprime DNA labelling systems.) following the manufacturer’s protocol. 1 shows the features of pANARGB2. and transferred onto nylon membranes (Hybond N . and CR133) using the Gigapack III XL packaging extract (Stratagene. nidulans ornithine carbamoyltransferase–encoding gene. cos site. 2. 2. nidulans transgenic strains was isolated as previously described [9]. Only cloning and diagnostic restriction sites are shown. blunt-ended by Klenow fragment.8-kb bluntended HindIII/EcoRI fragment containing the backbone of the cosmid cloning vector pMLF2 [12].) was carried out at 65 C [14].5.6. Southern Blot Analysis Genomic DNA from A. argB. ampicillin resistance–encoding gene. origin of DNA replication. ApR. Cosmid libraries were constructed for genomic DNA from five slow-growing strains of cranberry endophytic fungi (CR61. The DNA fragment was then ligated to a 5. ori. CR68.Expression of Cosmid-Size DNA of Slow-Growing Fungi 169 Figure 1 Features of pANARGB2. cos. 35. CR132. coli strain XL1-Blue MR. A. La Jolla. Amersham Corp. nidulans MF5999 using cosmid DNA isolated from pools containing ca. 4000 . CR70. CA). digested with BamHI. Fig.to 45-kb Sau3A1 partially digested genomic DNA fragments were isolated by sucrose gradient centrifugation and ligated into the BamHI site of cosmid vector pANARGB2 [13]. Briefly. Packaged cosmid clones were introduced into E. separated by electrophoresis on agarose gel. Amersham Corp. Generation of Transgenic Strains About 1000 transformants were generated for each of the five cosmid libraries by transforming A.

CT) and flasks. except for MF5999. soybean meal. 5 g. methG1 biA1. San Diego. cultures . (NH4)2SO4.. methG1 Wildtype Wildtype Wildtype Wildtype Wildtype Wildtype biA1. methG1 biA1. nidulans A. The cultures grown in LSF1 medium were incubated for 14 days at 25 C and 220 rpm on an orbital shaker with a 5-cm throw in Kuhner cabinets (Kuhner. After 14 days. 5 g. Ardamine PH. nidulans A. and pH adjusted to 7. PA. sodium citrate. tomato paste.0 with NaOH). Inc. methG1 Source of all strains was this study. 2 g. respectively. Fermentation Conditions A. nidulans A. nidulans transformants were grown in either 24-well microplates or 250-mL flasks. methG1 biA1. nidulans A. The medium used for liquid fermentations was LSF1 (per liter: glycerol. nidulans A. nidulans A. 10 g. methG1 biA1. methG1 biA1. Basel. methG1 biA1. nidulans A. nidulans A. nidulans Genotype biA1. nidulans A. nidulans A. The solid fermentation medium was yeast extract sucrose agar (YES) [15]. Individual L-arginine prototrophic transformants were transferred into wells of 24-well plates to verify the L-arginine prototrophic phenotype. methG1 biA1. A total of 2 mL and 50 mL of YES and LSF1 media was placed into each well of 24-well microplates and into 250-mL flasks. argB2. Milford.7. methG1 biA1.170 An et al. Philadelphia. Spores from A. which was from Larry Yager. Table 1 Fungal Strains Used in this Studya Strain Name MF5999 CR61 CR68 CR70 CR101 CR132 CR133 MF5999/7–2–1 MF5999/7–2–2 MF5999/7–3–1 MF5999/7–3–2 MF5999/7–4–1 MF5999/7–4–2 MF5999/7–5–1 MF5999/7–5–2 A09T0943 B07T0399 B09T0391 B09T0573 A09T0567 a Species Aspergillus nidulans Ascomycete Ascomycete Oomycete (?) Ascomycete Ascomycete Ascomycete A. methG1 biA1. nidulans A. 5 g. nidulans A. Transformants were stored at 4 C in 24-well plates on supplemented minimal media. methG1 biA1. methG1 biA1. Temple University. 75 g. clones of each library. Table 1 lists some of the fungal strains used in this study. CA) or sterile cotton-tipped applicators (Hardwood Products Company. methG1 biA1. 2. The cultures grown on YES medium were incubated under static conditions under the same incubation parameters as stated above. Guilford. nidulans (MF5999) strains were transferred from 24-well storage plates via replicating pins (V&P Scientific. MN) into wells of 24-well fermentation microplates (Whatman-Polyfiltronics. 2 g. Switzerland) at 85% relative humidity in the dark. respectively. glucose.

xip).12 to 0. generating a single corrected spectrum for each sample. As an internal standard for FIA-ESI-MS and TLC analyses. The few novel samples will have distinct spectra that are far from the main cluster and appear as individuals or small groups of individuals. …. and where i 1. 2.8. NC). xI (xII. This representative spectrum contained the abundance values reported as absolute abundance. Most of the samples not producing recombinant natural products should be similar and should yield similar spectra. 801 in our spectra.01% trifluoroacetic acid (TFA) at a flow rate of 400 L/min was used. A total of 1 mL of the top organic phase was removed and transferred to sealed glass vials (National Scientific Company) and stored at 20 C for downstream analyses. Time between each injection was 2 minutes. 2. Samples were introduced into the mass spectrometer by a flow injection analysis method and analyzed by electrospray (FIA-ESI-MS). Notation We denote the spectra of the n samples in the library by row vectors. The peak maximum of the total ion chromatogram was detected. with a peak width of 0. Research Triangle Park. Cultures grown on YES solid medium were extracted with 1. Inc. These spectra should therefore cluster relatively close together in this space. 2. An average spectrum at one-half height was determined and the background spectra were subtracted to produce a single representative spectrum per injection. Mass Spectrometry Instrument Conditions Mass spectral data were collected on an Agilent-MSD mass spectrometer with an APIelectrospray source.89 sec/cycle. Sample extracts were prepared using an automated liquid dispensing system (Tecan US. The instrument scanned from 1000 to 200 Da at a cycle time of 1. Therefore.8 mass units. background MS spectra were obtained from 0.10. griseofulvin at 10 mg/L was added to the MEK extracts. 2. Data Description In all cases. The combination of all spectra yields an n by p matrix X whose rows are the spectra of individual samples. homogenized for 30 seconds using a tissue homogenizer (Biospec Products.2-minute delay to allow for background determination. The abundance values were zero-filled.2 minutes. xIj is the absolute ion intensity (abundance) at the j-th m/z ratio. A total of 5 L of each extract was injected into the solvent stream after a 0. An isocratic solvent system of 90% MeCN water solution with 1.9. and shaken for 1 hour on a reciprocal shaker at room temperature. The m/z values were converted to integers by using a rounding point of 0. p is the number of m/z ratios.5 volume of MEK for 12 hours at room temperature. Identification of Novel Samples Each spectrum xi could be represented as a point in a p-dimensional abundance space. n.Expression of Cosmid-Size DNA of Slow-Growing Fungi 171 in LSF1 medium were mixed with 1.).11.2 min and a ramped fragmentor voltage of 50 at m/z 200 to 120 at m/z 1500.3-mM NH4-formate and 0. the problem of identifying novel samples becomes a problem of detecting outliers in p-dimensional .5 volume of methyl ethyl ketone (MEK). ….

since these channels would not contribute any useful information to the analysis. Because the two procedures are based on different models. of outliers in the data. (2) Agglomerative hierarchical clustering with average linkage was carried with each of the three matrices.05 in our analysis. We expected novel samples should be in small. kimin.17]. The procedure was performed as follows: (1) The Euclidean distances. We refer to assumption 2 above as the -criterion. While there are many methods for outlier detection in multidimensional data.172 An et al. a problem with using Mahalanobis distance for outlier detection is that the usual (least squares) estimate of the covariance matrix can be distorted by outliers so that the outliers are masked and therefore missed. of the data. space. A simple plot of Mahalanobis distances based on this covariance matrix was then used to identify the outliers by plotting them in descending order. Pearson correlations and rank correlation matrices were computed. Perhaps the most important feature of this procedure is how it gets around the difficult problem of selecting the right number of clusters by using step (3) described . none work satisfactorily in all situations.14. (3) For each distance/similarity matrix and each sample spectrum. 2. . and clusters that satisfy this criterion as -clusters. We therefore computed an outlier-resistant estimate of the covariance matrix using the minimum covariance determinant (MCD) algorithm [18]. isolated clusters (possibly having only one member). the data were preprocessed by removing channels (m/z) where no sample has ionization intensity exceeding 1000 (the detection limit). We have developed two procedures for our purpose: resistant Mahalanobis distances and a sequential clustering procedure (SCP). However. (4) The n samples with the smallest kimin (if there are ties in kimin. the smallest number of clusters. and there can be no more than n outliers in all. and (2) these outliers belong to clusters that contain at most a fraction. We expected both and to be small. We used 0. . Outlier Detection by Mahalanobis Distances and MD-PCA Mahalanobis distances and principal component analysis (MD-PCA) are widely used chemometrics methods for characterizing spectra and finding outliers [16.05 and 0. they should be able to identify outliers containing different features. xi.12. 2. and (5) The union of the three lists (one for each distance/similarity) was taken as the final list. Novel samples are defined based on two assumptions: (1) there is at most a fraction. Data were also visualized by plotting the scores of the first few principal components of the MCD covariance matrix. 2. Outlier Detection by Sequential Clustering The second method for determining novel extracts was based on cluster analysis [19]. the ties were considered as the outlying samples). was found such that xi belongs to a cluster with fewer than n samples. In other words. This step significantly reduced the dimension of the data by eliminating as many as 40% of the channels. Data Preprocessing Before the outlier detection procedures were applied. a sample must belong to a cluster that contain no more than n samples to be considered as an outlier.13.

The upper layer was subjected to semi-preparative RP HPLC on C8 as described above eluted with a gradient of 10 to . 2H). 2H). The material was further purified using preparative RP HPLC (Phenomenex Primesphere C8. 1.8. TLC. 1.5′. confidence values of the samples’ novelty were estimated. Spectroscopic data for this violaceol I isolate were consistent with that previously reported [21]. a portion (14 mg) of the MEK extract of a LSF1 fermentation of culture A09T0567 was partitioned between hexane: EtOAc:MeOH:H2O (1:1:1:1). we relied on the bootstrap method [20] to simulate what would happen if this were done.4 250 mm) using a mobile phase of 35% CH3CN/65% H2O containing 0. The details are 1. 20 mL. If the number of clusters is varied from k 2 to n.118 (s.16.0-mL fractions collected. were combined and concentrated in vacuo to yield violaceol I (1 mg).8.2% aq. where li is the number of bootstrap outlier lists that included sample xi and Bi is the number of bootstrap matrices containing that sample.5 mL/min. and dried over anhydrous Na2SO4 to yield a red residue. For violaceol I isolation and identification. equilibrated with MeOH.Expression of Cosmid-Size DNA of Slow-Growing Fungi 173 above. it will be a member of some -cluster at all k that are larger than kimin. This confidence value is an estimate of the probability that a sample xi would be called an outlier if the experiment that generated the data were repeated under the same conditions but with experimental variability yielding somewhat different data. and Structural Identification The MEK layer of the transgenic strain extraction was applied directly to pre-coated silica gel TLC plates and the plates were developed with toluene/ethyl acetate/formic acid (5:4:1) [15]. tr 16. H3PO4. the smaller kimin is.0915 (M H).145 (brdd. 5 m. YES fermentations of culture B07T0391 were extracted with MEK. 6. 6. Xb* b samples (rows) from the original matrix X with replacement. Therefore confidence values were used to prioritize samples for follow-up.0920 for C14H14O5 H. The residue was dissolved in EtOAc. The crude EtOAc extract was applied to a column of Sephadex LH-20. Secondary metabolites were visualized on TLC plates with UV and p-anisaldehyde spray reagent. calculated 263. B. For asperugin A. 9. were simulated by randomly selecting as follows. …. and step (4) above then yielded the outlier list.15. Thus we ranked the samples by their kimin values. 2. 0. Bootstrap matrices. 2. Outlier Confidence Values Once the list of novel samples is determined.6. 0. HPLC. 1 80 cm.394 (brdd. The MEK extracts were filtered and concentrated to dryness in vacuo. Fractions 32 to 35.9. 100 mL total. Because it is not feasible to repeat the experiment multiple times. This step used the monotonicity property of hierarchical clustering methods. Outliers with high confidence are thus more likely to represent real novelty. We followed up on those samples with confidence values above 0. Fractions 19–23 were combined and concentrated to dryness. and this solution was extracted with 0. 177 mg. HRFT MS-ESI: found 263. Another property of hierarchical clustering is that the more outlying a sample xi. 1H NMR (CD3OD) 2. 6H). B isolation and identification. b 1.1% (v/v) TFA at 3. B. giving rise to B overall bootstrap outlier lists Lb. The bootstrap confidence value i* is the ratio li/Bi. 20 mL. then once a sample xi becomes a member of a -cluster at some k kimin.2.…. The SCP is applied to each matrix Xb*. and hence appears as a potential outlier. The EtOAc layer was washed with H2O and brine. The column was eluted with MeOH at 2 mL/min and 8.

The five endophytic fungi (CR61. CR68. NMR. Outlier Identification in Library 7 Extracts of 3. nidulans that carry a defect in the ornithine carbamoyltransferase locus [23. RESULTS 3. the backbone and multiple cloning sites were from pMLF2.1. nidulans transformants generated with each of the four cosmids from library 7 (cosmids 7–2. Genomic DNA was prepared from the eight cultures before subculturing and after the seventh subculturing. Additionally. Transformation of A. MS. and CR133) grew slowly with colony diameters smaller than 5 mm in 1 month on YM agar. except transformant 7–3–2. Briefly. 7–3. nidulans MF5999 with pANARGB2 yielded arginine prototrophic transformants. which has been described previously [12]. DNA fragments of over 40 kb in size isolated from Sau3AI partial digestions by sucrose gradient centrifugation and fractionation were cloned into the BamHI site of pANARGB2. The small percentage ( 1. the hybridization patterns for the two independent transformants generated by the same cosmid were all different. nidulans–specific cosmid-cloning vector pANARGB2 was constructed for this study. The argB gene complements arginine auxotrophic strains of A. High-molecular-weight genomic DNA ( 50–245 kb in size as determined by field inversion gel electrophoresis) was isolated from all five strains.4% of the total samples) of wells with either no growth or contamination were excluded from subsequent analyses.550 A. 11. two independent A. 3. nidulans transgenic strains from four cosmid libraries (7. Stability of Heterologous DNA in Transgenic Strains Grown on Selective and Nonselective Conditions To test the stability of integrated DNA in heterologous hosts. 10. CR70. Peaks corresponding to asperugin A.7 mg) and asperugin B (2 mg). . 2). B eluted at 29. more copies of cosmid 7–2 were integrated in transformant 7–2–1 than in transformant 7–2–2 (Fig. 3. CR132. respectively.24]. 3.2.174 An et al.0′ and 26. Cosmid Vector and Libraries An A. 90 CH3CN containing 0. 7–4. The solvent was removed in vacuo to yield asperugin A (1. 2). Digestions of ten randomly selected clones from each library with various enzymes showed that all clones contained an average insert size of approximately 35 to 45 kb. and 12) were processed and analyzed by FIA-ESI-MS (Table 2). The BamHI digested genomic DNA was hybridized with the respective insert DNA isolated from the cosmids. and UV data for these isolates were consistent with that previously reported [22].1% TFA. Transgenic strains generated from library 7 were fermented on solid YES medium whereas all other transgenic strains were fermented in LSF1 liquid medium. in which an extra band was detected in the seventh transfer without selection (Fig.3.5′. The hybridization patterns of Southern blots of each transformant under both selective and non-selective conditions were the same and identical to the pattern obtained before the strain was subcultured. and 7–5) were single-spore subcultured seven times at 5-day intervals in the presence and absence of L-arginine in minimally supplemented media. For example.

. SCP. transformants were single-spore subcultured seven times on medium without arginine supplementation at 5-day intervals. Mahalanobis distance—principal component analysis. F7 selection. Table 2 Mass Spectrometry Outliers as Identified by MD-PCA and SCP in Four Fungal Libraries Library 7 Donor fungus Fermentation medium Mass spectral analyses MD-PCA outliers % of outliers by MD-PCA SCP outliers with 90% confidence value % of outliers by SCP Outliers by both MD-PCA and SCP % of outliers by both MD-PCA and SCP CR61 YES 764 48 6. nidulans transformants generated from four cosmid clones (p7-2. Parent..Expression of Cosmid-Size DNA of Slow-Growing Fungi 175 Figure 2 BamHI-digested genomic DNA of A.5 Library 11 CR132 LSF1 970 56 5.2 50 5.0 Library 10 CR101 LSF1 980 51 5.g. original transformant without subculturing.1 34 3.3 54 7. p7-4.2 43 5.8 63 6. p7-3.3 Library 12 CR133 LSF1 836 127 15. F7 No selection.6 MD-PCA.1 46 6. p7-2-1 and p7-2-2 are independent transformants generated from cosmid p7-2). Two independent transformants generated by each cosmid were included in the experiment (e.5 32 3. and p7-5) probed with insert DNA isolated from respective cosmids. sequential clustering procedure.1 28 3. transformants were single-spore subcultured seven times on arginine supplemented medium at 5-day intervals.

3%) and 54 (7. two were identified only by MD-PCA and remaining eight were identified by SCP (Table 3). The samples occurring before the break were selected as outliers. 3). 10. 46 (6. 4 also shows that the outliers were randomly distributed throughout the entire library. vertical lines are drawn at the break of the curves to denote the cutoffs for outliers. 10. In E–H. The difference between Figs. The library 7 SCP outliers are shown in Figs. 3E). Of the 10 nonoverlapping outliers.176 An et al. MD-PCA and SCP identified 48 (6. 4. Fig. 11 and 12 (A–D) and the confidence values of outliers in libraries 7. 3E and 4 as filled dots and those with a 90% or higher confidence value were above the horizontal line in the figures. Points to the left of the vertical lines have large Mahalanobis distances and are selected as outliers. Fig. . The Mahalanobis distance plot used to identify outliers in library 7 by Mahalanobis distance-Principal Component Analysis (MD-PCA) is shown in Fig. Points above the line have confidence values of 90% or higher. 3A. A total of 56 outliers were identified by either or both of two methods (Table 3). 11 and 12 that were detected by sequential clustering procedure (E–H). 3).1%) outliers. 3E and 4 is that the outliers were ordered by confidence values in Fig. The vertical line in the figure indicates where a break occurred in the plot. respectively. Figure 3 The Mahalanobis distances of all samples in libraries 7. The difference between the median spectrum and the spectra of 8 representatives of the 56 outliers in library 7 is shown in Fig. 5.0%) of which were outliers identified by both methods (Tables 2. The two unique outliers identified by MD-PCA (B07T0414 and B07T0790) were ranked 47th and 48th among the 48 samples identified by this method and they are the two least significant outliers (Table 3). a horizontal line is drawn at 0. In A–D. 3E and the outliers were shown in sample numeric order in Fig. All 54 outliers identified by SCP with the 5% cutoff criterion using three correlation measures have a bootstrap confidence value of at least 90% (Table 3.9 in each figure. from the 764 strains in library 7 (Tables 2.

6. In addition to asperugin A. Fig. and 5. 3. these constitute 3.1%. and 28 for libraries 10. 6. Outlier Identification in Libraries 10. respectively (Table 2). The horizontal dashed line shows the 0. respectively (Table 2). 11. and 12 were 5. B. 5.5. A09T0567.6% of the total samples tested for libraries 10. 604 strains (24 host cultures. respectively (Table 2. 3. TLC Analysis of Recombinant Fungal Extracts For TLC analyses. 11.2%. 32. TLC pattern for one of the 15 outliers. . The numbers of overlapping outliers as identified by both methods were 34. respectively (Table 2). and 12 The number of outliers identified by both methods from libraries 10. Fifteen of the 556 extracts showed distinctive TLC patterns as visualized with p-anisaldehyde/H2SO4 and UV. The percentages of outliers with a confidence value of 90% or higher as identified by SCP for libraries 10. and 12 were listed and shown in Table 2 and Fig. and 12 were 5. 11. 11.Expression of Cosmid-Size DNA of Slow-Growing Fungi 177 Figure 4 The distribution of the entire set of 764 samples in numeric order and based on their confidence values in library 7 as determined by the sequential clustering procedure.4. The percentages of outliers as identified by MD-PCA for libraries 10. 11. is shown in Fig. and 12.9 cutoff. and 15. 11. and 556 transgenic cultures) from library 9 (donor fungus CR70) were fermented in 2 mL of LSF1 medium in 24-well microplates and extracted with MEK.3%. and the remaining samples are marked with open circles. and 3.5%. 3). The outlying samples are marked with filled circles. and 12.2%.5%.8%. 3. subsequent RP HPLC analysis of A09T0567 revealed several additional components not observed in the median extracts. 3. 24 host/vector cultures.1%.

88 30.92 31.16 18.99 22.19 39.05 22.80 22.12 42.14 35.43 24.49 34.59 41.83 34.40 48.50 29.59 19.29 30. Table 3 Mass Spectrometry Outliers as Identified by MD-PCA and SCP in Library 7 kmin a Sample ID B07T0211 B07T0789 B07T0691 B07T0715 B07T0791 B07T0759 B07T0767 B07T0087 B07T0763 B07T0752 B07T0447 B07T0443 B07T0786 B07T0778 B07T0779 B07T0577 B07T0774 B07T0306 B07T0012 B07T0831 B07T0391 B07T0810 B07T0782 B07T0426 B07T0573 B07T0438 B07T0451 B07T0422 B07T0462 B07T0091 B07T0200 B07T0420 B07T0079 B07T0418 B07T0115 B07T0107 B07T0042 B07T0687 B07T0847 B07T0111 B07T0038 B07T0570 B07T0679 B07T0683 B07T0439 B07T0799 Euclidean Distance 5 2 2 2 2 2 2 5 2 2 5 5 5 5 2 2 2 5 2 5 4 5 5 5 2 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Pearson Correlation 6 5 2 2 2 2 2 11 2 2 11 11 9 9 2 4 2 11 2 11 7 11 9 9 7 11 9 9 11 11 15 15 11 11 11 11 11 11 11 11 11 11 11 11 11 11 Rank Correlation 5 5 2 2 2 2 2 19 2 2 19 19 17 17 2 4 2 19 2 24 16 24 17 17 5 19 19 17 24 29 18 18 29 29 29 29 29 24 24 29 29 29 24 24 29 24 Confidence Value 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.87 41.39 49.38 38.178 An et al.33 18.58 19.66 20.29 33.47 33.62 28.54 21.88 27.81 20.27 44.03 Method Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both Both (Continued) .76 56.94 36.56 51.04 50.53 39.87 49.99 1 Mahalanobis distance 67.23 32.13 31.37 44.77 22.53 51.92 53.71 50.59 26.02 32.

4.97 1 0.17 0.88 13.01 12. Two outliers from library 7 based on their FIA-ES-MS profiles and statistical analysis. 4. Both strains contained several new components not observed in extracts of the background strains (Figs. 6). CR68. these fungi were not readily identified. we have cloned at least 12 ketosynthase domains of polyketide-encoding genes from 11 of the 23 isolates tested [39]. Reverse-phase HPLC analysis of the two cultures also showed clear differences from the typical transgenic background (data not shown).88 11. 6). SCP. B07T0391 and B07T0573.62 11. Because of the lack of morphological characteristics.47 0.Expression of Cosmid-Size DNA of Slow-Growing Fungi 179 Table 3 Continued kmin a Sample ID B07T0414 B07T0790 B07T0347 B07T0822 B07T0827 B07T0753 B07T0707 B07T0658 B07T0811 B07T0807 Euclidean Distance 92 51 5 5 5 5 5 5 5 5 Pearson Correlation 54 36 24 11 24 24 24 24 24 24 Rank Correlation 100 100 29 24 81 57 100 29 81 100 Confidence Value 0. We found that it was still necessary to isolate DNA fragments larger than 40 kb by sucrose gradient centrifugation before packaging to obtain consistently high quality libraries. CR132. Donor Fungi The secondary metabolites produced by the cranberry endophytic fungi are unknown. Mahalanobis distance—principal component analysis. were analyzed further with TLC. 5. DISCUSSION 4. MD-PCA. One of the major components from B07T0391 was identified as violaceol I (Fig.98 0.97 0. 6). It is estimated that the genome size for most filamentous fungi is between 25 and 45 mb and with average size of about 35 mb [25]. Based on this estimation.26 15.98 0. The two strains had different TLC patterns when compared with the typical background and host strains (Fig.1. In a brief survey of evaluating the secondary metabolic potential of the cranberry endophytic fungi.04 10. and CR133 were unrelated ascomycetes and CR70 is most likely an oomycete. we screened about 1000 transgenic . cosmid library construction was relatively straight forward.97 0.to 51-kb recombinants. Libraries and Transgenic Strains Once high quality genomic DNA were obtained (50–245 kb).68 Method MD-PCA MD-PCA SCP SCP SCP SCP SCP SCP SCP SCP a min k in italics are not supported by the respective distance/correlation. but the potential for producing novel bioactive chemical structures by these fungi exists because of their unique ecological niches.97 0.72 11.97 Mahalanobis distance 16.82 13.2.39 9. The Gigapack III cosmid packaging extracts from Stratagene were designed to preferentially package 47. Phylogenetic analyses using SSU rDNA sequences suggests that CR61. sequential clustering procedure.

the median spectrum of what a typical sample should resemble is shown at the top of the column. . Figure 5 Mass spectra of 8 of the 56 outliers in library 7. For comparison.180 An et al. The median spectrum is obtained by taking the median of each channel across all samples in the library.

lane 1). it is not necessary to maintain selection during fermentation for stability of the heterologous DNA. One of the major new components from B07T0391 was purified from a larger scale agar culture using Sephadex LH-20 and preparative RP HPLC. and A09T0567 (lane 6). B07T0573 (lane 5). and a background level transformant (B07T00399. 4. This study further confirms that once DNA is integrated in the A. Two components were isolated from A09T0567 using preparative RP HPLC. The isolated compound was identified as violaceol I. lane 2). mitotically stable [24. DNA integrated in the genome of A. Three controls were also included: the host (MF5999.26–29]. the structures were elucidated from NMR and MS data as asperugin A (minor component) and B (major component). Microplate fermentation is relatively high . strains for each library to give approximately 1X genomic coverage of the donor fungus.3. lane 3). vector transformant (A09T00943. a 24-well microplate fermentation method was developed.Expression of Cosmid-Size DNA of Slow-Growing Fungi 181 Figure 6 TLC patterns and structures of components from B07T0391 (lane 4). in general. nidulans and other fungi are. nidulans genome. Fermentation and MS Analysis Because of the large number of transformants generated in this experiment.

and solid medium under static conditions whereas the other three libraries were fermented in a complex liquid medium with agitation. defined. Outliers identified by the two statistical models (MD-PCA and SCP) from library 7 overlapped significantly (82%). we relied on the graphical approach. and scale-up reproducibility testing with fungi producing known compounds. LC-MS tools have been found to be useful in taxonomy and secondary metabolite profiling of extracts. These numbers are much smaller than for library 7. Outliers identified by both methods are very likely true outliers. The usual approach for outlier detection using Mahalanobis distances is to use a critical value from the chi-squared distribution as the cutoff. and with low cross-contaminations observed ( 1%). 11. This is based on the assumption that the data come from a multivariate normal distribution. were conducted before it was used in this experiment. but the so-called marginal outliers identified by one of the two methods may be rechecked in broad screening programs. Smedsgaard and Frisvad [30] demonstrated that one could characterize the taxonomy and secondary metabolites of crude fungal extracts using FIA-ESI-MS methods similar to those described here. therefore. throughput and can be automated for downstream processing. five were supported by the Euclidean distance measure. Outliers in Libraries 10. such as addition of extraction solvent and other liquid transfer steps.4. are also marginal outliers. however.182 An et al. Previous studies have shown that . What was unique in our analysis was that we used the mass ion profiles to compare the overall similarities and differences among a large set of samples. respectively. A series of validation experiments. Of the eight outliers identified only by SCP. 37%. Therefore.5. FIA-ESI-MS was used to measure the natural products productivity of actinomycetes under various fermentation conditions [31]. Analyzing hundreds of genetically similar samples allowed us to define a background profile. well-towell contamination testing with different fungi. 4. suggesting they are probably marginal outliers. the two MD-PCA–unique outliers have the smallest Mahalanobis distance values among the 48 outliers identified by MD-PCA and they. including cover design to prevent evaporation. One of major concerns with microplate fermentation is its reproducibility from experiment to experiment and from microwell to scale-up. as evidenced by the number of spectra identical to controls. and 12 were 51%. It is not clear what caused the difference except that library 7 was fermented on a simple. and 12 The overlapping outliers identified by the two methods from libraries 10. Similarly. that the mass spectra data sets are far from this assumption. We found that the 24-well fermentation was highly reproducible. The vertical line defining the elbow in the Mahalanobis distance curve for this library can be readily recognized indicating that there was a clear boundary separating the small number of outliers (48) from the majority of background strains (716). 4. and then we exquisitely detected differences with respect to this background using statistical tools. 11. one of the three measures used in the SCP analyses. We know. Outliers in Library 7 An elbow existing in the Mahalanobis distance suggests that the small fraction of the samples deviating from the majority is not caused by random experimental variations. These results suggest that both methods are effective in identifying the most obvious outliers. More recently. which is less exact but more useful. and 20%. This is not surprising because Euclidean distance measure is more sensitive to experimental variations than that of the other two measures.

ranging from biological variability to variations in fermentation conditions and sample storage and handling to MS measurement variations. The structures of other HPLC fractions from these outliers have not yet been determined. the data were scattered irregularly. However. and 12. What we do not know is the importance of the outliers that have a confidence value higher than 90%. with no clear pattern defining nonoutlying spectra. There was a large overlap among subgroups. If a typical fungal genome is 35 mb [25]. there was no typical pattern among the bulk of these data. but a search of Chapman and Hall’s Dictionary of Natural Products revealed more than 35 secondary metabolites produced by A. physiology. There are several possibilities how a silent pathway may be activated. MD-PCA score plots of library 12 showed it to be a mixture of several—five or more—somewhat homogenous subgroups. It is interesting that asperugin A and B were isolated from a mutant strain of A. TLC patterns for most of the cultures are similar because only a small fraction of the transgenic cultures are expected to have an altered chemical profile. This is particularly true for library 12. Consequently. The approach described in this study generated a large number of transgenic strains. TLC Analysis and Structure Isolation As expected. We believe that the production of these metabolites in the transgenic strains is most likely caused by the activation of silent biosynthetic pathways for these metabolites in A. and chemistry of fungi [32].Expression of Cosmid-Size DNA of Slow-Growing Fungi 183 fermentation media and culturing conditions play a major role in the morphology. We think it is prudent in such circumstances to apply a variety of methods that consider the data from several different perspectives.6. but they are less defined than for library 7. Using the same 5% cutoff criteria used for library 7. rugulosus [34]. all the methods essentially found considerably different subsets that they considered unusual. Assuming the average gene cluster size for . A quick isolation of the dominant compound present in outliers B07T0391 and A09T0567 revealed structures that were not produced by the controls under the fermentation conditions used in this experiment. There could be many reasons for this. and hence no clearly atypical patterns. nidulans by the transformation process. Not surprisingly. and 12 than found in library 7 (Fig. In short. the number of outliers that have a confidence value above 90% and the percentage of these outliers of the total samples was similar to the respective value from library 7. This translates into about 1000 transgenic strains. 3). B and violaceol I are known metabolites of Aspergillus and Emericella species [21–23]. or activation of additional silent host pathways. It is difficult to estimate the number of secondary metabolic pathways for a given fungus. significantly more outliers were identified by the SCP method for libraries 10. about 1000 cosmid clones are needed to have a 1X coverage of a given genome. nidulans [36]. This was also reflected in the relatively few points that passed the 90% bootstrap confidence criterion. nidulans genome from the donor fungus and this transcription factor activated a silent pathway. These metabolites represent at least 12 active distinct biogenic pathways. Elbows are also evident in the Mahalanobis distance curves for libraries 10. It has been reported that some global transcriptional factors regulate the secondary metabolic pathways in fungi [35]. 127 outliers were selected by the MD-PCA method. however. For example. because both asperugin A. One can expect that they might be structures encoded by secondary metabolite-encoding gene clusters from the donor fungus. a transcriptional activator may have been introduced into the A. 11. or hybrid compounds synthesized by genes from both the host and the donor fungus. 11. 4.

J. a semi-automated electrospray mass spectrometry (FIA-ESI-MS) protocol for characterizing the fermentation extracts. The novel chemistry outliers subsequently have been included in a library for screening against a broad set of therapeutic targets. coli. Pouwels. CONCLUSIONS A transgenic approach was developed to capture the genetic diversity of slow-growing fungi for the production of secondary metabolites. Between 3% and 6% of the mass spectra of transgenic strains were identified as significantly different from the background spectra. Springer: Berlin.B. more than 1 Gbp of soil DNA was cloned in two BAC libraries and expressed in E. In our approach.. 1997.A. it might be more desirable to construct a mega library that contains multiple fungal genomes. In this proof-of-principle study. 12 biosynthetic gene clusters constitute about 1% to 2% of the fungal genome. such as BAC and YAC clones. and screening of the library resulted in the identification of several clones that expressed antimicrobial activities [38]. 249–268. 117–124. Gloer. Isolation of dominant components from two outliers revealed natural products that were not produced by the controls under the fermentation conditions used in this experiment. transgenic clones were screened for specific activities such as antimicrobial activities. M. 2. 5. some of the clones expressed antimicrobial activities [37]. If a typical BAC insert is 130 kb. REFERENCES 1.J. Oliver. several similar experiments were being conducted by other investigators for heterologous expression of bacterial natural products encoding genes from environmental DNA. nidulans and the resultant transgenic strains were screened for altered secondary metabolite profiles. The chances of having cosmid clones containing a complete large secondary metabolite-encoding gene cluster by screening libraries with only 1X genomic coverage is smaller than if libraries were large enough to cover the genome multiple times. 4. C. clones were first prescreened for novel chemical production potential based on their overall FIA-ES-MS profiles. 56.J. Gene 1987. While this study was being carried out. With the rapid sequencing of fungal secondary metabolite encoding genes and gene clusters. J. This biased library approach will significantly reduce the number of transgenic strains needed to evaluate. Eds.. Vol. In another study. cosmid-size genomic DNA from slow-growing fungi was cloned and introduced into A. P.184 An et al. BAC clones containing soil DNA fragments (5–120 kb) were expressed in E. van den Hondel.. Our strategy included the following key steps: cosmid library construction. fungal secondary metabolic pathways is 34 kb and the A.A. Briefly. Dingemanse. In both cases. one needs less than 1500 clones to cover a typical fungal genome five times. In future discovery programs.. would be preferable if available.P. Transformation of Aspergillus based on hygromycin B resistance marker from Escherichia coli. In one study. a 24-well microplate fermentation procedure. it is possible to prescreen libraries for clones that contain DNA fragments homologous to known natural product-encoding genes to create so called biased libraries. and two statistical methods for data analyses (MD-PCA and SCP). coli and screened for antimicrobial activities. we constructed libraries for individual fungi. but the increase of genome coverage would result thousands of more clones to screen. D.M. Punt. Libraries containing larger DNA inserts. Soderstrom..E. R. . nidulans genome is 30 mb. P. B. Environmental and Microbial Relationships Wicklow.H.T..

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S. Extracts of these smaller-scale fermentations are prepared and screened in biochemical screens designed and executed by biochemists. HARRIS Merck Research Laboratories. the large size of most industrial screening programs requires significant technical support from computer and automation specialists simply to generate the hundreds of thousands of extract samples and to track the data generated. The chemically guided discovery of new fungal metabolites relies on chromatographic and spectroscopic tools to detect new or unrecognized compounds.A. The natural products chemist. natural products chemists employ chemical separation methodology to isolate the active component of the crude extract. compounds that would be missed using the chemically guided approach. These low-titer and biologically active metabolites have a good probability of being new or even novel. This approach is most useful from a chemotaxonomic perspective where the interest is in what metabolite(s) a particular fungus produces. 187 . determines the structure of the metabolite. Rahway. On detection of a desired activity. in collaboration with specialists in organic spectroscopy. Industrial bioassay-guided natural products programs are multidisciplinary teams of scientists working in close association. In contrast.8 The Isolation and Structure Elucidation of Fungal Metabolites GUY H. U. Finally. the focus of industrial natural products programs is almost entirely biological activity rather than interest in chemical novelty or chemotaxonomy. The purpose of this chapter is to provide a fungal metabolite centric overview of the natural products isolation and structure elucidation chemistry portion of this process. Mycologists isolate and culture fungal strains from environmental substrates. The cultures are then passed to fermentation microbiologists for initial smaller scale culture. New Jersey. The discovery of new fungal metabolites has been driven by the search for either novel chemical structures or for a desirable biological activity. The power of the biological activity–driven approach is the detection of extremely low-titer metabolites.

Disruption of the mycelia and solubilization of the compounds of interest is generally required for bioassay and secondary metabolite isolation. is approached systematically. The trial-and-error nature of the bioactivityguided isolation can be reduced if the entire process. A detailed description of each separation and structure determination method used by natural products chemists is beyond the scope of this chapter and would require volumes just for a meaningful overview. it is a useful model to understand the fungal metabolite isolation process. The isolation portion of the bioassay-guided isolation and structure elucidation process is often downplayed. the bias of the chapter is toward the isolation rather than structure elucidation aspect of natural products chemistry. In contrast. have decreased both the time and quantity of compound required for de novo natural product structure elucidation such that it is now more or less routine. As will be shown from a literature survey of fungal metabolite isolations. and varying amounts of water. and portrayed as time consuming. The result is that those not skilled in the art have few sources of information about how to undertake the isolation of a fungal metabolite. One such systematic approach is described below. This will be followed with a similar discussion of the dereplication and an overview of structure elucidation of the purified compounds. sufficient NMR data can now be acquired in a single overnight period on 1 or 2 mg of pure compound to allow proposal of a new structure. an industrially biased. has been composed of three overlapping subdisciplines through much of its existence: isolation. This perception is reinforced in the natural products literature. This three-step approach is not novel and has many similarities to protein isolation. more so than in academia. Often this is less material than required for an initial evaluation of the biological activity of the pure compound. and perhaps synthetic modification of natural products. ISOLATION OF FUNGAL METABOLITES Industrial natural products chemistry. The difficulties encountered during the isolation of a new natural product or the strategy used for its bioassay-guided isolation are rarely discussed.1. 1.188 Harris The intended audience is the nonnatural products chemist with a need to either isolate a fungal metabolite/bioactivity present in a fungal extract or simply to understand the process better. the bioactivity-guided isolation of natural products has not seen such major advances and in many cases is done the same way it was 20 or 30 years ago. The bulk of most literature reports of new natural products describe the details of the structure elucidation with perhaps only one or two paragraphs describing the isolation if it is not simply relegated to the experimental section of the report. from crude extract to pure compound. media components. 1. Instead. Rapid advances in the past 20 years in the spectroscopy of organic molecules. As will be evident. specifically nuclear magnetic resonance (NMR) and mass spectrometry. structure elucidation. misunderstood. general three-step approach to the bioassay-guided isolation of natural products will be described as a framework to highlight aspects of natural products chemistry unique to fungal metabolites. Today the most challenging of the three is the bioactivity-guided isolation of the pure compound. For example. Usually this involves . Neither is the goal to provide a general description of natural product isolation. It is hoped that the nonnatural products chemist reader can use this information as an introduction to the fungal metabolite primary literature. aside from the development and improvement of modern HPLC column materials. Extraction from Fermentation Broths Fungal cultures are complex mixtures of mycelia.

1. media components. Clarification can be accomplished using either filtration or centrifugation. dried. and the filtrate further processed as required. The solids are removed by filtration or centrifugation resulting in a clarified aqueous extract. Laboratory scale continuous flow centrifuges are available and enable the rapid separation of solids from whole-broth volumes of tens to hundreds of liters. inorganic supports or agar. Bioassay-Guided Isolation Process Bioassay-guided isolation of fungal metabolites from fermentation samples has much in common with assay-guided protein purification [1].2. The solids can be further extracted but this is rarely necessary. this extract is well suited to solid-phase extraction (SPE) on polymeric resins as described below. 1. Submerged liquid fermentations can also be directly extracted with a water immiscible organic solvent. The remaining aqueous layer can also be further extracted with a water miscible solvent for the recovery of polar metabolites as described above. Centrifugation. especially into water-immiscible solvents. Alternatively. submerged liquid or solid substrate. Extraction with a water-miscible solvent results in a mixture of cell debris. Alternatively. .The Isolation and Structure Elucidation of Fungal Metabolites 189 treatment of the fungal culture with an organic solvent followed by clarification to yield an extract. can affect the recovery of fungal metabolites as well as their stability. the solids removed by filtration. the properties of the extract. This is particularly effective if the desired component(s) are associated with the mycelia and the fermentation media does not contain insoluble components. Analytical samples. and the solvent removed in vacuo to yield a residue suitable for column chromatography. The organic phase is removed. Ethyl acetate (EtOAc) is commonly used for this purpose and efficiently extracts typical fungal secondary metabolites. The efficiency of extraction of ionizable components. the mixture is stirred or shaken for several hours. the organic solvent can be removed in vacuo and the resulting aqueous solution extracted with a water immiscible solvent chosen according to the polarity of the target compounds. and organic solvent that generally must be clarified before any chromatographic separation. The crude activity is evaluated for selectivity. volumes up to a few milliliters. and the available equipment and is summarized in Fig. Unbuffered fungal fermentations tend to be slightly acidic. is useful for volumes up to around 10 L.1 to 100 L. washed with water. Both begin with the detection of a biological activity in a crude preparation. and solid substrate fermentations extracted with an aqueous based solvent. Solid substrate fermentations. Submerged liquid fermentations can either be extracted whole or clarified and the resulting solids and culture broth separately extracted. using a commonly available laboratory centrifuge. The details of this process are determined by the physical properties of the compounds of interest and the type of fermentation. After solvent addition. is pH dependent. The only potential pitfall to this approach is emulsion formation or compound instability on concentration to dryness. Whole-broth extraction by addition of one to several volumes of a water miscible solvent such as acetone or an alcohol is common. Vacuum filtration is suitable for extract volumes of 0. Solvent choice depends on the solubility of the compound of interest. The choice depends on the volume of the extract. such as those on grains. must be extracted whole since the mycelia and support cannot be separated. The pH of submerged liquid fermentations. Since it is aqueous based. can be clarified using a high-speed benchtop microfuge or using syringe filters. the whole broth can be clarified by filtration or centrifugation prior to extraction. The harvest pH of liquid fermentations should be noted prior to extraction and provides a reasonable starting point for component stability.

1. Fortunately this source of instability can be controlled by conducting extract fractionation at the pH of optimum stability for the desired compound or activity using a buffer. as required to determine interest. 1. The lipophilic character of most fungal metabolites can cause solubility problems in an aqueous bioassay. The most common of these for fungal metabolites is pH-dependent instability/stability. specific activity. Biological Assay Interferences Most of the reported fungal metabolites are generally soluble in organic solvents and aqueous-organic mixtures. etc. Although this phenomena has not been studied specifically for natural products and natural product extracts its occurrence is plausible. A sequence of separation methods for fractionation are chosen to maximize activity recovery while simultaneously increasing the specific activity of the preparation until a chemically or biochemically homogenous sample is obtained. in the isolation process is that the ionic form of any ionizable metabolites should be understood. Free fatty acids are ubiquitous . 1. Assay solubility problems can change as the active component is purified through the removal of impurities that can either aid solubility or themselves cause insolubility. The solubility of fungal extracts and fractions is not a general problem for chromatographic separations but it can be for bioassay. The disadvantage of using buffers for small molecule separations is that desalting of the final product is required prior to structure elucidation. A further advantage of the use of buffers.2. It has recently been shown that the activity of some nonselective and moderately active pure compounds commonly found in industrial compound collections was due to physical aggregation phenomena rather than true biological activity [2].2. potency. Fractionation of the crude preparation or extract is then guided by the assay.2. Crude fungal extracts may contain compounds that are not specifically active in an assay but interfere with bioactivity detection in some way.190 Harris Figure 1 Useful sample volume ranges for common extract clarification methods. Metabolite Stability and pH Control Protein purification and fungal metabolite isolation also share some frequently encountered difficulties (Table 1) that can result in loss of biological activity. especially if high concentrations of material are required for activity. or at least some form of pH control.

.g.g. One IC50 unit is the amount. unsaturated fatty acids may be real.e. The total number of units of activity in the crude extract can be measured by titration of the sample and determination of an IC50. PMSF) added to buffers Concentration to dryness Not commonly performed for proteins Usually not a problem Metal complexes of fungal metabolites are known Not a common problem since nonribosomal peptides are generally less suseptible to nonspecific proteases Presence of proteases in fungal crude extracts can interfere with peptidebased bioassays (i. 1. Strongly (UV) adsorbing and or flourescent fungal metabolites can quench or otherwise interfere with assay methodology involving detection of a fluorescent substrate. targeting protease inhibitors) Can be problem for reactive compounds (i. DTT) added to prevent oxidation of thiols Chelating agents (EDTA) added to remove nonspecific metals Protease inhibitors (e.2. Activity Quantitation Bioassay-guided isolation requires a system to quantitate activity.3.e. A.The Isolation and Structure Elucidation of Fungal Metabolites 191 Table 1 Common Problems of Protein Purification and Fungal Metabolite Isolation Problem pH Protein Purification Buffered for stability and optimum activity Detergents added to reduce hydrophobic interaction Fungal Metabolite Isolation No pH control Control of ionic form Buffered for stability Added agents not necessary—either organic or aqueous solvents can be used Solubility problems sometimes encountered when preparing samples for bioassay Aggregate effects? Oxidation not limited to thiols Solubilization Oxidation prevention Metals Proteases Reducing agents (e.. One such system is based on the IC50 of the crude extract and its various subfractions. A more detailed discussion of assay methology as well as some of these issues is presented elsewhere in Chapter 10. actives that can mask or hinder the detection of active secondary metabolites.001 to 1 or 2 mg/L or more in fungal extracts. Fungal enzymes. but undesirable. as follows: V Total IC50 units sample (1) IC50A . of a sample sufficient to be active at the measured IC50. Depending on the assay system. can lead to degradation of peptide substrates used for protease assays. particularly proteases. The total number of IC50 units can be defined for a single active component... polyacetylenes) and are found in titers ranging from less than 0. weight or volume.

For example. to the above equations. for a total activity recovery of 83. respectively. t. containing components A and B.020 mL WBE/ mL and 0. suppose the entire sample was subjected to column chromatography on silica gel. with IC50s of 0. The total activity recovered would be 50. The resulting activity recovery from the column would be 83.000. was sufficient to yield the an IC50 (IC50 0. initial. then the total IC50 units of the sample would be 100. extract volume. The above equations can be extended to handle unstable components. (1) becomes: (Total IC50 units sample)t [A]0 exp( kt) V IC50A (3) where the initial concentration of component A [A]0 k the rate constant for the decomposition of component A Similarly. Equation (4) is useful.333 IC50 units. B. and a relatively unstable compound.192 Harris where V the total.3%. if 0. As an approximation we can assume that an unstable component decomposes according to first order reaction kinetics and add a concentration term. consider a hypothetical mixture of a stable compound. The activity recovery from the column can be determined by measuring the IC50 of the combined active fractions (rich cut) containing component A and comparing it with the total IC50 units of activity with that applied to the column. A.333 IC50 units. at any given time. Eq. Next. In the above example. .: Total IC50 units sample V IC50A V IC50B … (2) As an extension of the above example.010 mL WBE/mL). suppose the silica gel fractionation of the extract resulted in two rich cuts.000 and 33. if the combined active fractions exhibited an IC50 of 0. a common occurrence.010 mL of a crude extract. can be treated by an extension of the same method. Measurement of total solids of each rich cut can provide information about the relative potency of the two components.012 mL WBE/mL the total activity recovered would be 83. Multiple active components.333 total IC50 units.030 mL WBE/mL. The total IC50 units present in the original extract is now the sum of the activity from each of the active components. (2) becomes: (Total IC50 units sample)t [A]0 exp( kt) V IC50A [B]0 exp( kt) V IC50B … (4) In reality it is not possible to know the rate constants for the decomposition of individual components of an active mixture. A. B. at least in the early stages of characterization. Thus. etc. especially when coproduced with other metabolites. of total volume 1000 mL (1000 mL WBE). respectively. in mL whole-broth equivalent (WBE) measured value for the extract in units of mL extract/mL assay volume IC50A For example. Eq. The specific activity enrichment can be determined from total solids measurements of both the crude extract and the column rich cut. to illustrate the difficulty of detecting and isolating unstable fungal metabolites. however.

Details of component stability. The corresponding IC50 of the mixture increases. by only 2-fold (Fig.3) that resolves the two activities. This is particularly alarming because a 2-fold loss of activity in many bioassays might be attributed simply to assay variation resulting compound B being missed entirely. or any natural product. and relative concentration are given in the text.The Isolation and Structure Elucidation of Fungal Metabolites 193 with the following properties: A Titer at t IC50 Half-life k Volume 0 0.1. Once resolved from compound A the instability of compound B should be easier to detect and then ways to control it can be explored. The solution to this dilemma is an effective initial fractionation of the crude extract (see section 1.001 mg/mL 3.2 1000 mL The titers and activities above were chosen so that A is present at a 10-fold higher titer than B but also so that B is 10-fold more active than A. 1).3.47 days 0. activity. fungal metabolites. 2 and 3. Fungal Metabolite Isolation as a Three-Step Process 1.3. The isolation of any fungal metabolite is thus a multistep process Figure 2 Observed time-dependent potency (in mL/IC50 unit) of a hypothetical mixture of unstable (A) and stable (B) components. The effect of the unstable activity of compound B on the potency and total activity of the initial mixture over time is shown in Figs. becomes less active. The Art of Fungal Natural Product Isolation As a general rule. 1. cannot be purified to homogeneity in a single step.1 mg/mL 0.01 mg/mL 347 days 0.002 1000 mL B 0. Complete decomposition of compound B only decreases the total activity units of the mixture by 2-fold (Fig.01 mg/mL 0. . 2).

three-step approach to the isolation of fungal metabolites is presented below. and maybe even unique. orthogonal (see below). Each of the steps has a predetermined purpose. involving several different chemical separation methods. a chromatographic peak may be homogeneous by UV adsorption detection but a mixture if analyzed with mass spectroscopic detection. components of a crude extract. as well as other natural products. If. and relative concentration are given in the text. Biochemists have developed generally applicable. If the major. to be a mixture of two or more components. Crude extracts are complex mixtures of primary and secondary metabolites as well as fermentation media components. An elegant natural product isolation might be one that employs two or three well designed. is similar to the isolation of proteins. The individual amounts of each can range from trace to 50% of the total solids. or the number of total steps used.194 Harris Figure 3 Observed time-dependent total activity (as measured in IC50 units) of a hypothetical mixture of unstable (A) and stable (B) components. however. As previously noted. the minor component(s) are as important as the major component until further resolved. easily reveal the major. Three distinct steps in the isolation of a fungal metabolite are described below: . is a relatively objective thing that can be measured by the time required. For example. chemically detected component is the one of interest then the minor components are generally of little consequence. Analytical separations with chemical detection. the overall yield from the crude extract. If one accepts that a fungal metabolite cannot be purified in a single step then it is instructive to use the same framework to examine the entire fungal metabolite isolation process. Efficiency in natural product isolation. multistep approaches to protein isolation. It is not uncommon for an apparently pure HPLC peak in a chromatogram of a crude extract. the isolation of fungal metabolites. separations resulting in a high yield of a relatively minor fungal metabolite. Detection of the minor components of such a mixture depends on the methodology applied to the sample. Details of component stability. and even intermediate concentration. Elegance in isolation is also analogous to elegance in natural product total synthesis and is a largely subjective thing. activity. There is not a correct way to isolate a specific fungal metabolite but there are processes that are more efficient and elegant than others. biological activity is the detector applied to such a mixture. A systematic. It is the low level and trace components that complicate purification of fungal metabolites. or other partially purified sample. just as in natural product total synthesis. such as UV adsorption.

2. Multiple active components. Reproducibility. The goals of the recovery and resolution step require only moderate chromatographic resolution. Complete recovery of the desired biological activity. selection criteria for methodology. Scalability. 2. This allows processing of relatively large numbers of active extracts to identify the smaller number of true interest for complete isolation and structure elucidation. and step 3. recovery and resolution. the separation methodology must be chemically gentle. a substantial increase in the specific activity of the active component(s) should result. purification. Step 1: Recovery and Resolution Goal The initial separation of a fungal extract is the most important step in the isolation process. Second. First. step 2. The purpose. 4. of each activity is extremely useful for characterization of an activity observed in a screening extract. solubility can be limited. Partially purified samples are generally more compatible with secondary evaluation assays than crude screening extracts. For example. even more importantly. Initial evaluation of bioactive fungal extracts is generally performed at a small probe scale (5–50 mL culture). As seen above. and conditions for optimal stability may be unknown or poorly defined. 1. The bulk of these solids are media components. which require removal prior to purification of the metabolites of interest. and commonly used separation techniques are discussed below and summarized in Table 2. To ensure the highest recovery of active component(s). 3. polishing. To use this information effectively the process must be reproducible. the process should provide a biological resolution of multiple active components. whether an active component is detected at all. are frequently produced by fungal fermentations. Methodology Constraints The recovery and resolution step is more demanding of separation methodology than the downstream isolation steps. . to allow isolation of native species. related and unrelated. first-step separation method should have the following characteristics: 1.3. resolution of an unstable activity from a stable coproduced one may be required to allow its initial detection. polymeric reverse-phase materials generally yield greater solute recovery than those based on silica. Chromatographic retention behavior in a standardized initial fractionation process can be exploited to group extracts containing the same component. Thus the choice of generally useful techniques is restricted since at the crude stage relatively little is known about the physiochemical properties of the active component(s). the extract volume to be processed is usually large. Moderate resolution. A biologically pure sample. partially fermented. or be compatible with buffers to allow control of the ionic form of components or for optimal stability as necessary. A general. This step has two purposes.The Isolation and Structure Elucidation of Fungal Metabolites 195 step 1. The separation methods should be effective at the extract pH. The degree to which these two goals are achieved often determines the difficulty of the remainder of the isolation process and sometimes. Fungal fermentations on complex substrates or in undefined liquid media yield extracts that are usually high in total solids. Solid-phase adsorbants should be chosen for maximum solute recovery. A typical primary screening assay yields tens to even hundreds of active extracts. containing only one active component. Direct scaleup of the probe methodology to volumes of several liters of culture eliminates the need to pilot a new process for the initial large-scale isolation.

Recovery and resolution Weight enrichment of activity Removal of media components and primary metabolites 2. physical properties of active component(s) 3. Purification Biological resolution of multiple active components Purification of active component(s) Associate activity with extract component(s) Reproducible Automation potential Orthogonal to step 1 Polarity.196 Table 2 Natural Product Isolation as a Three-Step Process Methodology Purpose High recovery of solutes/activity Scalable Moderate resolution Selection Criteria Commonly Used Techniques Step 1. Polishing Preparation of final pure compound Generally high resolution Sequential liquid partition Differential pH partition Adsorption/elution on polymeric adsorbants Countercurrent chromatography (CCC) Sephadex LH-20 chromatography Normal phase column chromatography Medium resolution reverse phase chromatography Ion exchange CCC (all modes) Sephadex LH-20 chromatography Preparative reverse-phase HPLC Crystallization CCC Harris .

Controlling the pH of a liquid–liquid partition also allows control of the ionic form of a metabolite. Typically. also known as . are usually obtained. The pH of the aqueous solution can be adjusted as necessary for more efficient extraction of ionizable metabolites. Particle size choice is based on the scale of the separation and the resolution desired during elution. Good increases in specific activity. Industrial screening programs typically yield tens to hundreds of active crude extracts depending on the size of the extract library.g. Repetitive execution of the initial isolation step for such large numbers of extracts is not only less tedious but also more robust and reproducible if it is automated. methylene chloride.The Isolation and Structure Elucidation of Fungal Metabolites 197 5. This is accomplished by either dilution of the extract with water or removal of the organic solvent component in vacuo. The distribution coefficient of ionizable fungal metabolites subjected to liquid–liquid partition can be altered by varying the pH. methanol or acetone. After sample loading the resin is thoroughly washed to remove unadsorbed impurities. Concentration of the organic phase is easy and the resulting residue is ready for column chromatography. The most useful for the general extraction of fungal metabolites from aqueous crude extracts are neutral polystyrenedivinylbenzene polymers. Smaller particle sizes favor resolution and are generally used for smaller samples and combined with gradient elution. The can be done in steps or with a gradient. The industrial use of these resins for solid-phase metabolite capture has been described. is removed by concentrating the crude extract in vacuo and then the mostly aqueous solution is extracted with an organic solvent. Advantages to it are simplicity. provides little resolving power and occasionally distributes a specific metabolite in more than one solvent. Even the use of a sequence of increasing polar solvents (e.. Automation potential. An aqueous alcoholic or acetone clarified extract is adjusted to a sufficiently low organic modifier strength to allow adsorption of the desired component(s) to the resin. hexane. high recovery. Multiple extractions at different pHs. A disadvantage is that little or no resolution of multiple active components is obtained. and is relatively polar. Larger particle sizes are typically used for larger scale processing of tens of liters or more. the pH of the aqueous phase can be lowered a pH unit or more below the pKa of the acidic moiety of the compound. A sequential series of solvents of increasing polarity and corresponding ability to extract metabolites can also be used [3]. The typical aqueous methanol or aqueous acetone clarified fungal extract is suited to SPE. Generally Useful Separation Techniques SOLID-PHASE EXTRACTION. Ethyl acetate is a good general solvent for liquid-liquid extraction since it is easily dried. Liquid–liquid extraction is not the perfect solution to the recovery and resolution step. LIQUID–LIQUID EXTRACTION. has a relatively low boiling point. followed by ethyl acetate). Polymeric adsorption resins fulfill all of the criteria for the recovery and resolution step listed above. The lipophilic nature of most fungal metabolites allows their recovery from aqueous methanol or aqueous acetone fermentation extracts by extraction into an organic solvent. The separation mechanism on a high-quality resin is almost entirely adsorption. when compared with the total solids of the aqueous methanol or acetone extract. the organic extraction solvent. Similarly. to favor the distribution of acidic compounds into an organic phase. and the chemically gentle nature. A wide range of particle sizes and polymer crosslinking is available. The desired components) or activity is then eluted with an increase in the organic solvent component. For example. the pH of the aqueous phase can be increased above the pKa of basic compounds to favor distribution into the organic layer.

Sephadex LH-20 has also been used in a similar fashion. Ion exchange resins function predominantly through ionic equilibria between solute and stationary phase and are either anionic. Both techniques are most effective when an organic extract of a crude fermentation is the feed to the separation since they require lower volume sample application. nonpolar samples soluble only in hydrocarbon solvents are poor candidates for reverse-phase separations and. such extremes are not the rule. components can have wildly different apparent polarity behavior in a separation method than might be expected from the gross solubility of the sample. METHODOLOGY CONSTRAINTS. High specific activity gains were achieved and the retention information was useful for dereplication (see below). the purification step. A bewildering number of chromatographic stationary phases exist for the purification fungal metabolites. These (see methodology descriptions below) have also been effectively used for recovery and resolution although literature reports of their use for this purpose is rare. . Few absolute constraints exist for the choice of which separation method(s) to use but three factors can be useful when evaluating potential methods: (1) sample solubility/component polarity. a much smaller sample volume. was effectively used in a parallel fashion to fractionate small ( 100 mg) samples of 2-butanone extracts of fungal fermentations [4]. A good portion of the art of bioactivity-guided natural product isolation lays in the careful selection of one or two separation methods for the purification step. solid supported stationary phases are either predominantly through partition or adsorption. Purification Goal Successful execution of the recovery and resolution step results in a sample with improved specific activity. A specific form of CCC. and (3) the orthogonality of the separation method to the one used previously in the recovery and resolution step. For example.2. dual-mode. Countercurrent Chromatography and Sephadex LH-20. can be a highly effective way of increasing the specific activity of ionic compounds. 1. Sample solubility in a solvent suitable for applying the sample to the separation method is a general requirement and can guide method choice. from plant crude extracts. conversely. a reverse-phase separation method or perhaps ion exchange (see below) might be the first choice.198 Harris differential pH extraction. (2) the presence of ionizable functional groups in the component of interest.3. or mixed mode. for nonpolar separations a normal phase mode technique such as silica gel column chromatography might be a reasonable choice. generally secondary or tertiary amines. Step 2. Thus. Both techniques fulfill most requirements for the recovery and resolution step with direct scaleup the only potential disadvantage. Both operate in either normal (polar stationary phase) or reverse-phase (nonpolar stationary phase) modes. Sample solubility and component polarity are loosely associated. Occasionally. Fortunately. Mechanistic interactions of solutes with neutral. cationic. How does one choose a sequence of separations? To simplify method selection it is important to remember that this large number of stationary phases exploit only a limited number of actual separation mechanisms. The goal of step 2. Similarly. This technique has been used for over a hundred years to prepare crude alkaloid preparations. more defined solubility properties. and probably better characterized biological activity. samples soluble only in water or alcoholic solvents are poor feed samples for silica column chromatography. for the polar sample. is to associate the biological activity of this active material with a specific component or components while simultaneously increasing its purity.

making a second purification step. Figure 4 Separation of a hypothetical mixture of components A–G (panel 1) using a nonorthogonal separation method. Such combinations of separation techniques can be viewed as having orthogonal mechanisms. panel 2 might result. and third overall isolation step challenging. 5.The Isolation and Structure Elucidation of Fungal Metabolites 199 Sequential combination of purification methods that exploit completely different separation mechanisms is the most effective strategy for fungal metabolite purification in the minimal number steps. 4 and 5. Examples of orthogonal pairs of separation methods are silica gel adsorption chromatography followed by reverse-phase partition chromatography or reversephase adsorption chromatography on polymeric resins followed by ion exchange. B. such as normal phase adsorption chromatography on silica gel. and E as well as C and is likely to be of lower specific activity than that obtained after the hypothetical RP HPLC step above. the hypothetical rich cut mixture shown in Fig. . If this mixture is then subjected to reverse-phase HPLC. the resulting active rich cut is likely to be similar to that illustrated in Fig. B. F–H. if the same rich cut from the resolution and recovery step is further fractionated with an orthogonal separation method. for the purification step. 4. Panel 1 of each figure illustrates a hypothetical rich cut obtained after a reverse-phase column chromatography Resolution and recovery step applied to a crude acetone fermentation extract (as analyzed using analytical reversephase HPLC). The effect of using a purification step separation method orthogonal to that used for the recovery and resolution step is illustrated using the example shown in Figs. Components A. This mixture contains a broader polarity range of compounds including A. the most difficult separation problem. a nonorthogonal separation method. and some of E were removed and the specific activity of rich cut is higher but the resulting rich cut still contains compounds D and some of E. Importantly though. in contrast. panel 2. Compound C is the target acompound. the most challenging separation problem for reverse-phase HPLC.

preparative thin-layer chromatography (TLC). Retention of solutes is controlled by the mobile phase polarity and the relative elution strength of various organic solvents is known as an elueotropic series. Most forms of silica chromatography were represented and included classical. Normal-phase adsorption chromatography on underivatized silica gel has been routinely employed for the isolation of fungal metabolites. This general utility correlates with the nonpolar to moderately polar. or a polar derivative of silica gel. Normal-phase mode chromatography on polar stationary phases is perhaps the most commonly used separation method for fungal metabolites. has been eliminated since the silica gel separation completely removed compound D from the active rich cut. organic soluble.to 63. is the most frequently used stationary phase and is combined with organic mode phases ranging in polarity from hexane to methanol. For example. approximately 60% of the new fungal metabolite isolations reported in the Journal of Antibiotics between 1997 and 2001 used silica gel chromatography at some point during the purification sequence. Exceptions are generally due to the presence of ionizable functional groups in the solute (carboxylic acids and amines). a second purification step and third overall isolation step using preparative reverse-phase HPLC should yield pure compound C. open-column. medium pressure liquid chromatography (MPLC). The reader .200 ´ Pelaez Figure 5 Separation of a hypothetical mixture of components A–G (panel 1) using an orthogonal separation method. Silica gel. Generally. the more polar the solute the stronger the adsorption. Thus. small molecule character of the majority of reported fungal metabolites. Adsorption interaction of solutes is predominantly through hydrogen bonding with the silanol groups of the silica gel. and HPLC. gravity-fed separation on 40.m particles. Generally Useful Separation Techniques NORMAL-PHASE ADSORPTION CHROMATOGRAPHY. compounds C and D.

increases with decreasing particle size of the packing material.01% to 1% of an organic acid. cost and resolution is often obtained using an intermediate particle size reverse-phase packing.or polycarboxylic acids. and the corresponding equipment demands. For the three-step strategy described herein the highest possible resolution is not always necessary for the purification step. Fungal metabolites present few unique problems for the application of typically practiced reverse-phase chromatography. a separation employing a gradient from 30% to 90% acetonitrile requires the feed sample to be dissolved in a relatively small volume of an aqueous solution of 30%. in step gradient mode (stepwise increases in percentage of organic solvent) or using a gradient (continuously. this may not be possible. acetonitrile. such as formic acid or trifluoroacetic acid to acidify the mobile phase.g. usually linear.. this is used in most fungal metabolite isolations.. mono. Its use has been reported for a small percentage of fungal metabolites (Table 4). The best combination of separation scale. Reverse-phase chromatography media are either a silica or a polymer bead derivatized with a lipophilic bonded phase. however. For typical preparative reverse-phase separations the sample is applied to the column in a solution of lower eluting strength than the initial mobile phase composition. and nonfunctionalized Amberchromes) are also used for reverse-phase chromatography. Hamilton PRP-1.The Isolation and Structure Elucidation of Fungal Metabolites 201 is referred to the numerous literature descriptions of the mechanism and practice of normal phase adsorption chromatography for complete information [5]. Silica gel is slightly acidic in character due to the presence of particularly active silanol residues. Depending on the amount of sample. its purity. The primary concern is the solubility of the crude or semipurified fungal extract. The simplest is to dissolve the sample in a more nonpolar but water miscible organic solvent such as tetrahydrofuran or DMSO. amines) in a molecule can result in stronger retention than expected from observed solubility. tetrahydrofuran and isopropanol are occasionally used. Selectively in reverse-phase separations can be controlled by changing organic modifier and the pH of the mobile phase.g. The presence of ionizable functional groups (e. Mitsubishi HP series. to use of a buffer such as potassium phosphate. For example. the Rohm and Haas XAD series. but the separation mechanism is adsorption rather than partition. An alternative is to dilute the sample to be . Solute retention in reverse-phase chromatography is determined by the percentage of organic cosolvent in the mobile phase. In one or more of its forms. and the solubility of the mixture components. speed. Large selectivity changes within a crude or semipurified sample can be affected by pH modification. The separation mechanism on bonded phases such as octyl (C8) or octyldecyl (C18) is generally partition. Diol silica can be an alternative for such problematic compounds. Two strategies exist for sample application in solubility limited situations. REVERSE-PHASE CHROMATOGRAPHY. or less. This can be as simple as adding 0. Aqueous acetonitrile or methanol is typically used as a mobile phase with eluent strength increasing with increasing organic content. The same mobile phases are used as for the bonded phase materials. Separations of ionizable fungal metabolites are generally more reproducible if the pH of the mobile phase is controlled. This solution is then diluted with water or buffer to an aqueous composition equivalent to the starting column eluent. Resolution. The commonly used organic modifiers are acetonitrile and methanol. Underivatized neutral polymeric resins (e. How the reverse-phase separation fits (its orthogonality) with the previous and subsequent steps in the isolation is of greater importance than resolution. This can lead to decomposition of acid sensitive metabolites and irreversible adsorption of solutes. Separations can be performed isocratically (a fixed mobile phase composition). Reverse-phase chromatography can be performed in forms ranging from low-resolution open columns and vacuum bed separations to high-resolution HPLC. increasing organic composition).

This powerful technique is widely used for the isolation of classical. The relative rarity of cationic fungal metabolites makes this a powerful purification method if the target compound contains an amine. these magic separations cannot be predicted. it is an extremely gentle technique and high recovery of solutes is typical. footnotes). compounds have occasionally been purified to near homogeneity from crude organic extracts after a single LH-20 fractionation. is below the fractionation range for such separations. ION EXCHANGE. in effect. Irreversible adsorption is uncommon and no reactive functionality is inherent to the polymer.202 Harris separated in a large volume of a solution with an organic modifier content significantly below the initial column eluent. However. The basic Sephadex polymer is a crosslinked polydextran typically used for gel filtration separation of proteins. Ion exchange can be a highly selective tool for the enrichment of families of fungal metaboites as was demonstrated by the use of the weakly basic anion exchanger AG4 4 to recover low levels of zaragozic acids from crude fungal fermentations [7. can be retained on various anion exchange resins. maybe more so. Commonly encountered mono-. Conversely. The column is then thoroughly washed to remove unretained material and then eluted normally. Almost . typically less than 1000 daltons. basic compounds are excellent candidates for retention on cation exchange resins. a crude organic extract can be passed through LH-20 with little or no gain in specific activity of the active component(s). COUNTERCURRENT CHROMATOGRAPHY. This solution is applied to the column using the column as a SPE. CCC has been referred to as the support-free liquid stationary phase [9]. a combination of partition and adsorption mechanisms dominate with the contribution of each mechanism for a particular compound loosely dependent on the number of polar functional groups and the mobile phase polarity [6]. Nevertheless. First.and tricarboxylic acids. The lack of water solubility can be overcome by the addition of an organic cosolvent to the ion exchange mobile phase. SEPHADEX LH-20. Rather than size exclusion. Classical gel filtration is rarely employed for the isolation of fungal metabolites because their relatively small molecular weight. Compound elution from LH-20 is typically broad as a result of the complex separation mechanism and the relatively large particle size. Mobile phases of methanol. This reveals one of the significant problems with its use. methylene chloride:methanol. as for silica based materials. Ion exchange–based separation methods can be exploited for fungal metabolites with a net anionic or cationic character. many natural product chemists view LH-20 as a no lose technique. packing the sample onto the top of the bed. The infrequent use of ion exchange for fungal metabolite isolation is likely due to the fact that it is regarded as a technique for water-soluble molecules and thus generally incompatible with the typical organic soluble fungal metabolite. water-soluble antibacterial agents from bacterial sources but it is rarely used for the isolation of fungal metabolites (see below). LH-20 has two advantages for the isolation of fungal metabolites. Just as frequently. the Sephadex LH-20 and LH-60 variants are unique in that they are hydroxypropyl ether derivatives of the basic Sephadex polymer to create a more lipophilic stationary phase suitable for use in organic solvents and have found wide application in natural product purification. the magic separation. That is. or methanol:methylene chloride:hexane are typically used (Table 4. The separation mechanism for small organic molecules on LH-20 is complicated and not well studied.8]. CCC is true liquid–liquid partition chromatography. Sample loading is generally lower than with classical partition or adsorption methods but guidelines based on rigorous study are not available. for want of a better terminology. and especially di. is sometimes obtained. Second.

Specialized instrumentation is required and the learning curve is relatively steep when compared to more classical forms of chromatography. dual-mode. Usually the target compound is characterized by at least a chromatographic retention and UV spectrum. CCC has general utility for all three isolation steps and has been underused for fungal metabolite isolation. An almost infinite number of biphasic solvent systems can be conceived of and the partition behavior of the bioactivity or compound of interest can be characterized before separation in the instrument. This coil is rotated in a planetary motion. Particle sizes range from 3 to 5 m spherical used for analytical (column diameters 2. a rotor containing numerous chambers interconnected by small channels is rotated in a centrifuge retaining one phase.3. within the centrifuge like instrument generating the hydrodynamic fluid mixing responsible for the separation. Two major types of instruments exist for this purpose: the multilayer coil planet centrifuge (MLCPC) and the centrifugal partition chromatograph (CPC). Preparative reverse-phase HPLC is the highest resolution separation method available for fungal metabolite isolation and is unquestionably the most commonly used last isolation step. In the CPC. This aids in optimization of the polishing step separation. This variation in surface chemistry can result in relative selectivity variations for a given mixture of components on different column materials. 1. Generally Useful Methodology PREPARATIVE REVERSE-PHASE AND NORMAL-PHASE HPLC. . which are no doubt responsible for its uncommon use for fungal metabolite isolation.3.1–4. CCC has some disadvantages. The reader is referred to the references herein for a detailed description of these techniques.The Isolation and Structure Elucidation of Fungal Metabolites 203 any biphasic mixture of two or more solvents can be used. hence the name. Hundreds of different reverse-phase HPLC bonded phases are commercially available. but the most commonly used for fungal metabolite isolation is either octadecyl (C18) or octyl (C8). as can be seen from Table 4. The variation in C8 and C18 bonded phases from different manufacturers derives from the physical characteristics of the silica used. and the particle size [12]. Polishing Goal The purpose of the polishing step is to provide pure compound for structure elucidation and biological evaluation. wound in several layers on a spool. and the mobile phase is pumped through it [10. the density of the carbon loading. Step 3. three modes of operation are possible: classical continuous elution chromatography.11]. It is extremely gentle because the problems of a solid stationary phase support are eliminated resulting in complete sample recovery. This selectivity variation can be exploited for the optimization of a particularly difficult separation. One of the two phases is retained as a stationary phase using centrifugal force within a centrifuge-like apparatus. the type of end-capping used to mask unreacted silanol residues. A solvent system is selected that results in approximately equal distribution of bioactivity or compound between the two phases. Finally.6 mm) and small quantity preparative separations to 12 to 15 m used for large scale preparative HPLC (column diameters 22 mm). hydrostatic. typically Teflon 1. Most biphasic solvent systems can be used in either normal or reverse-phase modes.68 mm inside diameter 100 m long. and pH zone refining. The column within the MLCPC consists of a long coil of tubing. CCC has many advantages for fungal metabolite isolation. This step is generally the highest resolution or most selective step in the isolation process and provides relatively modest gains in specific activity.

GR135402 [16]. and one water-miscible solvent. the solvent system can be optimized using small scale partition experiments prior to actual separation of the sample in the column [4]. the reported isolation procedures for sordarin. A wide range of natural product isolation chemistry has been described for this family encompassing the initial discovery isolation and structure elucidation of members of the family through the large scale production of sordarin for synthetic modification. were used for fermentation extraction in the eight examples. Sordarin is typically distributed to some degree between both the culture filtrate and mycelia at harvest.204 ´ Pelaez CRYSTALLIZATION. Crystallization is the method of choice for a polishing step in the isolation of a fungal metabolite. highly selective inhibitors of protein synthesis in fungi (see Chap. neosordarin [17]. and the process can be scaled.5. Pharmaceutical industry interest in this biological activity has lead to considerable medicinal chemistry effort to improve the potency and pharmacologic profile of this family. As a case study in practical natural product purification. all modifications of the sordarose carbohydrate portion of the molecule. assuming suitable crystals are obtained. Both examples using a water immiscible solvent were followed by a normal-phase recovery and resolution step whereas the six water-miscible solvent extractions were all followed by a reverse-phase recovery and resolution step. and an unnamed 3′-oxoketal sordarin [18] (structures shown in Fig. Obtaining a crystalline product can be challenging and is especially difficult with the initial. The use of various reverse. Disadvantages of CCC include scaleup. Case Study: Isolation of Sordarin and Related Derivatives Sordarin is a novel diterpene glycoside with good in vitro antifungal activity against a variety of fungal pathogens [13]. Crystallization is actively sought for process scale separations of fungal metabolites of high interest. For sordarins. and high recovery. 6) were analyzed in terms of the three-step isolation protocol described here (Table 3). and (3) pH zone–refining CCC. The single carboxylic acid moiety is itself unremarkable but the highly pH-dependent solubility of sordarin has . Advantages of CCC as a polishing step are the ability to use either normal or reverse-phase operational modes. have been reported.or normal-phase chromatography steps for both the purification and polishing steps of the eight compounds is also typical. Members of this family are potent. at least a dozen new sordarin analogues. Atypical for fungal metabolite isolation is the exploitation of the ionic character of the sordarin aglycone. 11). if grams of compound are required. few-milligram sample of a new compound. Fungal metabolites do not require special treatment when compared with natural products from other sources. Two water-immiscible solvents. and three different 3′-sugar derivatives. This is particularly true as a fermentation ages and some of the mycelial lysis occurs. SCH57404 [15]. This is more or less a typical workup for fungal natural products since most of the large scale capture methods rely on either polymeric or silica based reverse-phase materials compatible with aqueous alcoholic extracts. and the time required for development of a solvent system for the separation.3. For use as a polishing step. COUNTERCURRENT CHROMATOGRAPHY. since an analytical assay for the target compound presumably exists. This was exploited in three ways in the eight examples: (1) differential pH extraction. Since its discovery in 1970. (2) ion exchange. The more lipophilic 3′-derivatives are typically associated to greater amount with the mycelia than sordarin itself. the metabolite is obtained in a solid form. of allowing structure elucidation using x-ray diffraction. methylethylketone or ethyl acetate. methanol. This has several different operational modes as described above. 11-hydroxysordarin [14]. separation of the mycelia and culture broth prior to extraction is determined more by the initial isolation step than by the actual product distribution. Crystallization has the added benefit. ease of compound recovery from the mobile phase. Product purities are high. 1.

3’-oxoketal sordarin derivativel (5). 1. Literature Survey of Recent Fungal Metabolite Isolations The isolation procedures reported for approximately 350 novel fungal metabolites described in the recent (1998–2002) literature were analyzed in terms of the three-step process of fungal metabolite isolation described above. although hydroxylation of the diterpene reduced the methylene chloride solubility and the more polar solvent ethyl acetate was substituted. Ion exchange was extremely effective for the isolation of the 3′-oxoketal derivative. 11-hydroxysordarin (4). This pH-dependent solubility was recognized early and used in the initial isolation of sordarin. pH zone–refining CCC was effectively used to separate sordarin and 11-hydroxysordarin on a multigram scale [19]. SCH57404 (2). is soluble in various organic solvents including dichloromethane. Differential pH extraction was not useful for any of the 3′ester derivatives due to the reduced aqueous solubility and stability of ester. The salt form. Differential pH extraction of the 11-hydroxy analogue of sordarin was also possible. present under acidic conditions. This technique relies on the same pH-dependent solubility as differential pH extraction but in a zone-refining operational mode [20]. and methanol. has much reduced solubility in the above organic solvents and exhibits significant water solubility. ethyl acetate. This information is summarized in Table 4 and provides an unbiased sample of the actual methodologies currently being . The selectivity of the weak anion exchanger AG4 4 (formate cycle) was exploited to selectivity retain and separate diacids from monocarboxylic acids. been exploited for its purification.3. Finally. neosordarin (6).6.The Isolation and Structure Elucidation of Fungal Metabolites 205 Figure 6 Structures of sordarin and related natural products: sordarin (1). The free acid form of sordarin. sodium or potassium for example. a dicarboxylic acid sordarin analog. GR135402 (3).

initial workup Step 1. Resolution and recovery Reverse-phase capture Mitsubishi HP-21 Step 2. upflow mode Step 2a. initial workup Step 1. Polishing Countercurrent chromatography pH zone-refining mode Reverse-phase chromatography Preparative RP HPLC–C8 Reverse-phase chromatography Preparative RP HPLC–C8 Reverse-phase chromatography Preparative RP HPLC–C8 . Resolution and recovery Countercurrent chromatography (CCC) Normal-phase. Purification Differential pH extraction Ion exchange BioRad AG4 4 weakly acidic ion exchanger Step 2b. step gradient elution (CH3CN:H2O) Reverse-phase chromatography Partisil BioPrep P40 ODS-3 (500 g)–isocratic elution Normal-phase adsorption chromatography Kieselgel Step 3. dual-mode Ion exchange BioRad AG4 4 weakly acidic ion exchanger Reverse-phase capture Mitsubishi SP207. Purification Harris Step 3. Purification 500 Mycelia Methanol (2 200 L) Precipitation Reverse-phase chromatography Partisil BioPrep P40 ODS-3 (500 g)–batch adsorption.Table 3 Comparison of the Reported Isolation Processes for Sordarin and Some Sordarin Analogues Neosordarin 100 Culture filtrate 1 Mycelia EtOAc Normal-phase adsorption chromatography Silica gel Sephadex LH-20 CH2Cl2:CH3CN (1 : 1) SCH57404 Sordarin (original) Not reported Mycelia Methanol:water (9 : 1) Differential pH extraction 206 GR135402 Fermentation volume (L) Product location Extraction solvent Misc. Polishing Crystallization 11-Hydroxysordarin Whole broth Methanol Normal-phase adsorption chromatography Silica gel column chromatography Reverse-phase chromatography Preparative RP HPLC–Nucleosil C18 3 -Oxoketal derivative 12 Whole broth Methylethylketone Mitsubishi SP207 3 -Oxoketal derivative 56 Whole broth Methanol Fermentation volume (L) Product location Extraction solvent Misc.

3-dioxane 10-membered lactones 12-membered macrolide 12-membered macrolide 16 member macrolide 16-mer peptide 2-pyridones 3-carboxyindole acetylene acyclic polyketide polyol esters acyclic polyketide polyol esters acyclic polyketide polyol esters alkaloid alkyl amides alkyl amides alkyl amides alkyl amine alkyl citrate alkyl nonenolide alkyl phenol alkyl phenol anthracycline anthranilic acid anthraquinone anthraquinone anthraquinonepyrones anthraquinones -pyrone ester -pyrones 207 (Continued) . B. d Penicillium Xylaria Cladosporium Cladosporium Periconia Dendrodochium Cordyceps unident. B. [28] [29] [30] [30] [31] [32] [33] [34] [35] [36] [37] [37] [38] [39] [40] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] coruscol A multiplolide A. b. B cordypyridone A. nipponica catinus roseum ochraceous sp.-2 neobulgarones topopyrones A-D UCT1072M1–3 phomalactone ester 11G219 a. D TMC-205 unnamed roselipins TMC-171A-C TMC-154 circumdatin G peniamidienone viriditin O-methylviriditin piptamine CJ-15183 microcarpalide none integracin A.Table 4 Survey of Reported Fungal Metabolite Isolations 1997–2002 Type sp. c. C. b cladosorolide D cladosprolide D macrosphelide H. C seragakinone A NI 15501A BM2419-1. viridi-nulans viridi-nutans betulinus aculeatus The Isolation and Structure Elucidation of Fungal Metabolites sp. Clitocybe Gliocladium Gliocladium Gliocladium Aspergillus Penicillium Aspergillus Aspergillus Piptoporus Aspergillus unidentified Chrysosporium Cytonaema unidentified Penicillium Paecilomyces Neobulgaria Phoma Aspergillus Phomopsis unidentified pura 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1. multiplex Name Genus Species Ref Index byssoides sp. L integramide A.

-6 S-15183a S-15183b azaspirene none circumdatin A. E 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 aromatic polyketides aspochalasin asterriquinones azaanthraquinone azaphilones azaphilones azaphilones azaphilones azaphilones azaphilones azaphilones azaphilones azaspirone benzo[63]fluoranthen-3-one benzodiazepine benzofluoranthene benzofuran betaenone inermis inermis sp. Cordyceps Cytospora Cytospora Gerronema RP-1551-M1. B. B spirobenzofuran 10-hydroxy-18methoxybetaenone none(11) cytoskytin B cytoskyrin A gerronemin A. D. B. sclerotiorum sp sp Sphaeropsidales Sphaeropsidales Mycena Botrytis Penicillium Harris [68] [69] [69] [70] [71] [72] [72] [73] [74] [75] 48 49 50 51 52 53 54 55 56 57 bioxanthracenes bisanthraquinone bisanthraquinone biscatechol bisindolyl benzoid bisnapthyl bisnapthyl b-methoxyacrylate botryane lactones breviane spiroditerpenes cinerea brevicompactum . F oxasterriquinol D methyl ether sphaerolone dihydrosphaerolone 9-hydroxyoudemansin A brevione B. -2. D aspochalasin F. -M2 RP-1551–7 RP-1551–1. B. C hypoxlonol A. G scorpinone Name Genus Species 208 Ref Index candidus like multicolor multicolor multicolor [54] [55] [56] [57] [58] [58] [58] [59] [59] [59] [60] [60] [61] [62] [64] [65] [66] [67] Scytalidium Aspergillus Aspergillus Bispora Penicillium Penicillium Penicillium Penicillium Penicillium Penicillium Zopfiella Zopfiella Neosartorya Cladosporium Aspergillus Hypoxylon Acremonium Microsphaeropsis sp. -4. C. D. C. -3. C. cladosporoides ochraceous truncatum Pseudomildis itaris sp. sp. E. sp.Table 4 Continued Type scytalols A. -5.

globosum globosum sclerotiorum virens unguis pallidoroseum chlamydosporia sp. B alutacenoic acid A. D The Isolation and Structure Elucidation of Fungal Metabolites kobayashil jesteri jesteri sp.trichocarane A. B. Hirsutella Pestalotiopsis Pestalotiopsis Pestalotiopsis Suillus Trichoderma Eupenicillium Aspergillus Phomopsis Metarrhizium Rhinocladiella anisoliae sp. B zygosporin D un-named Trichoderma Fusarium Termitomyces Tolypocladium Xylaria Bombardioidea Bombardioidea Trichothecium Fusarium Glomospora Nigrosabulum Nigrosabulum Aspergillus Ustilaginoidea Gliocladium Emericella Fusarium Diheterospora Fusarium Beauveria unident. D bombardolide C roseocardin N-methylsansalvamide glomosporin pseudodestruxin A pseudodestruxin B scleramide ustiloxin F argifin unguisin C apicidin B. D virens albuminosus extinguens sp. anartia anartia roseum sp. B TMC-169 phenochalasin A. C. granulatus hamatum flavipes [76] [77] [78] [79] [80] [81] [81] [82] [83] [84] [85] [85] [71] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [95] [95] [96] [97] [98] [99] [100] [101] [102] xyloallencide A bombardolide A. C. B. termitomycesphins A. C diheleropeptin JM47 beauveriolideill SCH218157 hirsutellide A jesterone hydroxyjesterone ambuic acid suillusin pentenocin A. (Continued) 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 carotane sesquiterpenes carotenoid glycosyl ester cerebrosides chromone cinnamoylcyclopeptide -lactones -lactones cyclic depsipeptide cyclic depsipeptide cyclic depsipeptide cyclic depsipeptide cyclic depsipeptide cyclic peptide cyclic peptide cyclic peptide cyclic peptide cyclic tetrapeptide cyclic tetrapeptide cyclic tetrapeptide cyclodepsipeptide cyclodepsipeptide cyclohexadepsipeptide cyclohexanone epoxide cyclohexanone epoxide cyclohexanone epoxide cyclopentabenzofuran cyclopentanone cyclopropenone cytochalasin cytochalasin cytochalasin cytochalasin 209 . B.

F none virescenoside M. N. Penicillium Penicillium Chrysosporium Tolypocladium Nectria Penicillium Penicillium Aspergillus Stereum Phoma Oldiodendron Acremonium Sarcodon Stachybotrys Stereum Leptosphaeria Aspergillus Pezicula unidentified unknown [103] [104] [105] [106] [106] [107] [108] [109] [110] [111] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 cytochalasin cytochalasin decalactone decalin polyketide decalin polyketide decaline polyketide depside depsipeptides depsipeptides dihydroxanthones dihydroxanthones dihydroxybenzone quinones diketopiperazine diketopiperazine diketopiperazine diketopiperazine diketopiperazone dimethylchromene dioxopiperazine diterene diterpene glycoside diterpenoid diterpenoid epidothiopiperazinedione epipolysulfanyldioxopiperazine epoxylactone equisetin derivative equisetin derivative equisetin type Harris . B clavariopsin A. B. B polanrazine B. D CJ-17665 sterin A. C decumbenone B decumbenone A phomopsidin agnodepside A. Z2. N1 nafuradin CJ-17572 CJ-21058 LL-49F233a Pyrenophora Pseudeurotium Penicillium Penicillium Penicillium Phomopsis unidentified Clavariopsis unident. B. Z3 cytochalasin X. Y. M1. D. C. Z Xestodecalactone A. B SCH378161(4) F390B F390B semichliodinol A. C. N scabronine B. D. C. E. E. F atranone A-G epicorazine leptosin M.Table 4 Continued Type Name Genus Species semeniperda zonatum montanense decumbens decumbens 210 Ref Index aquatica merdarium haemalococa fellutanum ochraceus hirsutum lingam griseum striatisporum scabrosus chartarum hirsutum sp. B Sch56396 haematocin fellutanine A. niger sp cytochalasin Z1.

E. B1. C TMC-120A TMC-120C ustus ustus (Continued) 211 .TC1068 bassiana bassiana bassiana bassiana sp. Aspergillus Aspergillus Aspergillus 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 ergostane farnesyl hydroquinone fatty acids furopyridine furosesquiterpene -lactone glycolipid glycolipid glycosylated terpene hexacyclic illudane-illudane bissesquiterpene hexaketide lactone hexaketide lactone hexaketide lactone hexaketide lactone hydroxybenzaldehyde ilicicolin H related ilicolin H like ilicolin H like ilicolin H like ilicolin H like illudalane indole diterpenes indole diterpenes isobenzofuranone isochroman isocyanocyclopentanes isonitrile isonitrile isoquinoline alkaloid isoquinoline alkaloids isoquinoline alkaloids iso-cladospolide B seco-patulolide C pandangolide 1 pandangolide 2 FR198248 TMC-69 pyridomacrolidin pyridovericin pyridomacrolidin pyridovericin gloeophyllol A–D nodulisporic acid B.B L970843 L970844 TMC-120A.B isopestacin CJ-12373 MR566A. C. G. CJ-15696 tsugicoline F. unident. B blazei pfeifferi clavipes varium tsugicola modonium Agaricus Ganoderma Clitocybe Cladoboytrum Laurilla Talaromyces Coryneum unidentified Irpex Bovista sp. F ganomycin A. ochraceous microspora harzianum ustus [140] [140] [140] [140] [141] [142] [143] [143] [144] [144] [145] [146] Z [147] [148] [149] [150] [151] [151] [152] [153] [153] unidentified unidentified unidentified unidentified Aspergillus Chrysosporium Beauveria Beauveria Beauveria Beauveria Gloeophyllum Nodulisporium Aspergillus Pestalotiopsis Penicillium Trichoderma unident. H rasfonin corynecandin emmyguyacin A. B. B irpexans bovistol [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] 119 120 121 122 123 124 125 126 127 128 The Isolation and Structure Elucidation of Fungal Metabolites terreus sp.blazeispirol B. B2 stephacidin A.

E CP225917 CP263114 benesudon PF1092A.Table 4 Continued Type TMC-120B stachyflin acetylstachyflin ustus sp. misc. 4 preaustinoid A. misc.d. mulitpspora roseus roseus pulchella sanguinea LL-23G227 Harris . misc. misc. misc.e YM-170320 arborcandins A–F CJ-12950 CJ-13357 pandangolide 3. C. concinna lindtneri oceanicum oceanicum Name Genus Species 212 Ref Index verticillata verticillata herbarium sp.C pterulinic acid pterulone gelastatin A. sp. B aranochlor A.B. B xyloketal A. misc. parasiticus lipohexin lipohexin FR901469 15G256c. B pulchellalactam hongoquercin A. Hypoxylon Hypoxylon deuteromycete unident unident. B aranochlor A. B. misc.e 15G256c. misc. B benesuada oblatum [153] [154] [154] [155] [156] [156] [157] [158] [158] [159] [160] [161] [161] [162] [163] [164] [165] [166] [166] [167] [168] [169] [169] [170] [171] [171] [172] [173] [174] Aspergillus Stachybotrys Stachybotrys Ganoderma Paecilomyces Moesyzia unident.d. misc. misc. misc. misc. D. misc. B sequoiatone A. Mortiercella Mortiercella Cladosporium Penicillium Aspergillus Xylaria unidentified unidentified Mollisia Penicillium Pterula Pterula Westerdykella Pseudoarachniotus Pseudoarachniotus Corollospora Peniophora unidentified 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 isoquinoline alkaloids K-76 decalins K-76 decalins lanostanoids linear lipohexapeptide linear lipohexapeptide lipodepsipeptide lipodepsipeptides lipodepsipeptides lipopeptide lipopeptide macrocyclic lactones macrocyclic lactones macrolide meroterpene misc.

misc. misc. misc. misc. misc. B 10-hydroxy-18-N-2-napthylN-phenylaminobetaenone TAN-1813 cis-fumagillin cis-fumagillin SCH420789 topopyrones A–D phellinsin A xanthoepocin 10-norparvulenone waol B 8-O-methylsclerotiorinamine bisabosquals bisabosquals Phoma Penicillium Penicillium unidentified Penicillium Phellinus Penicillium Microsphaeropsis Myceliiophthora Penicillium Stachybotrys Stachybotrys (Continued) . misc. acremolactone A erinacine E KM-01 KM-01 coprophilin NG-061 NG-061 diazaphilonic acid alutenusin memnoconol menobotrin A. misc.roseum ramosum avenae coccineus minoluteum minoluteum flavus echinata echinata echinata griseofulvum [175] [176] [177] [177] [178] [179] [179] [180] [181] [182] [182] [182] [183] [184] [184] [185] [186] [186] [67] Acremonium resupinatus Drechlera Pycnoporus unidentified Penicillium Penicillium Talaromyces Pencillium Memnoniella Memnoniella Memnoniella Pencillium Penicillium Penicillium Trichopezizella Aspergillus Aspergillus barbata terreus terreus 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 misc. misc. misc. misc. D arisugacins C. misc. misc. misc. misc. misc. misc. misc. misc. misc. misc. misc. misc. D F-12509A kodaistatins A. misc. misc. B memnoconone SCH351633 arisugacins C. misc. misc. misc. misc. misc. The Isolation and Structure Elucidation of Fungal Metabolites simplicissimum lutea multicolor 213 [187] [188] [188] [189] [50] [190] [191] [192] [193] [194] [195] [195] 198 199 200 201 202 203 204 205 206 207 208 209 misc. B kodaistatins A.

misc. B CR377 epoxyquinol A CR377 cladosporoide B–D clonostachin longibarchin LGB II. longibrachiatum ampullosporum Harris [195] [195] [196] [196] [197] [198] [199] [200] [201] [202] [203] [204] [39] [205] [206] [207] [208] [209] [210] [211] [212] [213] [212] [214] [215] [216] [217] [218] [219] Stachybotrys Stachybotrys Eupenicillium Eupenicillium Pencillium unidentified Panus unidentified Polyozellus Myrothecium Pencillium Guanomyces Penicillium Aureobasidium Phoma Laccaria Cytospora Metarrhizium Aspergillus Acremonium Fusarium unidentified Fusarium Cladosporium Clonostachys Trichoderma Sepedonium Sepedonium Emericellopsis 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 misc. E Mer-f3 aspergillimides UCS1025A. misc. napthopyranone napthopyranone napthoquinone nonenolide o-benzoquinone octaketide pyrone ovalicin-like paraherquamide-like pentacyclic polyketides pentaketide pentaketide pentaketide tricarbonyl pentanorlanostane peptaibol peptaibol peptaibols peptaibols peptaibols bisabosquals bisabosquals 4 oxomacrophorim A 4 oxomacrophorim D phenylpyropene C V214w panepophenanthrin pyrrocidine A. misc. misc. misc. II laccaridiones A. c. mixed terpenoid modified hydroxyquinone modified octapeptide mycophenolic acid deriv. sp. herbarum amethystea sp. B polyozellin MS-681a. B cytopyrone D. b. III ampullosporin peptaibolin berofungins B–D donezkii . d F13459 unnamed penidilamine aureoquinone herbarumin I.Table 4 Continued Type Name Genus Species 214 Ref Index crustaceum crustaceum griseofulvem rudis multiplex polthrix sp. sp. misc.

60065.donezkii neofelleus tubakii tubakii atroviride atroviride montagnei oryzae anisopliae niger sp grisea lecanii lecanii lecanii lecanii lecanii velutina hepatica oceanicum oceanicum oceanicum The Isolation and Structure Elucidation of Fungal Metabolites [219] [220] [221] [221] [222] [222] [223] [224] [225] [226] [163] [227] [228] [229] [230] [230] [230] [230] [230] [231] [232] [233] [233] [233] [234] Emericellopsis Tylopilus Acermonium Acermonium Trichoderma Trichoderma unidentified Apiospora unident. B humicolone vertilecanin A vertilecanin A. Acremonium Aporpium Stachybotyrs tunicata murorum caryae parvispora (Continued) 215 .2B.2C destruxin A.B 15G256a-1 15G256a 15G256b SCH60059.B. B TMC-95A. 60057. 60063. methyl ester vertilecanin C CJ-12954 cinnatriacetins A. 64878 massarilactone A. C neoatroviridin A. B. B. D dictyonamide A. B FR191512 F-11334As.Bs unnamed stachybotryin C Massarina unident. 64879. 60061. B funalenone sculezonone A. Aspergillus Metarrhizium Aspergillus Penicillium Humicola Verticillium Verticillium Verticillium Verticillium Verticillium Phanerochaete Fistulina Hypoxylon Hypoxylon Hypoxylon Acremonium 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 peptaibols peptaibols peptaibols peptaibols peptaibols peptaibols peptide peptide derivative peptide-aib peptides peptides phenalene phenalenone phenolic tetralone phenopicolinic acids phenopicolinic acids phenopicolinic acids phenopicolinic acids phenopicolinic acids phthalides polyacetylene polyesters polyesters polyesters polyhydroxyisoprenoid [235] [236] [237] [238] [239] 264 265 266 267 268 polyketide C4 polyphenolic ester prenylated hydroquinones prenylated indole prenylated phenols aa berofungins B–D tylopeptins cephaibols cephaibols atroviridin A.C SCH466457 (2) TMC-2A. methyl ester vertilecanin B vertilecanin B. C.

-6 kynapcin-12 none akanthomycin militarinone A sequoiatone C. unident. F trichodion actofunicone Name Genus Species 216 Ref Index citrinum indusiata sp. L curtisans A–D leucomentin-5. -5. B zopfiellamide A. D.B CJ-16264 CJ-16367 quinolactins dictyoquinazol A. -4. B. K. -6 leucomentin-5. Pencillium Dictyophora Pencillium Penicillium marine fungus Aigialus Aigialus Aigialus Aigialus 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 prenylated phenols#Plaa prenylated phenols aa prenylated quinone preussomerin p-terphenyl p-terphenyl p-terphenyl p-terphenyl pyridine pyridone pyridone alkaloid pyrone pyrone pyrone pyrones pyrrolidinone pyrrolidinone pyrrolizidine terpene pyrrolizidinone pyrrolizidinone pyrroloquinolone quinazoline quinolone quinolone quinone resorcylic acid lactone resorcylic acid lactone resorcylic acid lactone resorcylic acid lactone Harris . parvus parvus parvus parvus [239] [240] [241] [242] [243] [244] [244] [245] [246] [247] [248] [249] [250] [251] [252] [253] [253] [254] [255] [255] [256] [257] [258] [259] [260] [261] [261] [261] [261] zopfiellamide A. -6 clavilactone D preussomerin J. C quinolactins A–C pencillazine chlorogentisylquinone aigialomycin D aigialomycin C aigialomycin E aigialomycin F Stachybotyrs Stachybotyrs Clitocybe unidentified Paxillus Paxillus Paxillus Polyozelles Marasmiellus Akanthomyces Paecilomyces Aspergillus Trichosporiella Talaromyces Penicillium Zopfiella Zopfiella Acremonium unident. B UCS1025A. E.Table 4 Continued Type parvispora microspora clavipes curtisii panuoides panuoides multiplex gracilis militaris parasiticus flavus waksmanii lateipes lateipes parvisporin SMTP-3.

C. B. mellea arcularius ciliatus virens leightonii maculata [261] [261] [261] [262] [263] [264] [265] [266] [267] [267] [268] [269] [270] [270] [76] [271] [272] Aigialus Aigialus Aigialusz Phoma Chrysosporium Chloridium Xylaria Omphalotus Phoma Phoma Armillariella Polyporus Polyporus Trichoderma Resupinatus Collybia 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 resorcylic acid lactone resorcylic acid lactone resorcylic acid lactone resorcylic acid lactone resorcylic acid lactone salicylic acid tetramer sesquiterpene sesquiterpene sesquiterpene sesquiterpene sesquiterpene esters sesquiterpene esters sesquiterpene ethers sesquiterpene ethers sesquiterpene-carotane sesquiterpenes sesquiterpenes aigialomycin A aigialomycin A aigialomycin B L-783277 queenslandon CJ-21164 xylarenal A. B omphadiol phomapentenone A phomadecalin A. 6 aspergilloxide mangicol A–G YW3699 dihydrocarolic acid penitricin D diheteropeptin trichodenones A. C trichodenones A. sp. B. C WF14861 CRM-51005 WF14865B WF14865A 1 hydroxysordarin Aspergillus Fusarium Codinaea Aspergillus Aspergillus Diheterospora Trichoderma Trichoderma Colletotrichum unidentified Aphanoascus Aphanoascus Acremonium Sordaria 217 fulvescens fulvescens sp. B.parvus parvus parvus queenslandicum sp. persicaria illudens sp. D melleolides K–M dictyopanines A–C cryptoporic acid isocryptoporic acid trichocarane A–D The Isolation and Structure Elucidation of Fungal Metabolites heterosporum simplex niger niger harzianum harzianum [273] [274] [275] [276] [276] [277] [278] [278] [279] [280] [281] [281] [282] [17] 315 316 317 318 319 320 321 322 323 324 325 326 327 328 sesterterpene sesterterpene sesterterpene small acid small acid small cyclic peptide small lactones small lactones small peptide deriv small polyketide small pseudopeptide small pseudopeptide small quinone sordarin unnamed collybolides 4a–c. araneosa (Continued) . 5.

Table 4 Continued Type Name Genus Species araneosa putredinus putredinus minioluteum concentrica concentrica 218 Ref Index [17] [283] [16] [284] [285] [285] [286] [286] [287] [288] [289] [290] [291] [292] [293] Sordaria Graphium Graphium Penicillium Daldinia Daldinia Mycena Mycena Mycena Ganoderma unidentified Memnoniella Aspergillus Aspergillus unidentified Sarcodon asterric acid. C cryptocin ascosalipyrrolidinone A. D talaroconvolutin A. C. B. D talaroconvolutin A. B talaroconvolutin A. B. D tetrachloropyrocatechol strobilurin M strobilurin O. 3-Cl and 3. D concentriol B. D talaroconvolutin A. C. B. C. C. lactone guanacastepene memnopeptide terprenins galericulata lucidum echinata candidus candidus [294] 344 leucopus longicolla quercina salicorniae convolutus convolutus convolutus convolutus [295] [296] [297] [298] [298] [298] [298] [299] [300] [301] [301] 345 346 347 348 349 350 351 352 353 354 355 terphenyl-diketopiperazine hybrid tetrahydroxanthone tetramic acid tetramic acid tetramic acid tetramic acid tetramic acid tetramic acid tetramic acid tetramic acid tetramic acids tetramic acids dicerandrol A. D lucilactaene ravenic acid (7Z)-fusarin C (5Z)-fusarin C Phomopsis Cryptosporiopis Ascochyta Talaromyces Talaromyces Talaromyces Talaromyces Fusarium Penicillium Nectria Nectria Harris coccinea coccinea .5diCl sarcodonin 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 sordarin sordarin type sordarin type sordarin type squalene triterpene squalene triterpene strobilurins strobilurins strobilurins terpene terpene terpene peptide terphenyl terphenyl terphenyl ether neosordarin GR135402 GR135402 BE-31405 concentriol B. B. C. C. P lucidenic acid O. B.

M curvularol cytonic acid A. microspora coccinea [301] [302] [303] [303] [304] [304] [305] [306] [307] [307] [308] [309] [310] [310] [310] [311] [312] [313] [314] [315] [315] [316] [317] [318] Myrothecium Curvularia Cytonaema Colletotrichum Colletotrichum Aspergillus Stachybotyrs Aspergillus Aspergillus Aspergillus Preussia Preussia Daedalea Penicillium Humicola Humicola Cordyceps Penicillium Phomopsis sp. B2 NF00659A3 NF00659A1.-2 NF00659B1. B colletotric acid colletotric acid aspergillamide A.Nectria unidentified Myrothecium Myrothecium verrucaria verrucaria sp gloeosporioides gloeosporoides sp. A2 S19159 S19159 12 unnamed anicequol fuscoatroside fuscoatramide cordytropolone PF1163A. B phomoxanthone A. 47525 8-acetoxyroridin H 8-acetoxyroridin E verrucarin M roridinL. B SMTP-1. (Continued) aemulans aemulans quercina aurantiogriseum fuscoatra fuscoatra 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 tetramic acids trichothecene trichothecene trichothecene trichothecene trichothecene trichothecene-like tridepside tridepside tridepside tripeptide triprenyl phenol triterpene triterpene triterpene triterpene triterpene triterpene triterpene triterpene triterpene tropolone tyrosine lactone xanthone dimers fusarin C YM-47524. B The Isolation and Structure Elucidation of Fungal Metabolites 219 .

7 Yieldh (mg) 220 Indexa F/Tb L/Sc Vol.6 62. 11. 70. 11.3 30.5 3.4 22.5–148.2 Harris 23 F L 10 Filtrate .d (L) Locatione 1 F L 10 Mycelia 6 4 4 90 Filtrate Filtrate Mycelia Filtrate WB Filtrate RP HPLC 39 RP HPLC 72 RP HPLC 64 EtOAc LH-20 (2) LH-20 (1) SiO2 LH-20 (2) SiO2 RP HPLC 57 RP HPLC 56 CC RP 17 SiO2 RP HPLC LH-20 (1) CC RP Prep TLC SiO2 CC RP 17 LH-20 (3) LH20 RP HPLC 15 7 2 3 4 5 6 7 8 9 Acetone nBuOH nBuOH CHCl3 SiO2 HP-21 HP-21 EtOAc RP HPLC 42 RP HPLC 42 SiO2 RP HPLC 60 RP HPLC 60 EtOAc F F F T F F F F L L L L S L L L WB CHCl3:MeOH (1 : 1) EtOAc XT EtOAc Acetone EtOAc XT MEK EtOAc xtn nBuOH EtOAc 5.g Step 2 SiO2 CC RP 4 LH20 RP HPLC 57 RP HPLC Step 3 Step 4 Step 5 Step 6 Seq liq-liq partition SiO2 12.1–10.15–11. 3. 23 mg/L 85.8 50.7.2 3.7 10 108 48 35 32.2 18 10 11 12 13 14 15 16 17 18 19 F F F F F T T F T F L S S L L L L L L L 4.5 2 10 16 16 10 20 4 WB WB WB WB Filtrate Filtrate Filtrate WB Filtrate 20 21 22 EtOAc MEK CHCl3:MeOH (1 : 1) ODS BuOH xtn F F F S L L 1 10 WB WB Mycelia EtOAc XT SiO2 CC RP 5 LH-20 (1) Prep TLC SiO2 SiO2 LH20 HP-20 Repeated normal phase chromatography SiO2 LH-20 (1) EtOAc XTn RP HPLC 71 RP HPLC 70 CC ODS Gel filtration 80 260. 2.1 7. 36 6. 90 2..Table 4 Continued Extraction Solvent Step 1f. 1.5 171 2.8 3. 285.4 1.

28. 2. 4.1. 12.9 9. 18 30 hexane xtn CCC SiO2 RP HPLC 27 Crystallization SiO2 RP HPLC 51 T L 19 Filtrate HP21 SiO2— Lichroprep Si60 EtOAc:hexane SiO2— (1 : 1) XT BiotageFlash75 KP-Sil EtOAc partition hexane:water partition EtOAc XT SiO2 31 32 T F L L 55 40 Mycelia WB EtOAc XT EtOAc 33 F L 2 Mycelia 46.5 0. 73 57 787. 37. 9.4–8. 37. 31.0. 22.6 1 WB Filtrate Acetone: MeOH nBuOH HP20 28 T L 50 Mycelia MeOH 29 SiO2 RP HPLC 24 SiO2 Prep TLC SiO2 SiO2 RP HPLC 26 T L 300 Mycelia Acetone 600.8.6 0. 2 RP HPLC 28 31–7500 RP HPLC 24 T nBuOH pptn HPLC Hex: EtOAc xtn SiO2 RP HPLC 37 CC RP 19 L 20 Mycelia Acetone 25 T L Mycelia 26 27 F T L L 2. 2.7. 47. 10. 20.6.5–23 282.5 12–1400 (Continued) 221 . 3. 4.5 34 T 20 WB CHCl3: Prep TLC MeOH (2 : 1) EtOAc SiO2 The Isolation and Structure Elucidation of Fungal Metabolites RP HPLC 51 RP or NP HPLC RP HPLC SiO2 RP HPLC RP HPLC SiO2 RP RP HPLC RP HPLC 35 36 37 38 39 SiO2 SiO2 HP-20 HP-20 HP-20 T F T T T L L L L L 2 20 200 8 8 1 EtOAc XT EtOAc XT EtOAc partition EtOAc XT SiO2 SiO2 SiO2 CC KP-Sil WB WB Mycelia Mycelia Mycelia EtOAc EtOAc MeOH MeOH MeOH Toyopearl HW40 RP HPLC Xtal SiO2 SiO2 SiO2 40 41 42 43 T F F T L L L L 1 15 50 WB WB MMycelia Mycelia Acetone Acetone Acetone Acetone: MeOH.24 UV rxn 3. 12.2 97.1. 3.5 40 3. 3 2250 47. 32. 6.8. 6 140 20 83. MeOH RP HPLC 43 RP HPLC 43 RP HPLC 37 Xtal RP HPLC 48 24. 6.

2. 1.Table 4 Continued Extraction Solvent Step 1 SiO2-Lobar LiChroprep Si 60 RP HPLC Prep TLC f. 4.5 Harris 62 F L 170 Mycelia/ filtrate Filtrate EtOAc 50.0. 24.6 3. 2.. 0.5.g 222 Index F/T a b L/Sc (L) Vol.5. 2. 5. 4. 20 5.d Step 2 Step 3 Step 4 Step 5 Step 6 Location e Yieldh (mg) 33. 2. 4 Prep TLC 57 58 59 60 EtOAc xtn EtOAc xtn EtOAc xtn nBuOH partition SCC C18 SiO2 F F F S S L 10 4. 7. 11.9–377 48 49 F F L L 10 FB WB Mycelia/ filtrate Mycelia WB MeOH EtOAc 50 RP HPLC 34 LH-20 (9) LH-20 (4) LH-20 (4) SiO2 NP HPLC F L WB EtOAc 51 52 53 54 55 56 T F F F F 20 Mycelia sclerotia L S L L L L 5 29 WB WB EtOAc XT Kupchan partition Kupchan partition SiO2 LH-20 (3) SiO2 SiO2 RP HPLC SiO2 SiO2 SiO2 32. 150 EtOAc .4.8.7 120.3. 7. 6. 7 10. 14. 25.4 15 17.8 1. 6. 4 44 AgA WB EtOAc 45 46 47 SiO2 CC RP 4 CC RP 4 successive precipitation RP HPLC 73 LH20 RP HPLC RP HPLC 28 F F L L 360g 20 SiO2 LH20 SiO2 CHCl3 EtOAc EtOAc NP HPLCLiChrocart LiChrospher Si 60 RP HPLC SiO2 CC RP 3 17.3 1 mg/L 3 mg/L 35 5. 100.2 11. 8.9.8.3 kg WB WB Mycelia FB MeOH CHCl3 EtOAc EtOAc EtOAc EtOAc (NaCl sat broth) Acetone Acetone Acetone EtOH SiO2 SiO2 SiO2 CC RP 13 repeated diol SiO2 Prep TLC RP HPLC 7 SiO2 RP HPLC 13 61 F L 0. 19 4.

2 RP HPLC 45 5.1 46. 3.8 0.6 1. 2.7 L L L L 4.5 mg/L 8.2 3.4 72 F DiaionSK1B HP20 CIE Hexane:80%Me CC RP 4 OH partition L WB Filtrate Filtrate Sclerotia 500g False smut balls Mycelia 8 EtOAc XT EtOAc XT HP-20 EtOAc/ CH2Cl2: MeOH (1 : 1) MeOH EtOAc XT EtOAc XT CHCl3 Water 205 9.8 15 20 Filtrate Filtrate WB Mycelia/ filtrate RP HPLC Crystallization RP HPLC 73 CHP20P SiO2 CC RP 11 Dowex1 RP HPLC 29 67 68 69 70 71 F F F F EtOAc XT SiO2 SiO2 LH-20 (3) ODS nBuOH XT RP HPLC 1 RP HPLC 1 LH-20 (9) SiO2 S L L S 2.5 CHP20P RP HPLC 42 20 G-10 2.4 2. 3 : 5:3 : 5 LH-20 (1) SiO2 SiO2 SiO2 seq Liq-liq partition . 4.63 64 65 66 T F F F SiO2 SiO2 EtOAc xtn CC RP 4 LH-20 RP HPLC SiO2 LH-20 (11) NP HPLC SiO2 RP HPLC 4 RP HPLC 14 55.7 9.1 61.4 ? 74 75 76 SiO2 LH-20 (7) SiO2 SiO2 CC ODS NP HPLC— YMC PVA-Sil F F L/S L S RP HPLC RP HPLC 37 SiO2 2 WB Filtrate WB EtOAc: CHCL3: MeOH (3 : 2:1) MEK EtOAc EtOAc The Isolation and Structure Elucidation of Fungal Metabolites 77 78 F L 10 WB WB Acetone EtOAc F F F F SiO2 SiO2 SiO2 Crystallization SiO2 L L L L 5 1 1 1 79 80 81 82 83 Mycelia Filtrate Filtrate Filtrate FB CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 MeOH 18 20 120 15 LH-20 (1) (Continued) 223 LH-20 (1) SiO2 Hexane:90%Me OH partition EtOAc XT CCCH:E:M:W.8 4.2 MeOH 73 F S WB 1.1.

5 70.3.5. 1.2 92 F L 93 94 95 96 SiO2 SiO2 SiO2 CH2Cl2:MeOH xtn EtOAc XT LH20 HP-20 EtOAc xtn Fractogel TSK HW-40 SiO2 SiO2 SiO2 Hex/MeOH partition SiO2 RP HPLC 12 EtOAc/H xtn F F F F L L L L 10 10 1 1 Mycelia/ filtrate Mycelia/ filtrate Filtrate Filtrate WB WB? 97 98 99 F F F L L L 6 4 2.8 Mycelia WB Filtrate MeOH/Et OAc EtOAc EtOAc Acetone Freeze dried Acetone EtOAc 70 86 12 0. 480 12. 1.g Step 2 LH-20 (14) CC RP 1 RP HPLC64 LH20 RP HPLC11 Step 3 Step 4 Step 5 Step 6 HP20 Yieldh (mg) 5.d (L) Locatione F L 2 WB EtOH 84 85 86 87 88 89 EtOAc XT EtOAc XT SiO2 CC RP 4 LH-20 (3) CC RP 4 LH-20 (1) RP HPLC 69 CC RP 5 RP HPLC 64 RP HPLC RP HPLC 69 Prep TLC Crystallization SiO2 F T F F S L L L 20 10 1.5.3 91 F L 0.8 1.CH2Cl2 seq liq-liq partition CC RP 4 diol RP HPLC 15 Crystallization RP HPLC RP HPLC 42 RP HPLC CH2Cl2 xtn CC RP 4 CC RP LH-20 (7) SiO2 CC RP 15 RP HPLC 34 RP HPLC 4 LH-20 (19) CC RP 12 Prep TLC RP HPLC 55 SiO2 27 22. MeOH Hex.9 0. 11.5 WB WB Filtrate Filtrate 90 F S WB nBuOH Acetone EtOAc EtOAc. CH2Cl2 55% aq. 20 2. 2.2.Table 4 Continued Extraction Solvent Step 1f. 75 Harris 100 F L 1 Mycelia Acetone 101 T L 200 WB EtOAc . 10.8 5.6 EtOAc 2.1.1. 13.3 2. 3. 50 0.4.6.9 224 Indexa F/Tb L/Sc Vol. 19..5–100mg 1. 1.

7.7.7.8 22. 12.9 3 4 WB Filtrate Filtrate EtOH HP-20 EtOAc 109 T 110 F L S 12 WB WB 3.4 22.9. 4.5 3 Filtrate Filtrate Mycelia EtOAc CHCl3 Aq. 93.5.7 21. 18. 6.8.102 F pptn-hexane CHP20P LH-20 (2) CHP20P YMC PVASil 12.5. 1. 25.5 RP HPLC 35 RP HPLC 25 Prep TLC 18 L 8 WB EtOAc 103 F 104 F 105 T SiO2 SiO2 EtOAc XT Prep TLC Prep TLC SiO2 SiO2 Crystallization LH20 LH20 LH-20 (17) CC RP 8 SiO2 EtOAc partition SiO2 SiO2 Prep TLC SiO2 RP HPLC 60 RP HPLC 42 SiO2 RP HPLC 10 RP HPLC 42 PEI silica SiO2 SiO2 L L L 5 55. 5. 12. 53. acetone 106 F 107 T 108 F S L L 0.1 trace–12 50. 27.6 5.5.2.9.5 13. 1.5 WB EtOH S 117 F 118 NR L 119 F Mycelia MeOH 120 FB CH2Cl2 2. 11. 22.7 RP HPLC 43 LH-20 (16) LH-20 (20) 1. 14. 4. 2. 11.9 Mycelia Filtrate Acetone HP-20 RP HPLC 59 100. 20 225 (Continued) . 1 3.7–20. 58.1 18 41.5.4 kg FB 112 F S WB EtOH EtOAc XT CHCl3:MeOH SiO2 (2 : 1) MeOH Hex:EtOAc:wat er partition CHCl3:MeOH CHCl3 xtn (1 : 1) The Isolation and Structure Elucidation of Fungal Metabolites 113 F 114 SiO2 LH-20 (2) RP HPLC 33 SiO2 RP HPLC Hexane XT CC RP 8 CC RP 8 CHCl3 xtn SiO2 LH-20 (13) LH-20 (5) SiO2 Hex:MeOH:H2 O partition RP HPLC 59 L L 10 90 WB Mycelia EtOAc MeOH 115 F 116 F L L 10 0.5 30.7. x. 190 x. 2. 14 111 1.

9.2 6.CHCl3 seq liq-liq partition Hex.105.CHCl3 seq liq-liq partition SP207 LH-20 (2) HP20SS RP HPLC 67 CC RP 8 47 43.4.5 700 115.CHCl3 xtn SiO2 RP HPLC 23 60.2 3 126 A 3 EtOH EtOAc Acetone Acetone: MeOH MeOH:H2O 127 T L 20 Mycelia 100.6 20 RP HPLC 34 SiO2 128 129 T F L L Filtrate WB Acetone: MeOH EtOAc EtOAc RP HPLC 42 130 F SiO2 L WB EtOAc RP HPLC 42 1.140 3.5 30 WB WB WB Mycelia CC RP 8 CC RP 4 SiO2 CPC HP-20 SiO2 EtOAc XT CH2Cl2 trituration Hexane.d (L) Step 2 Step 3 Step 4 Step 5 Step 6 Location e Yieldh (mg) 121 FB F 3 122 123 124 125 CC RP 4 RP HPLC F T S S S L 0. 4.CHCl3 seq liq-liq partition Hex..1–100 120 7.5 132 F L WB EtOAc SiO2 RP HPLC 42 0.6 Harris 133 134 1.CHCl3 seq liq-liq partition Hex.6 131 F L WB EtOAc SiO2 RP HPLC 42 2. 4.g 226 Index 85% EtOH a F/T b L/S c Vol. 4.Table 4 Continued Extraction Solvent Step 1 CHCl3/H2O partition RP HPLC LH-20 (1) RP HPLC 59 RP HPLC 37 SiO2 SiO2 f. 3. 20(70% pure) 1.9.3 WB Acetone nBuOH SiO2 Hex.0.3 .

5 2 WB WB WB nBuOH nBuOH nBuOH Acetone 49.2. 7.9 17 The Isolation and Structure Elucidation of Fungal Metabolites 152 F S 2 Acetone 4200 153 LH-20 (1) RP HPLC 50 HP20 SiO2 Hexane pptn HP20 Hexane pptn EtOAc XT FB EtOH SiO2 SiO2 SiO2 hex:90%aq. 1.L L CEtOAc XT EtOAc XT SSiO2 SiO2 34 41 18 18 Mycelia Mycelia Acetone Acetone SRP HPLC 44 RP HPLC46 RP HPLC 44 RP HPLC 46 135 136 137 138 139 HP-21 SiO2 RP HPLC 34 5.5 F F NR NR T L 20 Filtrate L RP HPLC LH-20 (1) LH-20 (19) pptn SiO2 CC RP 4 LH20 Crystallization ML LH-20 (3) LH-20(3) SiO2 SiO2 Crystallization CC RP 4 RP HPLC RP HPLC Xtal Xtal RP HPLC Crystalliz 20 ation Prep TLC SiO2 RP HPLC 58 SP207 EtOAc xtn SiO2 EtOAc XT RP HPLC 68 LH-20 Crystallization SiO2 EtOAc xtn SiO2 EtOAc XT EtOAc XT EtOAc XT EtOAc XT HP-20 HP20 1 140 141 142 143 F A F T L L ? 12 WB WB Filtrate WB MeOH MeOH CH2Cl2 70% Acetone T F F L L L 6 40 40 Filtrate Filtrate Mycelia Acetone 144 145 146 147 148 149 150 151 T T T F L L L S 44.5 44. 5.6 22 365.9 22. 0. 2. 62. 3.2. MeOH partition EtOAc XT hex:90%aq. 18. MeOH partition EtOAc partition SiO2 LH-20 (18) Prep TLC SiO2 5.5 44. 4 120–150 CC RP 7 RP HPLC 42 SiO2 CC RP 7 HP20 72 2000.5 ? –180 0.9. 24 28000 c 154 155 156 157 158 T T T T L L L L 40 75 300 300 Filtrate Mycelia WB Filtrate Mycelia EtOAc MeOH Acetone EtOAc XT MeOH 227 (Continued) .7.

5 F lyophilized 160 T L 600 T 161 F L 8 F Acetone: MeOH EtOH RP HPLC 3.3. CHCl3 LH-20 (7) Sequential liquid partition–hex.9 2.. 6 162 F L 8 F EtOH 163 F L F EtOAc seq Liq-liq XTN SiO2 SiO2—prep TLC LH-20 (7) Hibar-NH2 CC Hibar-NH2 CC RP HPLC 42 164 F S F MeOH 165 F L 1 F MeOH NP HPLC 1.5 166 Differential pH xt T L 170 T EtOAc CHCl3 xtn CCC Filtrate/MeOH xtn solids SiO2 167 T L 15 T EtOAc RP HPLC 2 1900. toluene. 5.g 228 Index F/T a b L/S c Vol.2 Harris . toluene. 2300. 3. 23. 50 31 168 Differential pH xt T L 15 T EtOAc LH-20 (7) Sequential liquid partition–hex.16 32.9 42 34–711 89 4.3.d (L) Step 2 Step 3 Step 4 Step 5 Step 6 Location e Yieldh (mg) 159 F S 3. CHCl3 RP HPLC 2 18. 30.Table 4 Continued Extraction Solvent Step 1 50% Acetone EtOAc xtn HP20 SiO2 SiO2 CC-C18 SiO2 CCC CC RP 4 RP HPLC 4 RP HPLC 4 RP HPLC 58 SiO2 CC ODS flash CPC HP20 HP-20 HP-20 f.

2 The Isolation and Structure Elucidation of Fungal Metabolites 173 174 175 176 177 178 179 180 181 182 183 184 185 186 T F L S 30 iltrate WB Mycelia Mycelia WB Filtrate Mycelia WB 187 188 F F L S 0.3 SiO2 LH-20 (1) S 171 T L WB 6 kg medium Filtrate 16 EtOAc RP HPLC 21 HPLCLiChrogel PS1 xtal 60. acetone EtOAc MeOH→ CHCl3: MeOH→ CHCl3 229 (Continued) .4 20.169 F HP-20 EtOAc xtn 51 SiO2 L 8 Filtrate 170 F SiO2 HP-21 SiO2 9.4 RP HPLC 42 MeOH pptn 41 209 16 90. 15 3 600 600 3 350g FB EtOAc XT Acetone EtOAc MeOH F F LH-20 (1) L L A Xtal SiO2 SiO2 SiO2 LH-20 EtOAc xtn SiO2 SiO2 RP HPLC 4 3 1 1 1 EtOAc Aq.6 mixture 90170 25.9 Filtrate WB Acetone 67% aq. 30 172 T SiO2 L 16 Mycelia MeOH:acet one HP-20 SiO2 MPLC SiO2 RP HPLC49 Prep TLC SiO2 HPLC— LiChrosorb Diol SiO2 EtOAc xtn LH-20 (1) RP HPLC 57 NP HPLC— LiChrosorb Diol RP HPLC 24 16 T T T F EtOAc CCC EtOAc XT SiO2 RRP HPLC SiO2 L L L L Filtrate Filtrate Mycelia 4. 49. EtOH Acetone Acetone MEK SiO2 EtOAc xtn ETHER xtn ether xtn SiO2 HP-20 HP-20 EtOAc XT SiO2 PEI column RP HPLC 36 Prep TLC CC— SiO2 Whatman (Chromatot LPS-1 ron) 90 30 20.

8 200 WB Filtrate Mycelia WB Filtrate Mycelia 197 F L 198 199 SiO2 SiO2 T T RP HPLC 66 CC RP 4 L L 105 30 Mycelia/fil EtOAc trate WB EtOAc Filtrate EtOAc Methylation SiO2 Prep TLC HPLC YMCPak PVASil 2100 15 T CC L 30 Mycelia WB Acetone EtOAc EtOAc Acetone EtOAc RP HPLC Prep TLC CC RP 4 LH-20 (6) CC RP 4 RP HPLC 46 RP HPLC 59 Harris 200 201 202 203 204 205 SiO2 EtOAc XT SiO2 F F F L L L 10 5 6 Mycelia WB WB 6 12 3.2 .0 189 F S WB 190 F S WB PEI column MeOH→ CHCl3: MeOH →CHCl3 MeOH→ CHCl3: MeOH→ CHCl3 EtOAc CH2Cl2 xtn EtOAc EtOAc XT CHP20P SiO2 Fractogel HW 40 CC RP 3 MeOH Acetone XAD 7 30% MeOH 3.7 100 3.g 230 Index a F/T b L/S c Vol.8.d (L) Step 2 Step 3 Step 4 Step 5 Step 6 Location e Yieldh (mg) 22.3 200 RP HPLC 22 11–120 8.4–109. 25..1 191 192 193 194 195 196 T T F T L L L 2 36 36 0.Table 4 Continued Extraction Solvent Step 1 PEI column SiO2 (Chromatot ron) SiO2 (Chromatot ron) RP HPLC 62 SiO2 RP HPLC 9 RP HPLC 22 RP HPLC 37 f.

LH20 Prep TLC LH-20 (18) SiO2 206 207 208 209 210 211 212 xtal SiO2 RP HPLC 42 RP HPLC 37 T F T T T T F EtOAc XT SiO2 SiO2 EtOAc XT SiO2 SiO2 Seq liq-liq XTN SiO2 10–17 50 91 67 146 4130 145. 85. 30.2 L Agar Filter Paper 700g 218 FBc MeOH 387 219 F XAD-8 LH20 SiO2 Crystallization Prep TLC CC RP 4 Acetone CH2Cl2 EtOAc XT SiO2 SiO2 EtOAc EtOAc Prep TLC SiO2 SiO2 SiO2 SiO2 LH20 SiO2 SiO2 EtOAc L 15 Mycelia MeOH Seq partition —EtOAc HP-20 CHCl3 XTpH12 RP HPLC 47 RP HPLC 67 The Isolation and Structure Elucidation of Fungal Metabolites 220 F 221 F L L 10 12 21.8 25 25 25 25 Filtrate WB Filtrate Mycelia Mycelia WB WB HP20 Acetone EtOAc Aq.1 S 2 L 290 L 9.1 13–17 (Continued) 222 F 223 F 224 F L L L 10 60 40 225 F L 30 Mycelia Mycelia/ filtrate Filtrate WB Mycelia/ filtrate WB 231 . Acetone EtOAc EtOAc EtOAc 213 214 215 216 217 EtOAc EtOAc XT HP-20 MeOH SiO2 SiO2 EtOAc xtn RP HPLC 58 RP HPLC 58 LH-20 (1) Crystallization SiO2 SiO2 RP HPLC 58 pptn RP HPLC-43 F F T F WB Filtrate Filtrate Mycelia 20.8. 3.5 120 135 21. 22. 27 55 5 to 20 3 60 12. 8 L L L L L L S 30 4.8.

7.8 21. 0. 225 .2–87.5 20 40 10 60 60 T L 70 Mycelia Filtrate WB WB Filtrate Mycelia FB Filtrate ?? 6. 5. 1.5–13 RP HPLC 17 26 3.d (L) Extraction Solvent Step 1 f.8. 4 4–520 242 243 T F L L 70 12 RP HPLC 38 RP HPLC 38 RP HPLC 244 F L 12 245 F 10 Mycelia Mycelia/fil trate Mycelia/fil trate Mycelia 12.1.Table 4 Continued 232 Index F/T EtOAc CC RP 4 RP HPLC 42 SiO2 CCRP 4 EtOAc XT Acetone LH20 HPLC-SiO2 RP HPLC Kupchan partition SiO2 NP HPLC NR 10 11 a b L/S c Vol. 1.2.5 130 12 234 235 236 237 238 239 240 241 MeOH EtOAc SiO2 SiO2 EtOAc F F F F F F L L L S L L 2.8 5.4.6 5 Hexane SiO2 Filtrate WB 229 230 231 232 233 SiO2 SiO2 SiO2 SiO2 SiO2 LH-20 (1) LH-20 (1) RP HPLC 42 RP HPLC 30 SiO2 RP HPLC 19 RP HPLC RP HPLC 49 SiO2 NP HPLC HP-20 LH-20 (1) SiO2 SiO2 Hexane CHCl3MeOH Acetone nBuOH EtOAc XT EtOAc EtOAc XT EtOAc XT MeOH SiO2 SiO2 CHP20 RP HPLC 49 RP HPLC 38 Fractogel TSK MW-40s LH-20 (1) LH-20 (1) SiO2 RP HPLC 56 F L WB F F L S ? WB WB ? 2. 15 Harris 246 247 T L 29..5 4 Filtrate Filtrate CH2Cl2: MeOH HP20 XAD-16 nBuOH partition SiO2 HPLC – PLRP-S 4.4 2.1.g Location e Step 2 Step 3 Step 4 Step 5 Step 6 Yieldh (mg) 226 F L WB 10 227 228 F A L S 4.

12. 0.8 2.4 RP HPLC 8 MPLC C18 15. 5.7 18.2.5 7. 380.3. 10. 10. 560 50.2. 560 420.4. 50.4 2 7. 70 960 (Continued) 233 . 15 34. 380. 380. 0.21. 560 420.1 3.2 7.6 RP HPLC 37 CC RP 5 1600. 4 xtal or SiO2 CH2Cl2 Acetone EtOAc EtOAc EtOAc RP HPLC 73 EtOAc RP HPLC 73 RP HPLC 73 RP HPLC 73 RP HPLC 73 LH-20 (1) Toyopearl HW-40 EtOAc EtOAc EtOAc EtOAc Acetone EtOAc SiO2 RP HPLC 54 RP HPLC 54 RP HPLC 54 CHP20P RP HPLC 12 SiO2 SiO2 EtOAc LH-20 (1) 85% aq acetone SiO2 SiO2 LH-20 (15) LH-20 (15) LH-20 (15) LH-20 (15) AmberliteT RA-400 EtOAc XT SiO2 SiO2 AmberliteIR1 SiO2 20 CC RP 16 LH-20 (1) NP HPLC LiChrospher LH-20 (15) SiO2 L 85 Filtrate 249 F L Filtrate 250 F 251 F 252 F L L L 3 1 ? Mycelia Filtrate Filtrate 253 F S WB 254 F S WB 255 F S WB 256 F S WB 257 F S WB 258 T 259 L ~10 WB FB Hex:MeOH partition Hex:MeOH partition Hex:MeOH partition Hex:MeOH partition Hex:MeOH partition EtOAc xtn EtOAc XT 260 ? L 5 Filtrate The Isolation and Structure Elucidation of Fungal Metabolites 261 ? L 5 Mycelia 262 ? L 5 263 F L 1 WB 264 265 T EtOAc Acetone SiO2 EtOAc XT L 8 20 WB LH-20 CC SILICAR CC-4 RP HPLC CC RP 8 CC RP 8 420. 6.248 T HP-20 SiO2 RP HPLC LH-20 (21) RP HPLC 58 97.1 2.5.

9.3.2. 21.Table 4 Continued Extraction Solvent Step 1f.6 279 MeOH F L ? Mycelia SiO2 Prep TLC SiO2 LH-20 (8) SiO2 SiO2 SiO2 LH20 CCC (HEMW— 1 : 1:1 : 1) CC RP 4 9.g Step 2 SiO2 NP HPLC— SenshuPak silica4251-N RP HPLC 9 Step 3 Step 4 Step 5 Step 6 Aq.6.0.. 3.9.5. 4 268 269 270 T T F L L L 6 6 4. 15.7 10.5 62 17 . 12.9. 23. 8.9 Filtrate Filtrate Filtrate F 271 272 S S 6 WB 9.2 20 4.7 7.1–15 3 110 16.2 3. acetone EtOAc XT 7–34 Yieldh (mg) 234 Indexa F/Tb L/Sc Vol. RP HPLC 43 SiO2 RP HPLC 38 CC RP 4 273 274 275 276 277 278 F F L L 4 ? FB FB FB FB WB Filtrate SiO2 EtOAc XT EtOAc XT EtOAc XT nBuOH XT Kupchan partition LH-20 (1) Prep TLC LH-20 (7) LH20 CC RP 4 RP HPLC RP HPLC 53 MeOH MeOH MeOH MeOH EtOH CH2Cl2 1.2–76.7 Mycelia 267 EtOAc EtOAc HP-20 SiO2 SiO2 SiO2 RP HPLC 44 RP HPLC 44 EtOAc EtOAc SiO2 SiO2 F SiO2 Prep TLC SiO2 LH-20 (12) LH-20 (12) RP HPLC 16 L 2 Filtrate EtOAc 2. 7.5.6 280 MeOH F L Mycelia SiO2 RP HPLC 281 282 HP21 Acetone T T L L 20 Filtrate Mycelia Harris Solvent partition (HEMW) CH2Cl2/H2O LH-20 (7) partition pptn SiO2 CC RP 10 EtOAc partition RP HPLC 37 4.7 1. 15.d (L) Locatione 266 F L 0.

2 13. 6. acetone 290 HP-20 LH-20 (17) LH20 RP HPLC 52 Acetone EtOAc EtOAc EtOAc HP20 SiO2 SiO2 LH-20 (2) RP HPLC RP HPLC RP HPLC RP HPLC Xtal LH-20 (2) LH-20 (2) LH-20 (2) LH-20 (2) LH-20 (2) LH-20 (2) SiO2 SiO2 LH-20 (1) EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc MEK HP-20 EtOAc XT EtOAc partition SiO2 FB MeOH RP HPLC 18 RP HPLC 18 Chiral HPLC— Chirex— NGY RP HPLC 61 4. 1.5 Filtrate Filtrate WB Filtrate 303 F L 1 Filtrate LH20 MeOH trituration SiO2 SiO2 SiO2 MeOH trituration RP HPLC 29 RP HPLC 29 RP HPLC Prep TLC SiO2 RP HPLC 59 235 .4. 6.5 (Continued) 295 296 297 298 F F F F L L L L 14 14 14 14 Filtrate Filtrate Filtrate Filtrate 299 300 301 302 F F T F L L L L 14 14 ? 7.2.85.3 25.8 106.7. 5.9.0 WB The Isolation and Structure Elucidation of Fungal Metabolites 291 292 F 293 F 294 F S L L L 5 20 5 14 Filtrate Filtrate 9–138 12 59–103 33 28 25 7.5 283 95 101 ? 16.7.7. 111.5 23.3 131.283 RP HPLC L SiO2-MPLC 23 Filtrate EtOAc LH-20 (2) 284 T 285 T RP HPLC 31 RP HPLC 31 SiO2 LH20 LH20 LH20 Xtal RP HPLC 35 RP HPLC 35 RP HPLC 58 SiO2 CC RP 8 CC RP 8 SiO2 L L 16 16 Filtrate Mycelia SiO2 SiO2 5.8 25. 3. 23. 418. 3. 1.5. 4.9 90 286 287 288 289 T F F F L S S S 30 2 2 Filtrate WB WB WB EtOAc MeOH: acetone HP20 EtOH EtOH Aq.

43 4.5–19 311 F L Filtrate 312 F S WB SiO2 SiO2 RP HPLC 42 RP HPLC 42 EtOAc XT T L 16 313 314 315 316 SiO2 SiO2 CC RP 4 CC RP 4 LH-20 (11) LH-20 (9) F F L L 5 20 Filtrate FB WB Mycelia EHP20 EtOAc EtOAc CHCl2: MeOH EtOAc XTn MeOH: water (2 : 1) EtOAc EtOAc EtOAc XT EtOAc CCC CCC SiO2 LH-20 (2) LH-20 (1) NP HPLC SiO2 RP HPLC 317 T L 70 Mycelia RP HPLC 20 NP HLCLiChrosph er RP HPLC 28 RP HPLC 28 LH-20 (7) SiO2 9. 1.5 70–226 11–30 51.15% total batch) 2. 37 2.0.Table 4 Continued Extraction Solvent Step 1f.. 6. 8.5. 11 32. 2.5. 2.8 Harris . 7.0. 1. 3. 3. 35.5 20. 10.6.d (L) Locatione 304 305 F T S L 1 100 WB WB? 306 307 SiO2 SiO2 LH-20 (10) LH-20 (10) RP HPLC 1 RP HPLC 1 RP HPLC 5 LH-20 (7) Prep TLC MeOH EtOAc EtOAc EtOAc Acetone SiO2 RP HPLC 24 Prep TLC F F S S WB WB EtOAc EtOAc 308 309 310 F F F L L L 5 20 Mycelia WB Filtrate 5. 3.3 5.8 318 319 320 321 F F F T L L L L 3 3 2 300 WB WB Filtrate Filtrate RP HPLC 43 SiO2 rep RP HPLC 40 50 10 NR 18. 7.6 10 (0.0.9 NP HPLCHibar Si60 5. 8.3 72.5.g Step 2 RP HPLC Step 3 Step 4 Step 5 Step 6 CC RP 4 SiO2 MEK EtOAc Yieldh (mg) 236 Indexa F/Tb L/Sc Vol.9. 42. 2.

9 12 14 L The Isolation and Structure Elucidation of Fungal Metabolites 337 F 338 339 F 340 T MeOH Acetone Hexane MeOH EtOAc EtOAc XT CC C18 CHP20 EtOAc Acetone L L 200 Mycelia FB WB Mycelia SiO2 20.7. 8. 5.8. 2.2 140 341 F L 2 Mycelia Batch BioPrep P40 ODS-3 EtOAc:MEK xtn LH-20 (7) LH-20 (7) RP HPLC NP HPLC LiChrosorb Diol SiO2 SiO2 RP HPLC Fractogel EMD SO3 SiO2 342 T Acetone L 50 Mycelia hex:CH2CL2 SiO2 xtn RP HPLC 3 pptn Xtal 70.3. 800 2710 237 (Continued) . 90.3 95. 23 5. 2.3 11 14 16 27 1990 41600 CC RP 9 SP207 CC RP 7 HP20 CC RP 9 CC RP 4 SiO2 RP HPLC 34 RP HPLC 19 Xtal Xtal RP HPLC pptns SiO2 SiO2 RP HPLC 24 CC RP 9 CC RP 9 NP HPLC RP HPLC 34 LH-20 (18) CC RP 9 SiO2 rep RP HPLC 40 MeOH Amberlite IRC-50 SiO2 HP20 HP20 SiO2 SiO2 SiO2 pptn HP-20 L LH-20 (2) 300 Filtrate EtOAc 323 T L 25 Mycelia 324 T 10 T T F T T MeOH MeOH L L L L L L L 320 40 7 100 100 Mycelia/ filtrate Mycelia Mycelia WB Filtrate Filtrate EtOAc/ acetone H2O MeOH EtOAc HP-21 HP-21 325 326 327 328 329 330 331 F L 500 Mycelia 332 T L 220 Mycelia 333 334 335 T 336 T EtOAc EtOAc HP-20 MeOH→ acetone Prep TLC SiO2 RP HPLC 32 SiO2 RP HPLC L L 80 20 FB FB Filtrate Mycelia SiO2 SiO2 SiO2 SiO2 28. 1000.4 120 RP HPLC 57 132 CC RP 9 HP20 64. 70.6.322 T SiO2 7.

6.5 Filtrate flash SiO2 Kupchan Hexane xtaln SiO2 RP HPLC SiO2 NP HPLC–Nu cleosil 6. EtOAc SiO2 polyamide a F/T b L/S c Vol.6 348 349 350 351 352 353 EtOAc xtn SiO2 F F F F T F S S S S L L NP HPLC SiO2 NP HPLC SiO2 Crystallization NP HPLC RP HPLC37 LH-20 (1) SiO2 SiO2 SiO2 SiO2 SiO2 CH2Cl2/H2O part HP-21 30 1.7.d (L) Extraction Solvent Step 1 f.9. 0.1 HPLC–LiCh rogel PS 1 5.. 69.85 Harris . 9 343 344 F S 9 WB FB 345 F L Kupchan partition CH2Cl2 xtn SiO2 SiO2 SiO2 SiO2 RP HPLC 49 1.6 2.g Location e Step 2 Step 3 Step 4 Step 5 Step 6 Yieldh (mg) 7. 5.5.Table 4 Continued 238 Index Acetone hex. 10.2 356 HP-21 T L 19 Filtrate EtOAc xtn 81 357 358 F L L EtOAc XT EtOAc 2.8 63 8.8 6 4 72 9 50 8.5.5 Filtrate EtOAc LH-20 (1) CC LiChroprep Diol CC RP 4 SRP HPLC NP HPLC LiChroprep Diol RP HPLC 42 346 347 F Agar S 1 220 14.6 Filtrate Mycelia/ filtrate WB WB WB WB Mycelia Mycelia MeOH MeOH EtOAc CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 Acetone CH2Cl2: EtOH 354 T L 19 Filtrate 355 HP-21 EtOAc xtn T L 19 Filtrate SiO2 HPLC–LiCh rogel PS 1 SiO2 HPLC–LiCh rogel PS 1 HPLCLiChrosorb diol HPLCLiChrosorb diol 2.

27 21.2–5. 18.93 SiO2 HW40 CC RP 10 NP HPLC SiO2 LH-20(7) LH-20 (7) SiO2 26 134 39.1. 9 6 5–55 55 367 368 369 370 371 372 373 FB 374 F L 20 Mycelia The Isolation and Structure Elucidation of Fungal Metabolites 375 F 376 F 377 F S S L 5 WB WB Filtrate Hex.9.7 49 22.6 46 7. 3 CC RP 10 2. MeOH 60% aq. acetone EtOAc EtOAc EtOAc 6. CH2Cl2.5 2.1 EtOAc XT SiO2 SiO2 CCC RP HPLC 29 SiO2 SiO2 RP HPLC 14 RP HPLC 63 359 360 F 361 F 362 T 363 L L L L S 10 10 15 Filtrate Mycelia Filtrate WB 364 F L 100 WB Acetone EtOAc XT CH2Cl2:MeOH (1 : 1) EtOAc 365 F 366 F L L 100 20 WB? Mycelia F F F T SiO2 CHP20 LH-20(1) LH-20 (1) SiO2 HP-20 SiO2 Crystallization RP HPLC 14 RP HPLC 14 L L L L 6 5 5 5 14 SiO2 SiO2 SiO2 SiO2 SiO2 RP HPLC17 CHP20 CHP20 CHP20 SiO2 Filtrate Mycelia Mycelia Mycelia Mycelia MeOH reflux? EtOAc SiO2 CH2Cl2:MeOH CC RP 4 (1 : 1) HP-20 MeOH:acetone SiO2 MeOH:acetone SiO2 MeOH:acetone SiO2 80% acetone EtOAc xtn RP HPLC 42 88.2 382 378 379 F SiO2 LH-20 (1) S L 5 WB Mycelia EtOAc MeOH SiO2 SiO2 EtOAc trituration SiO2 EtOAc XTn LH20 LH-20 (1) RP HPLC 29 300830 64. 3.7 (Continued) 239 . 3.1.

240 a Index 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 MODCol C18 NovaPak NovaPakC18 NovaPakHR C18 Nucleosil 100-7 C18 HD Nucleosil 100 C18 Nucleosil 100 C18AB Nucleosil 100-5 C18 Nucleosil C18 ODS Fluofix IEW Z25 Pegasil C8 Pegasil ODS PhenomenexMAXSIL C18 Prep Nova-Pak HRC18 Prep Shim-Pak PREPODS RP HPLC RP HPLC .ODS Senshu Pak Senshu Pak ODS H-4251 Senshu Pak PEGASIL 120-5 Senshu Pak PEGASIL ODS Senshu-Pak ODS-N Shandon hyperprep or Novaprep C18 60A Spherisorb ODS-2 Spherisorb RP18 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 SSC ODS-SH Supelcosil ABZ Supelcosil C8 Whatman CCS/C8 or Kromasil C18 YMC AM312 YMC D-ODS-5-B YMC D-ODS-AM YMC ODS YMC ODS AM SH-343-5AM YMC ODS AM-343 YMC ODS H80 YMC ODS S-5 YMC OSA AM-323 YMC-Pak D-ODS-5 YMC-Pak D-ODS-AM YMC-Pak S-363 YMC-R-ODS-5B Zorbax C8 Supelcosil SPLC -18 Cosomosil C18 Zorbax Rx C8 Varian Bond Elut C18 Ultrasphere C18 RP HPLC Column 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Alltech Hyperprep 100 BDS Bondpak RCM C18 C18 Bond-Elut Capcell Pak C18 Capcell Pak UG Capcell Pak Carotenoid C30 Chemcosorb 5ODS-UH Cosmosil 5C18-AR Cosmosil 75C18-OPN Daiso Pak C18 BP DeltaPak C18 Develosil ODS-10 Dynamax Eurospher C-18 Inertsil ODS Inertsil PREP-ODS J’spher ODS H80 Kromasil C8 LiChroprep RP-18 LiChrosorb LiChrosorb RP-select B LiChrospher WP300 RP18 LiChrosphere RP18 Metasil ODS Harris .

LH-20 (9) hexane:toluene:MeOH (3 : 1:1). LH-20 (8) CHCl3:MeOH (2 : 1). L/S: L liquid fermentation.Index 8 9 10 11 12 13 YMC gel ODS-AM 120 S50 YMC ODS-AM Pegasil ODS Pegasil Prep ODS-7515-12A Cosmosil 140C18-OPN C18-Cosmosil 75 KP-Sil LiChroprep RP-18 ODS-Senshu Sci ODS-Senshu SS 1020T SILICAR CC-4 YMC ODS-AQ-50 14 15 16 17 18 19 Column Chromatography RP Packings 1 2 3 4 5 6 7 C18-Develosil WhatmanLPS-1 Lobar RP-18 ODS ODS-YMC-A60 RP YMC ODS b c F/T: F flask fermentation. LH-20 (13) MeOH:H2O (2 : 1). mycelia mycelium after clarification. LH-20 (3) CH2Cl2:MeOH (1 : 1). LH-20 (21) EtOH. h Yield: isolated yields of reported compounds g 241 . LH-20 (2) CH2Cl2:MeOH. LH-20 (17) 70% aqueous MeOH. filtrate filtrate/mycelia components separately extracted and then extracts combined after extraction clarified culture broth. e Location: portion of fermentation extracted to obtain reported compound. reverse phase column chromatography packings listed in footnote a (above). LH-20 (7) CHCl3:MeOH (1 : 1). S solid fermentation. LH-20 (15) hexane:CH2Cl2 step gradient. d Vol: fermentation volume harvested. The Isolation and Structure Elucidation of Fungal Metabolites LH-20 solvents: LH-20 (1) MeOH. WB whole broth. LH-20 (6) CH2Cl2:MeOH (2 : 1). LH-20 (5) hexane:CH2Cl2 (2 : 7) increasing to MeOH. LH-20 (11) isooctane:toluene:MeOH. f Steps 1–6: isolation step. A = agar. LH-20 (14) 25% MeOH. LH-20 (16) CH2Cl2:MeOH (4 : 1). RP HPLC column used as reported in literature also listed in footnote a. FB = fruiting body. LH-20 (18) hexane:CHCl3:MeOH. LH-20 (12) hexane:CH2Cl2:MeOH. LH20 (20) acetone:CH2Cl2 (2 : 1). LH-20 (19) aqueous MeOH. LH-20 (10) hexane:CH2Cl2:acetone. LH-20 (4) acetone. T tank or fermentation.

242 Harris employed to isolate fungal metabolites in a random mix of industrial. and any in between as purification. and SPE (including low-pressure reverse-phase chromatography)—accounted for the firststep isolation method for 87% of the compounds. fungal taxonomy (if identified). reverse-phase (HPLC and low-pressure columns combined) and LH-20 accounted for 73% of the total isolation steps reported for the 379 fungal metabolites (Fig. respectively (Fig. polishing. Most (56%) of the isolations used reverse-phase as the final. . 9). Three general types of information are included: 1. isolation step. Extraction information–type of fermentation (solid or liquid. although it is doubtful that most were designed this way. Figure 7 Frequency of the number of required purification steps for fungal metabolites described in Table 4. and LH-20. and 80% used between two and four steps (Fig. Isolation process—the reported sequence of isolation steps Most of the individual isolation processes fit the three-step general model described. The data in Table 4 clearly support this. The techniques used were further broken down to those reported for the first and last isolation steps to compare with the concepts of resolution and recovery and polishing steps. 8). Silica gel is a commonly used first isolation step in the literature but is generally less commonly employed as such for industrial bioactivity-guided isolation. the volume of the fermentation. liquid–liquid partition. Source information—trivial name. Thirty percent of the isolations were accomplished in three steps. extraction solvent. location of product (mycelium or filtrate). The first reported isolation step was equated with recovery and resolution. and a general description of the structural type 2. flask or tank). The inherent bias of practicing natural products chemists is that most fungal metabolites can be isolated using some combination of reverse-phase HPLC or column chromatography. academic and government laboratories. the final step with polishing. 7). silica gel chromatography. Three separation methods—silica gel. and isolated yield 3. Silica gel.

The Isolation and Structure Elucidation of Fungal Metabolites 243 Figure 8 Frequency of usage of various separation methods reported for the isolation of fungal metabolites described in Table 4. . Figure 9 Comparison of the reported separation method usage for recovery and resolution step and for polishing step for fungal metabolites described in Table 4.

or comparison with an authentic sample if available. STRUCTURE ELUCIDATION Structure elucidation is the process of proposing a chemical structure for an isolated fungal metabolite.1. peptaibols) requires less time and effort than an entirely novel compound since interpretation of the spectroscopic data is aided by comparison with that reported for known analogs. stereochemical.g. it is helpful to determine just how novel it is. Both types of ionization can be used in positive ion mode. but a general knowledge of the common families of fungal metabolites is useful. ideally. LCMS is typically used for low-resolution mass spectra of natural products because it allows correlation of the major chromatographic component of the sample with a mass spectrum. Aside from dereplication. In many cases this is less material than required for evaluating the biological activity of the isolated compound. This quantity of material is sufficient to allow the required datasets to be rapidly generated in 1 to 2 days or less. Both are chemically gentle and generally yield a strong molecular ion with little fragmentation. Thus.5 to 1 mg of pure compound. A general outline of the structure elucidation process is shown in Fig. or other positively charged adducts such as [M Na]. [M H2O H]. trichothecenes. sordarins. is sufficient to establish the structure. . A new member of a known fungal metabolite family (e. The amount of time and effort required for the structure elucidation of a fungal metabolite depends on its degree of novelty. Low resolution mass measurements are easily obtained using standard mass spectrometry methods or from HPLC mass spectrometry (LCMS) data. As a result of these advances. mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). and as mentioned above. Establishment of the Molecular Weight and Molecular Formula Establishment of the molecular weight and. structure elucidation is no longer the most challenging or time consuming part of natural products chemistry. 10 and will be further described below. or in negative ion mode to measure [M H]. If it is known then a comparison of its physiochemical properties with those reported in the literature.244 Harris 2. very little of this process is unique to fungal metabolites. Positive ion mode is more general but the presence of a negative ion spectrum suggests the presence of an acidic or other negatively charged functional group. Modern structure elucidation relies heavily on two techniques of organic spectroscopy. 2. For a new compound. Physiochemical Characterization 2. a molecular formula for an isolated fungal metabolite is essential information for determining the novelty of an isolated compound. Electrospray ionization and atmospheric pressure chemical ionization (APCI) are the commonly used ionization techniques for LCMS. Entirely novel metabolites require extensive spectroscopy to deduce the planar. Advances in these two techniques currently allow routine structure determination of new natural products using only 0. cytochalasins. Comparison of the physiochemical data for the isolated metabolite with the known structures of the same molecular formula leads to rapid recognition of a known or conversely.1. The molecular formula can be used to search natural products and other chemical databases to determine if the metabolite is known. the first step for an isolated metabolite is to ascertain whether the compound is new or known—a process called dereplication.1. to measure a protonated molecular ion [M H]. can strongly suggest that the isolated metabolite is novel.. and absolute structures.

.The Isolation and Structure Elucidation of Fungal Metabolites 245 Figure 10 Outline of structure elucidation process.

melting point (if crystalline). It is not unusual. 2.246 Harris Exact mass measurements obtained with high-resolution mass spectrometry are extremely valuable and are used to suggest or a possible molecular formula or support a proposed formula. If no natural products are known actives in the screen then dereplication is not necessary. optical rotation. Following isolation of this first active component the remaining extracts require dereplication to rule out the presence of this compound. Reference libraries of UV and mass spectra are constructed and used to recognize knowns from new sources. Fourier transform mass spectrometry is becoming increasingly available and allows accurate mass measurement to within 0. general dereplication strategies for natural product extracts have been reported and were recently reviewed [21]. and 1H and 13C 1D NMR spectra for the isolated metabolite. electrospray ionization and APCI.2. however. The tens of active extracts may actually contain only a few different active components. Greater accuracy in the measurement leads to a shorter list of potential molecular formulas. 2. Comparison of the suggested molecular formula(s) with the other physiochemical data available for the compound is required to establish the correct formula for a novel metabolite. Structure elucidation relies predominantly on NMR and mass spectrometry. . It also allows the chemical examination of many more extracts than would otherwise be possible. This can be directly determined from the number of resonances observed in the 13C NMR spectrum if sufficient sample is available. but this additional physiochemical data is useful for supporting or refuting the presence of certain functional groups in the unknown. the first natural product isolated from the active extracts is of interest.5 ppm. Early stage recognition of knowns saves the costly time and effort required for complete isolation and structure elucidation. Classically high-resolution electron impact or fast atom bombardment ionization techniques were used for high-resolution mass measurements. These are commonly based on reverse-phase HPLC with diode array and mass spectrometry detection.2.1. Dereplication is ideally applied to the crude extract or semipurified fraction as obtained from a well-designed recovery and resolution step (above). For a recent isolate of a metabolite reported before the mid-1970s. UV and infrared spectra reveal functional groups rather than the atom-to-atom connectivity derived from NMR spectra. Most useful is a count of the total number of carbons present in the molecule. For example. Numerous. to begin structure elucidation of a new metabolite without a rigourously confirmed molecular formula. An indirect carbon count is also possible from a combination of HMQC/HSQC and HMBC (see below) twodimensional (2D) NMR data. This is an iterative process as the number of known natural product actives increases. this physiochemical data maybe the only way to establish a correlation between the two compounds. It also has the advantage of being compatible with the atmospheric pressure ionization techniques. highthroughput screening of crude natural product extracts in a biological screening assay might yield tens of quality hits. Absorption Spectra and Other Physiochemical Data Complete physiochemical characterization requires the recording of UV and infrared absorption spectra. Dereplication Dereplication is the process of recognizing known natural products. The known natural product(s) depends on the target of interest. Exact mass measurements are used to suggest a potential molecular formula for isolated fungal metabolite. The complexity and wavelength(s) of the absorption maxima in the UV spectrum indicate the extent of conjugation present in the metabolite.

Structure Elucidation of New Metabolites 2. 2D NMR pulse sequences are designed to provide either 1H-1H. the observation of a cross-peak depends on the scalar coupling. The most generally useful of these sequences is heteronuclear multiple bond correlation (HMBC).3. 1H-13C or 13C-13C correlation information. dereplication in this example had 476 knowns. The target for the system was simply the recognition of new fungal metabolites in a few genera of microfungi of interest to the authors. Thus. The multiplicity. of each carbon is determined using various polarization transfer pulse sequences such as distortionless enhancement by polarization transfer (DEPT) or insensitive nuclei enhanced by polarization transfer (INEPT) [23]. The first type provides one bond 1H-13C heteronuclear connection information (i. and heteroatoms. attached to a heteroatom) present in the molecule. Data were reported for a reference collection of 476 fungal metabolites analyzed using the method. and occasionally four-bond correlation information. In this experiment. methyls.e. three-. UV.The Isolation and Structure Elucidation of Fungal Metabolites 247 Dereplication can also be a more general process. which protons or protons are attached to which carbon). olefins. Similar information can also be derived from the totally correlated spectroscopy (TOCSY) experiment. Structure elucidation is the process of connecting and correlating the atoms of the compound. known as the J-coupling. olefinic. Planar Structure Initial examination of the chemical shifts of the resonances present in the 13C NMR spectrum of a new metabolite provides a general indication of the types of carbons (e. carbonyl. a general standardized system has recently been described for fungal metabolites and mycotoxins based on liquid chromatography. this coupling has an angular dependence so that just being separated by three bonds does not guarantee observation of a correlation. Quaternary carbons are not observed using these sequences since they do not have an attached proton. Just as for 1H-1H coupling. One of the first and simplest of these sequences. aromatic. Thus. the carbons to which the protons of the spin systems established from COSY information can be determined. The second type of 1H-13C correlation information provides two-. the presence of aromatic rings. From this correlation information the various spin systems of the metabolite can be established resulting in one to several partial structural fragments. In addition. between the 1H and 13C nuclei. cross-peaks can be observed from a proton to carbons that are two and three bonds removed. For example. HMBC spectra are able to see quaternary carbons if correlated with one or more protons in the molecule. which is derived from heteronuclear multiple quantum coherence (HMQC) or heteronuclear single quantum coherence (HSQC) experiments. In constrast to COSY and HMQC/HSQC spectra. a cross-peak is observed for each pair of directly coupled protons generally indicating that they are either geminal or vicinal in the molecule.1.. and mass spectrometry data [22]. Two general types of 1H-13C correlation information can be obtained. cross peaks are observed for all of the protons of a spin system.. the chemical shifts of the protons in the 1H NMR spectrum reveals the number of methyl groups. Obtaining this correlation information relies heavily on 2D NMR spectroscopy [24]. In the HMBC spectrum. Similarly. The data from these experiments allow the correlation of every carbon-bonded proton with a carbon in the 13C NMR spectrum. This pulse sequence is extremely useful for the connection of the isolated spin systems derived from COSY and HSQC data.3.g. or the number of directly bonded protons. In a COSY spectrum. correlates protons that are directly coupled. 2. 1H-1H correlation spectroscopy (COSY). HMBC spectra are narrowly optimized for a particu- .

Difficulties arise for acyclic molecules. The general procedure is to first establish the relative stereochemistry followed by the absolute stereochemistry although if the compound is crystalline both can often be obtained using x-ray diffraction. Stereochemistry Complete structure elucidation of a new fungal metabolite requires the determination of the relative 3D orientation of each atom with respect to the other atoms of the molecule and. COSY. Large and complex metabolites or those with extreme overlap of multiple proton and carbon resonances may require additional work or more selective pulse sequences. the geminal protons of a cyclic methylene exhibit a large. The above methodology for stereochemical assignment is most applicable to conformationally rigid fungal metabolites. The classic. HSQC. Through-space interaction between the protons of a molecule also is observed. Numerous 1D and 2D NMR pulse sequences have been described to obtain nOe information. the absolute chirality of the compound.2. the coupling constant. . Analysis of these multiplets can reveal the magnitude of the J-coupling and thus their relative orientation in the molecule. and still useful. chemical derivatization. For example. Since this is a through-space interaction the observation of a nuclear Overhauser enhancement is not limited to individual spin systems. The most commonly used 2D NMR pulse sequences for observation of nOes are known as NOESY (nuclear Overhause effect spectroscopy) and ROESY (and variants). is dependent on the relative angles of the C–H bonds of the two protons. COSY-45. each optimized for a different mulitple bond JCH. Combination pulse sequence experiments such as HSQC-TOCSY are particularly useful for triterpenes and peptides that can have a large amount of resonance overlap is a small region of the spectrum making unambiguous interpretation difficult. or long-range COSY (LR-COSY)—exist that allow better interpretation of complex cross-peaks or crowded regions of the spectrum. such as polyketides.3. In many cases. Through-bond J-coupling of vicinal and geminal protons results in the splitting of the resonances observed for individual protons in a 1D 1H NMR spectrum into multiplets. 1D method for the observation nOes is the known as nOe difference spectroscopy. or molecules with an acyclic portion containing a chiral center.g. A typical set of basic 1D and 2D NMR experiments for a new fungal metabolite includes DEPT. The magnitude of this interaction depends on the relative proximity of the protons within a molecule and occurs as a result of the nuclear Overhauser effect (nOe). The magnitude of the J-coupling. respectively. Stereochemical determination relies on NMR spectroscopy.248 Harris lar multiple bond JHC. 2. and occasionally circular dichroism spectroscopy. For example. typically 10 to 12 Hz. and HMBC. and it is typical to run two or three HMBC experiments. ideally. Determination of the relative stereochemistry of a fungal metabolite relies heavily on the analysis of the through-bond and through-space coupling between protons observed in 1D and 2D 1H NMR spectra. these experiments are sufficient to propose a planar structure for a new compound. A guide to the selection of appropriate pulse sequences for such natural product structure elucidation problems has been presented [25]. This J-coupling is also observed in some forms of 2D 1H-1H spectroscopy such as double quantum filtered correlation spectroscopy (DQF-COSY) and J-resolved experiments. Variants of the COSY sequence—e. x-ray crystallography.. These are known as relative and absolute stereochemistry. coupling constant and vicinal proton coupling constant typical range from below 1 to 10 Hz. double quantum filtered COSY (DQCOSY). usually ranging from 4 to 10 Hz.

Ersson. the development of new biological assays to detect their presence.The Isolation and Structure Elucidation of Fungal Metabolites 249 numerous fungal metabolites possess linear. organic chemist approach to natural products isolation has continually demonstrated that the challenge and art of modern natural products chemistry is in the isolation of the compound. In Protein Purification. L. as well as the complete structure.Ryden. 3. Ryden. 1998. attached to a cyclic core. J.. High Resolution Methods. 4.. REFERENCES 1. ACKNOWLEDGMENTS The author wishes thank his mentor. Janson. . 2nd Edition. which are then examined using NMR methods [26]. Principles. and friend Dr. Most methods require chemical derivatization followed by spectroscopic analysis of the resulting derivative. FUTURE PROSPECTS Interdisciplinary research at the interface of biology and chemistry is an area of increasing interest to chemists. Stereochemical assignment of the core is using the above methods is usually possible but assigning the stereochemistry of the chiral center in the acyclic portion can be difficult.and S. x-ray analysis of suitable crystals containing a heavy atom. The Mosher ester method is the most widely applied and is based on formation of R. of one of the chiral centers present in the molecule. such as a Br or Cl derivative. The power of collaboration between these two disciplines has long been evident to those involved in the bioassay-guided discovery of novel fungal metabolites. The absolute stereochemistry for a fungal metabolite of known relative stereochemistry can be determined by establishing the absolute chirality.-C. The effect of the MTPA ester on the chemical shifts of the neighboring protons of the alcohol (natural product) generally allows the determination of the absolute chirality of the starting alcohol. J. The challenge to industrial natural products chemists is to increase the efficiency of the isolation of novel fungal metabolites.-C. Finally. R or S. This will require the development of automated. of a fungal metabolite.-trifluoromethylphenyl acetic acid esters (MTPA) of sec-alcohols (or amines). 3–40. such as a secondary alcohol. While industrial interest in fungal metabolites is cyclical. Ken Wilson who has been critical to the formulation of many of the ideas expressed herein. Analysis of exciton coupling in the circular dichroism spectra of a metabolite with distinct but coupled chromophores.. Introduction to Protein Purification. Janson. their potential as sources of new biological active compounds remains undiminished. Wiley-Liss): (New York..to 18-carbon aliphatic appendages containing one or more chiral centers. and Applications. or typically a di-benzoate ester derivative. B. Eds. and the ability of natural products chemists to isolate them. and high-throughput separation methods and strategies that are as robust and as elegant as a single isolation.-methoxy. can be used to determine the absolute orientation of the two chromophores and thus the absolute stereochemistry of the metabolite [27]. His rigourous. L. parallel. off-and-on supervisor over the years. The author also wishes to thank past and present members of the Merck natural products chemistry group for countless valuable discussions over two decades. Continued discovery of novel fungal metabolites depends on the ability of mycologists to isolate and culture the new fungi that produce them. can be used to unequivocally establish the absolute stereochemistry.

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9 PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers in High-Throughput Screening for Bioactive Compounds from Fungi* MARC STADLER and VERONIKA HELLWIG PH-R Enabling Technologies Natural Products Research. Previous efforts of PCR fingerprinting to identify redundancies in collectives of morphologically similar fungi are summarized. and * Dedicated to Prof. Correlations between HPLC profiling. the introduction of high-throughput screening (HTS) technologies afforded considerable changes in paradigms to keep the workflow for NPs competitive with that of synthetic and combinatorial chemistry. Germany 1. PCR-based applications. Bayer HealthCare AG. the reduction of redundancies in NP-screening libraries became more important than ever before. INTRODUCTION Even though natural products (NPs) in general and fungal metabolites in particular still play an important role in modern drug discovery. As highly automated prefractionation and subsequent random testing have widely replaced the traditional extract screening. Wuppertal. alternative methods need to be established to reach this goal with fungi. Dr. 269 . While classical chemotaxonomy constitutes a powerful tool to predict metabolic diversity in plants.(em) Wolfgang Steglich on the occasion of his 70th birthday. The application of chemotaxonomy and polymerase chain reaction (PCR)-based data in a preselection strategy for fungal strains to enhance the quality and structural diversity of the resulting sample pools is discussed.

many companies regard screening NP and synthetic products in concert to be more efficient. target enzymes. the synthetic pools available for testing drastically increased at the same time. High-performance liquid chromatography (HPLC) profiling [6. requirements for quantities and purity of NPs for structure determination have been steadily decreasing. Robot-assisted miniaturized functional assays. 1. such as when a company is doing synergistic lead structure research in agricultural and in pharmaceutical indications.7] allows for the identification of known compounds from crude extracts in the nanogram range. Screening times and costs. involving reporter cell lines. . Even for assessment of preclinical in vitro structure–activity relationships. or receptors. mass spectrometry. Wherever possible. Microarrays appear promising to even further reduce the requirements for sample quantities [5]. and sample preparation are additional features to accelerate the process of the screening workflow. Because of the developments in combinatorial chemistry and parallel synthesis. the process of NP screening underwent some major changes.1. as well as amounts of test samples and biological assay kits needed for primary screening. Alternative readouts such as fluorescence resonance energy transfer and fluorescence polarization are readily available for automated recording of test data [2–4]. The same capacities for generation of NP extracts. chromatography.e. can now be performed in 384-well microtiter plates (or in so-called ultra-HTS using 1536well microtiter plates). and liquid chromatography coupled with spectral techniques. in vivo studies. and pure compound libraries may even be shared by several business units. rendering the work on NP highly compatible and competitive to HTS of substance pools from chemical synthesis or combinatorial chemistry. have decreased considerably. Meanwhile. automated techniques were introduced for downstream processing of extracts as well (Fig. 1). The identification of an appropriate lead structure from natural extracts therefore lasted several months or even years.270 Stadler and Hellwig classical morphology are exemplified by the outcome of a recent polyphasic study on Daldinia and allies (Xylariaceae).. Change of Paradigms: Why Are Natural Products Compatible with High-Throughput Screening? The current chapter will address the role of NPs—and fungal metabolites in particular—in the HTS procedures that were inaugurated in pharmaceutical and agricultural lead structure research [1]. Technologies relating to automated preparation and distribution of samples for random biological testing are being constantly refined and optimized. Because of adjustments following the introduction of HTS. which usually precedes the large-scale preparation of lead compounds for evaluation in second-line test models (i. making use of the same logistic prerequisites. Automation of extraction. These efforts were paralleled by adaptations of data management. endowing the revolutionary developments in computer technologies. fractions. The relationships between chemotaxonomy and molecular phylogeny are illustrated by some examples of Boletales. Structure elucidation of novel NP can now be done by using less than 1 mg of the enriched compound. A large number of preliminary test data were needed during bioassay-guided fractionation before the active compounds were finally identified. The traditional workflow led from biologically active crude extracts via bioassay-guided fractionation to the identification of lead compounds. Even though special NP-adapted screening approaches appear feasible for HTS. due to further developments in the fields of nuclear magnetic resonance spectroscopy. Future perspectives for dereplication and screening of fungi in HTS are discussed in relation to the characterization of secondary metabolite gene clusters. This prolonged the time for evaluation in screens with high hit rates and/or low sample throughput.

PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 271 Figure 1 Schematic workflow involving the discovery of bioactive natural products in highthroughput screening. .

272 Stadler and Hellwig etc. Galiellalactone from Galiella rufa and papyracillic acid from Lachnum papyraceum and its unprecedented fibrinogen-lowering adduct. respectively. On the other hand. (From Refs. easily accessible main metabolites appear helpful to complement the diversity of synthetic compound libraries. the purity rather than the quantity of a given NP represents a limiting factor. It did not share the reactivity of the parent compound but instead showed potent fibrinogen-lowering Figure 2 Compatibility of natural with combinatorial chemistry and high-throughput screening.) . 2) resulted from the reactive fungal metabolite papyracillic acid as a side product of a conventional acetylation procedure. 9 and 23. Such libraries of representative. many NPs are readily available in gram scale and can thus be utilized as synthons to be modified by directed or random chemical derivatization. For example. such chemical entities would be difficult to obtain from random isolation programs aimed at the purification of prominent compounds only. a novel N-heterocycle (Fig. Several industrial and academic groups specialized on the provision of technology platforms to create large libraries of prepurified NPs [8]. resulting in ‘‘chimera’’ of natural and synthetic products with unprecedented biological properties. this approach is not likely to replace the traditional and advanced extract screening. Nonetheless. Evidently. Data on selectivity and potency can now be obtained using minimal amounts. More than 50 years of experience with NPs clearly showed that the most interesting bioactive NPs are frequently formed as minor metabolites by particular organisms.).

While test capacities are no longer limited.. like kinases [13]..g. The endowment of NP scaffolds and pharmacophores to produce large semisynthetic libraries for enhancement of the chemical diversity in synthetic pools is feasible [11. In cases of synthetic compounds and pure NPs. the handling of many untapped and ‘‘creative’’ but slow-growing fungi from which unprecedented leads may be expected in future (e. that is. strain preservation and fermentation (and refermentation of hits) will constitute the major bottlenecks. Screening hits can thus be clustered according to their structural properties and selectivity.and cost-intensive follow-up assays now have to be used in a second line of biological evaluation. or in parallel reporter cell assays using similar signal transduction cascades. and. Refermentation. Frequently. These circumstances imply that false positives need to be excluded at an early stage.g. optimization of production. which usually show similar growth characteristics and can be maintained easily. Thus. are required to make use of the automated techniques by working simultaneously on several projects to keep pace with the steady flow of newly generated test data. These will eventually be flagged as nonspecific (false-positives). for example.12].PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 273 activities [9. HTS also means a high throughput of assays and lead evaluation projects. usually not allowing for more than 6 months from reconfirmation of primary hits. Such a procedure is practical. with batches of randomly isolated soil isolates). as well as microbiologists and NP chemists. it is important to focus the work-up procedure on those samples that contain novel compounds and to avoid redundancies within one screening project. Therefore. combinatorial chemistry and NP research should actually be seen as complementary rather than competitive approaches to increase the probability of success. let alone automate. derivatization and mimetic syntheses to improve selectivity and potency of the lead compound and establish structure–activity relationship (SAR). the timelines have become rather narrow. all remain necessary. only small-scale cultures are usually prepared for primary screening to enhance the throughput of fermentation.10]. until a decision about the most promising lead structure is made. Still. Even the best hit in HTS will be useless if the producer organism is no longer available. as well as microbiological know-how. information on chemical structures and previously noted bioactivities can be compiled in a database. and they frequently overproduce mycotoxins. These are sufficient for testing over a period of several years. If large strain collectives of fungi are used in this process. Because the initial sample amounts required for HTS decreased. and only a few selected hits can usually be evaluated intensively. However. In contrast. possibly. Screening laboratories. thus prohibiting their further evaluation. These organisms sometimes need special culture media and may show highly varying growth rates. HTS of NP extracts requires considerable adaptations and improvements of the quality and the quantity of samples. the . This selection process is best performed prior to the screening. such strains are already well investigated on their secondary metabolites. scale-up. this implies that cultures used for screening have to be preserved well. Therefore. the time. However. Such information is very useful to establish selectivity profiles of screening libraries. and their evaluation is therefore difficult to synchronize. Some may produce secondary metabolites only after several weeks. after identification of lead candidates from primary hits. basidiomycete cultures) can hardly be automated but requires skilled staff and mycological. such as in the course of a search for specific inhibitors of pharmacologically relevant enzymes. Parallel fermentation of a large number of fungal cultures will be less of a problem (e. Adequate database management will allow for exclusion of samples showing. general cytotoxic effects in cellular screens. Hence. the number of hits from HTS is reduced by statistical approaches or dereplication by comparison of activities in several parallel biochemically related assays.

Imperatively. NP research thus appeared less competitive with combinatorial and other synthetic approaches [15]. the acquisition of NPs for bioprospecting has been rendered more complicated by the implementation of the Convention of Biological Diversity (CBD). or other means of sample preparation. especially in the era of modern HTS. The impact of these aspects was recently discussed in the course of a conclusive analysis as to the perspectives of the exploration of tropical microfungi in industrial screening [14] (see Chap. once the biological function of orphan genes have been identified [18]. Powerful and selective natural inhibitors of cellular signal transduction were already found in fungi. the identification of the potential lead structures will be prevented by unacceptable primary high hit rates. If screening results on natural and synthetic products are evaluated in concert by statistical methods. there are good reasons to continue with NP research. the number of structurally unique biochemical tool compounds from nature that constitute potent modulators of enzymes and receptors is steadily increasing [21]. In fact. Such discoveries clearly constitute a benefit for the intellectual . At times. Nonetheless. unless prefractionation techniques are employed. Fractionation. phosphate) may at times interfere with particular readouts. this compound was initially found as an inhibitor of a herbicidal target [23]. An increasing number of new ‘‘screenable’’ pharmaceutical targets are evolving from genomic approaches aimed at the identification of orphan genes encoding for pharmaceutically relevant receptors and proteins. especially in the field of chemotherapeutics and antibiotics [19]. interacting rather specifically with the IL-6–induced JAK/STAT pathway [22]. This applies to aforementioned cytotoxic agents and to those that disturb fluorescence-based readouts because of their physicochemical properties. The number of valid targets is thus expected to increase from the present 500 to several thousands. the probability of finding a follow-up lead in a NP extract will consequently increase with the overall percentage of NP samples in the screening. In these cases. Historically. However. several pharmaceutical companies substantially reduced or even terminated their efforts on microbial screening in a belief that newly evolving synthetic technologies would make the NP approach expedient. is also necessary for some commonly used HTS assay formats that are not practicable for crude extracts.17]. no estimate of the percentage of currently accepted pharmaceutical targets that were found by NP screening approaches has ever been made. ergosterol. crude extracts will sooner or later be flagged as nonspecific.274 Stadler and Hellwig primary screen may already allow for the deduction of preliminary SAR. element-driven reporter gene in the nanomolar range. Finally. 2) from the sarcosomataceous ascomycete Galiella rufa inhibited the expression of an IL-6–responsive. employing the new generation of reporter cell screens. Moreover.[Ando]). If such compounds are present in abundance in the sample libraries. cometabolites with specific bioactivities present in the same extract may not easily be found. galiellalactone (Fig. Even ubiquitous primary metabolites and constituents of fermentation broths (fatty acids. there are countless examples where the elucidation of the biochemical mode of action of a particular natural compound—rather than a synthetic chemical—led to new targets. indicating that they should be used in concert to increase the chances of the lead finding process [16. To our knowledge. some classes of secondary metabolites frequently disturb the screening as well. statistical evaluations using computerized comparisons of structural moieties of synthetic/ combinatorial versus natural libraries showed that both pools are complementary. For instance. A recent overview further emphasized the importance of NPs as templates for drug discovery with focus on anticancer agents [20]. Therefore. Interestingly. which is obviously not the case with NP extracts or their fractions. even if they contain merely one nonselective or cytotoxic agent. Several hundred thousands of chemicals may now compete with a fairly limited number of materials from natural sources.

However. even these powerful methods will fail if the redundancies in the sample libraries become too high. Hence. Chemotaxonomy of Plants and Fungi for Preselection In agreement with the procedures used for synthetic chemicals. new target-based screens are easier to establish if a standard inhibitor is already available as biochemical tool. Such information can be linked to proprietary and literature data on biological activities. since new targets and assays are patentable. and activity profiles of these fractions are stored in a database. and so forth. This will even be enhanced by the increasing number of screening samples per strain. and the efforts of the intensified evaluation can be focused on hits containing yet unknown components. Therefore. including mycotoxins. Capacities and manpower for fermentation. database management. As discussed further below. has become a valuable tool in most pharmaceutical and agricultural companies dealing with HTS of NPs. Also. A literature survey for information on the taxonomy of species to identify taxa that are particularly interesting with regard to their secondary metabolism appears promising as a precondition to decide on their use in screening programs. which multiply by HTS-adapted prefractionation. The ratio of producers of nonselective agents and duplicate strains thus needs to be minimized at an early stage to increase the predictability and efficiency of the screening process. It is therefore unavoidable that such samples cause undesired redundancy. Dreyfuss and Chapela [27] established a creativity index. As the individual metabolite patterns can hardly be predicted. It is difficult to envision how modern pharmaceutical and agricultural lead structure research could do without them! 1. Still. relying on previously published information on fungal secondary metabolites. To meet the requirements of HTS. basidiomycetes and loculoascomycetes were not among the most creative taxa they identified because these groups had been widely neglected at that time. NP-adapted HTS can be performed in the following manner: the automated preparation of ‘‘standardized’’ and prefractionated samples from crude extracts replaces the traditional screening of extracts. secondary metabolites and other features of the current pools constitute an important prerequisite for preselection of biological sources to be acquired. Then again. The full metabolic capabilities of microbial organisms are only expressed upon considerable variation of culture conditions. automated fractionation. several samples are prepared by different fermentations from each strain. some secondary metabolites. can thus be used more efficiently. along with HPLC-based dereplication of crude extracts. Databases containing information on the taxonomy.2. The isolation and structure elucidation of known nonselective compounds becomes expedient if they are found by early-stage dereplication. it appears crucial to reduce the surplus in microbial strain collections even prior to fermentation and extraction. handling of samples.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 275 property situation in pharmaceutical companies. As many as 250 screening samples result from each strain if it is propagated in five-culture media and each of them is processed into 50 semipreparative HPLC fractions. may be formed in a broad range of culture media. This ultimately allows for comparison of hits in several assays in conjunction with a spectral library to verify the identity of the bioactive constituents already on the stage of the crude extracts by physicochemical dereplication [24–26]. One challenge of microbial HTS is the deliberate increase of the metabolic diversity in a given screening library by adding new extracts from sources that are complementary to those already available in the NP pools. just counting numbers of previ- . the adaptation of NPs to meet the requirements of HTS appears more promising once again.

It is virtually impossible to exclude them by prefractionation. their producer organisms are best to be avoided. recent reviews on fungal metabolites include fungal pigments [28]. alkaloids. For reasons discussed above. they were employed to search for entries on various taxa. In contrast to BNPD.. and the Vinca alkaloids [36]. The value of chemotaxonomy for preselection of biological sources is best demonstrated with seed plants.’’ ‘‘nematicidal. The data contained in this free-format.’’ ‘‘anthelminthic. camptothecin. which also means a high degree of redundancy. while others only interfere with the screening process. data on bioactivities and taxonomy are provided in DNP as comments in free-text format. volatiles).’’ ‘‘nematocidal. BNPD contained no chemical structures and was not compatible with modern software applications. Metabolites may be sorted according to their chemical types. which frequently are contained in the crude extracts as mixtures of congeners with varying polarity. However. it does not contain entries on compounds from higher plants or marine organisms. polyketides. It provided valuable features. such as the clustering of entries according to reported bioactivities. polyphenols and other aromatic compounds will usually show broad and nonspecific biological activity profiles. In contrast. Table 1) resulting from queries on entries for bioactive plant metabolites in BNPD are used to illustrate this preselection strategy at subclass and family levels. a search in DNP for entries on metabolites from a certain taxon frequently yields a lot of primary and other irrelevant metabolites (sugars. such as Antibase [32]. the fact that BNPD contained entries only on biologically active compounds was of particular advantage for queries related to the process of drug discovery. the patterns of biogenetic diversity may be restricted to the production of several congeners of the same type (rather than the simultaneous biosynthesis of manifold chemical types!). exemplified by the anticancer agents. a query for nematicides from fungi in DNP involves searching for ‘‘nematode. a kind of darwinistic chemotaxonomic approach appears feasible to achieve survival of the fittest taxa in the screening libraries. or nematicidal and insecticidal agents [31]. which could be searched using well-defined identifiers. While particular subclasses of Angiospermae [35] are extremely rich in polyphenols (e. Many HTS readouts are sensitive to these nuisance compounds. taxol. Most of these have been studied thoroughly for secondary metabolites and chemotaxonomic affinities. 3). In many cases. Bioactive Natural Products Database (BNPD). text database were meanwhile incorporated to some extent into DNP. basidiomycete cultures [30]. respectively. The BNPD was once the only comprehensive computer database on NP.’’ ‘‘antiparasitic. chemical types of secondary metabolites. Polycyclic triterpenes—and. lignans). amino acids. however. In the current study. Thus. Several classes of NPs have proved to be suited for evaluation as lead structures. The latter chemical types include numerous examples for lead structures. and it should be more than sufficient to keep characteristic representatives in the screening libraries. Moreover. and metabolites of mixed biosynthetic origin (Fig. DNP and Antibase include chemical structures and provide various different search features and browsing functions. Thus. others are known to contain unique terpenes. that is.’’ ‘‘nematocide. among those.276 Stadler and Hellwig ously isolated metabolites from a given taxonomic group does not necessarily suffice to achieve sound preselection criteria. The most comprehensive information of this kind is contained in commercially available NPs databases. and Dictionary of Natural Products (DNP) [33]. and taxonomy of biological sources. compounds from loculoascomycetes and pyrenomycetes [29]. the most comprehensive database of this kind [34]. Some examples (Fig. especially the steroids and the saponins—are other examples . flavonoids. In contrast.’’ and various expressions circumscribing fungi before all entries have been found as desired.g. Antibase focuses on microbial metabolites. in some cases including invalid and formerly misapplied synonyms. 3.

Liliopsida have not been further divided into subtaxa. since it is intended only to illustrate a general strategy to classify plants according to chemotaxonomy and potential in pharmaceutical lead structure research. The same is true for some alkaloids (e. in which a particularly high chemical diversity may be expected from the numbers and chemical types of previously reported bioactive secondary metabolites. Their occurrence was in basic agreement with morphological features—and even with molecular data as inferred from analyses of the rbcL gene.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 277 Figure 3 Classification of the major subclasses of Angiospermae (Magnoliophytina) according to quality of predominant secondary metabolites as lead structures. the results of Wink and coworkers [37] demonstrated the presence of metabolite families in various groups of Leguminosae.g. families such as Rubiaceae and Asclepiadaceae contain many structurally unique alkaloids. which are preferably found in Caryophyllales). many taxa are included in Asteridae s. of NPs that from experience appear less promising as lead candidates. Examples for families and orders that are exceptionally rich in ‘‘drugable’’ secondary metabolite classes in otherwise less interesting taxa are printed in bold. (Table 1) contains several families that preferably produce alkaloids. and the criteria should be refined by searching for respective data at the generic and subgeneric levels. polycyclic aromatic benzylisoquinolines that are prevailing in Ranunculidae and Magnolidae except Laurales. In contrast. Within this subclass. lat. The Leguminosae would be regarded as rather unsuited for HTS in the currently presented preselection process. Several taxa that preferably contain unique alkaloids or different flavonoid types were thus identified in evolutionary . the number of species per family should be considered. the Rosidae s. the polyphenols are predominant. while the Asteraceae are among the richest sources of terpenes and polyketides. [35]. considering the large amounts of flavonoids and other aromatic compounds known from this large superfamily. while. lat.. and the betains. Queries were made in BNPD (on bioactive metabolites) and DNP (on all entries of secondary metabolites) [33].. The taxonomy follows the system presented in Sitte et al. In comparison. However. the basic Asteridae such as Ericaceae (7 alkaloids and 60 aromatic compounds in BNPD) or the families of Cornaceae and Verbenaceae (no alkaloids but 25 aromatic compounds in BNPD) appear less interesting. in other groups. For instance. The scheme is simplified. Evidently.

950 1.S. quinones) 40 25 70 1 None 86 19 13 434 7 7 84 28 29 53 139 28 5 9 7 3 25 10 17 Family Anacardiaceae Burseraceae Clusiaceaeaa Crassulaceae Cucurbitaceae Euphorbiaceae4 Fagaceae Geraniaceae Leguminosaeb Loranthaceae Melastomataceae Moraceae Myrtaceae Rhamnaceae Rosaceae Rutaceae Salicaceae Sapindaceae Saxifragaceae Simaroubaceae Tiliaceae Thymelaeaceae Violaceae Zygophyllaceae 4 Estimated No. of Alkaloids None None None 2 None 2 None None 60 3 None 2 1 9 None 82 None None 1 21 2 None None 5 No.100 1..500 760 7. b BNPD allows for search only on the superfamily “Leguminosae. taxonomy according to Ref.700 435 1. [35]. that are underrepresented in existing libraries. New acquisitions should focus on those taxa for which many structurally unique metabolites are known. lat. of Other Metabolites. A continuous check of the current libraries against potentially interesting taxa to be newly attained can be done using chemotaxonomic criteria. polyketides) 15 (terpenoids) None 18 (terpenoids) 20 (terpenoids) 3 (macrolactams) 13 (triterpenes) 15 (terpenoids) 5 (terpenoids) 8 (triterpenes) None 144 (terpenoids) 2 26 (terpenoids) 7 (terpenoids) None AU: significance of bold in this table? Previously reported bioactive secondary metabolites from some important families of Rosidae s. basic and in advanced tribes of Leguminosae. a Including Hypericaceae. flavonoids polyphenols.g. it was concluded that the value of secondary metabolites as taxonomic markers may be limited by the reticulate nature of their metabolic expression.. sorted according to chemical types.278 Stadler and Hellwig Table 1 Results of a Database Search in Bioactive Natural Products Database No.000 1. Such criteria may be used for further acquisitions and for prioritization of stock materials to be used for screening and prefractionation.” actually consisting of the families now included in the order Fabales.350 1.000 720 16. Source: M. From the outcome of these studies. of Aromatic “Nuisance’’ Compounds (e./BNPD.000 875 3.280 3. and that .300 450 725 720 830 250 No. of Species 850 540 1.000 940 4. (prevailing type) None 12 (triterpenes) 24 (aromatic terpenes) 5 32 triterpenes 153 (terpenoids) None 3 (triterpenes) 31 (terpenoids.

relying on samples from the Bayer extract pools. These examples clearly reveal the taxonomic significance of the unique terpene structures.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 279 have not been studied much for secondary metabolites.46–50] give further examples as to the potential taxonomic relevance of certain basidiomycete metabolites. Even extraction protocols and prefractionation methods may be modified to enrich the basic alkaloids in a given plant and separate them from their undesired acidic phenolic co-metabolites prior to screening (the latter may be discarded).45]. fruitbodies of many species may not be easily collected in sufficient amounts to meet the requirements for stock samples necessary for follow-up work after HTS. Chemotaxonomic relationships in basidiomycetes have been investigated for several decades. and Trichoderma. In the first place. entries on metabolites from some genera of plant-associated and soilinhabiting conidial fungi are compiled from Antibase. Within the ascomycetes. the fruitbodies of most basidiomycetes. Some chemotaxonomic correlations already . have been reported to contain different secondary metabolites.000 plant species that are now believed to exist [38]. The latter genus is now under revision and many species have been removed from it [52]. and none or only a few of their secondary metabolites are known. HPLC profiling was carried out according to a previously described method [51]. Penicillium. only the Boletales are treated in detail further below. At least the fruitbodies of some species may be made available for screening. as well as some ascomycetes [40]. restrictions concern some groups of fungi or their respective life cycle stages that are to be excluded for practical reasons. This practice facilitates selection and adequate handling of the most promising ones out of the approximately 422. An analogous taxonomic preselection of fungi as sources for HTS is perhaps more difficult. Stable cultures of obligate plant and invertebrate pathogens and many mycorrhizal basidiomycetes can apparently not be established. Many other genera appear underrepresented in these databases. Fruitbodies and cultures should thus be regarded as complementary sources for fungal secondary metabolites. all of which have rarely or never been encountered in other biological sources. Several hundreds of compounds are known from each of the genera Aspergillus. and more than a hundred were reported from Alternaria and fungi named Verticillium. but studies on other secondary metabolites resulted in the discovery of a surprisingly high diversity of new carbon skeletons and other unprecedented chemical moieties. Fusarium. The results have been mainly based on their pigment chemistry. Perhaps because of changes in differential gene expression during morphogenetic events. and the pleosporaceous Alternaria species [44. A comprehensive overview on the state of the art in fungal chemotaxonomy was given by Frisvad et al. Out of this context. mediated by signal transduction processes that are not yet fully understood [39]. For instance. Table 4 gives some examples for the chemotaxonomic significance of fungal terpenes as judged from data presented in Antibase. [41]. chemotaxonomic surveys were mostly carried out in Xylariaceae [42] and in mycotoxin-producing taxa and important groups of plant pathogens such as the Trichocomaceae (see [6]). despite their importance in other biotechnological applications. In Table 2. and our own experience.30. and it appears worthwhile to focus on both. DNP. However. Several reviews [28. or their saprophytic relatives can be used for screening of mycelial cultures. the genus Fusarium within the Hypocreaceae [43]. Aside from these aspects of general availability and suitability. most taxa of higher fungi can still be used for screening purposes and can be assessed on their chemical diversity. ascomyceteous yeasts and zygomycetes (but certainly not basidiomycetes as stated by Dreyfuss and Chapela [27]) are indeed known to be rather poor in secondary metabolites.

Gliocladium roseum) Corynespora Cristulariella Curvularia Cylindrocarpon Cylindrocladium Dendryphiella Dictyochaeta Embellisia Epicoccum Fusarium Geotrichum Humicola Memnoniella Microdochium Acremonium (incl. Gliocladium virens) Trichothecium Ulocladium Verticillium No. Cephalosporium p.p. Gliocladium roseum) Corynespora Cristulariella Curvularia Cylindrocarpon Cylindrocladium Dendryphiella Dictyochaeta Embellisia Epicoccum Fusarium Geotrichum Humicola Memnoniella Microdochium a Genus Mycoleptodiscus Mycosphaerellaa Myrothecium Nectriaaa Nigrospora Paecilomyces Paracercospora Passalora Penicillium Phaeoisariopsis Phaeoramularia Phialophora Phymatotrichopsis Pseudocercospora Pseudocerocosporella Ramularia Scolecobasidium Stachybotrys Stemphylium Trichoderma (incl. [33]. Source: Ref. Gliocladium virens) Trichothecium Ulocladium Verticillium Mycoleptodiscus Mycosphaerellaa Myrothecium Nectriaa Nigrospora Paecilomyces Paracercospora Passalora Penicillium Phaeoisariopsis Phaeoramularia Phialophora Phymatotrichopsis Pseudocercospora Pseudocerocosporella Ramularia Scolecobasidium Stachybotrys Stemphylium Trichoderma (incl.280 Stadler and Hellwig Table 2 Entries on Secondary Metabolites from 46 Anamorphic Genera of Plant-Associated Ascomyceteous Fungi No.p. Those genera which have yielded more than 100 compounds are printed in bold. . Cephalosporium p.) Alternaria Aspergillus Botryosporium Botrytis Cercospora Chalara Cladosporium Clonostrachys (incl. of Reported Metabolites 0 28 72 49 8 42 None None 609 None 0 17 None 15 None None 2 43 20 243 31 2 172 0 28 72 49 8 42 None None 609 None 0 17 None 15 None None 2 43 20 243 31 2 172 Corresponding teleomorphic genus.) Alternaria Aspergillus Botryosporium Botrytis Cercospora Chalara Cladosporium Clonostrachys (incl. of Reported Metabolites 69 110 753 None 58 50 3 48 10 1 None 63 28 11 19 1 None 10 290 3 18 1 3 69 110 753 None 58 50 3 48 10 1 None 63 28 11 19 1 None 10 290 3 18 1 3 Genus Acremonium (incl.

These may be selected from diverse ecological habitats and geographic localities in an attempt to increase the metabolic diversity in the screening pools by tapping the higher biodiversity to be expected from a broad range of isolation strategies and sampling techniques [55]. compactin. could not be classified at all. most compounds previously obtained from Trichoderma are oligopeptides of the peptaibol type ( 200 entries in Antibase). it had been in use by mycologists until 1975 [54].. respectively. Aspergillus. Aside from such name changes. which will be discussed in detail further below.. It would be of special interest to focus on such species of these genera that do not or not only produce mycotoxins. the fungi now classified under Hohenbuehelia and Clitopilus were still included in Pleurotus in the 1950s. Therefore. many industrial companies have so far relied on large numbers of non-identified strains for screening. This problem of strain-specificity may be due to inadequate species concepts. Penicillium. With some exceptions reviewed further below. Myrothecium is notorious for its macrocyclic trichothecenes ( 50 entries in Antibase). Even if all reports on the taxonomy of producers ever published in the course of NP discoveries had been correct. and Fusarium species are well-known producers of mycotoxins. Such criteria should definitely be regarded as valid for preselection prior to HTS. Singer already recognized in 1951 that the generic name Drosophila was an invalid synonym for Psathyrella. mevinolin. Despite the fact that new evidence was obtained in the meantime to change the taxonomic position of the producers.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 281 become fairly evident at the generic level. the nematophagous conidial basidiomycete Nematoctonus robustus [53].’’ Another example given in Table 3 (Omphalotus and illudins) is discussed further below. which. a given taxonomy can even be misleading because of morphological similarities that are not paralleled by secondary metabolite production. The genus Hohenbuehelia and its anamorph. These substances cause membrane damage in mammalian cells and will thus disturb cellular reporter screens. drastic changes now inferred from molecular taxonomy and phylogeny would still have to be dealt with. which interfere with the biosynthesis of macromolecules. A database-aided retrospective chemotaxonomy becomes even more complicated because many secondary metabolites were reported several decades ago. not even the genus of the producer strains was reported along with new interesting metabolites. unpublished). Sometimes. there are several cases of misidentifications of producer organisms in the literature. Indeed. reports on novel biologically active agents do not necessarily include the taxonomy of the producers at species level. too. neither the trivial names nor the respective database entries were updated according to the current knowledge. Pleuromutilin and derivatives were repeatedly found in Clitopilus spp. until recently. Accordingly. For instance. it is no wonder that NP chemists named several compounds from Psathyrella species ‘‘drosophilins. such as cyclosporin.S. are the only organisms so far known to produce antibiotics of the pleurotin type. in the current sense do not contain such compounds. the database entries from these genera and Trichoderma also include useful bioactive agents. and further unique chemical entities that were never obtained from other biological sources. It is now well-established that Pleurotus spp. Evidently. In several other cases. As mentioned before. penicillins. which are not discussed in detail here. including the nonpleurotoid Clitopilus prunulus (M. their secondary metabolites were named pleurotin and pleuromutilin. Still. they relate to the taxonomic approach . Some of them were obtained from fungi growing as sterile mycelia in culture. Because of their pleurotoid habit. Albeit R. not all researchers dealing with the discovery of new fungal metabolites are also experts in the taxonomy of the organisms they deal with. Some of the first antibiotics discovered in basidiomycetes are compiled in Table 3.

and other Basidiomycetes Drosophilin C Drosophila Psathyrella spp. the collection to which they shall ultimately be added—will constitute a . drosophilin B Current (and originally reported) Taxonomy Pleurotus Clitopilus spp. the morphological dereplication of such large strain contingencies hardly appears feasible in a cost-efficient way. Cannon and Simmons [56] recently reported that of more than 2500 samples collected from 12 tree species in Guyana. and associated anamorphs (Nematoctonus) Drosophilin A Drosophila Psathyrella spp. such strain contingencies will usually contain a high degree of redundancy as well. biotopes. However. Drosophila Psathyrella subatrata Pleurotin Pleurotus Hohenbuehelia spp. or hosts. duplicates among a series of cultures grown from the same plant or soil sample may be recognized by morphological or cultural characteristics. whereas a further 52 of their isolates were characterized as sterile mycelia. In the course of the isolation procedure from the natural habitat. almost as many endophytic fungal cultures were obtained. Illudin M Clitocybe illudens Omphalotus olearius Pleurotus japonicus Lampteromyces japonicus because adaptive radiation of species is driven by ecological factors and geographic isolation. but those belonged to only 64 morphotypes.e.. Assessing their novelty with respect to the existing strain contingencies—i. For instance. As prerequisite for HTS. when isolated in large numbers from similar habitats.282 Stadler and Hellwig Table 3 Some Antibiotics of Basidiomycetes: Current and Outdated Names of Their Producers Structure Trivial Name Pleuromutilin.

They proved especially helpful in differentiating groups of fungi that are poor in morphological characters. Fujimori and Okuda [67] compared 74 strains of Trichoderma spp. Anamorphs of creative secondary metabolite producers in the Clavicipitaceae. the number of mycologists involved is not likely to increase as rapidly as the number of newly obtained uncharacterized strains to meet the requirements of HTS. They noted correlations between secondary metabolite profiles and RAPD data but stated that only a part of their species that they identified by morphological methods as T. Brief [64] and comprehensive [65. Hypocreaceae. on their ability to produce isonitrile antibiotics and their random amplified polymorphic DNA (RAPD) profiles.html) and related databases is growing rapidly.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 283 problem. will once facilitate the discovery of new nature-derived therapeutic agents. who ¨ studied 64 isolates identified as Chaunopycnis alba for their secondary metabolite profiles in conjunction with growth characteristics and other characters. Such techniques have also found broad application in plant pathology and for indirect characterization of mycotoxin producing fungi [63].3. and the role of taxonomy and mycology as a core disciplines in NP screening is nowadays widely accepted [60]. The first applications of PCR fingerprinting in relation to industrial screening date back less than 10 years. Complementary methods should be endowed to achieve a preselective classification similar to the one described above for the chemotaxonomy of higher plants. Molecular methods frequently confirmed hypotheses that were based on ecological. morphological. RAPD patterns. Some companies have actually employed mycologists and spend great efforts to enhance the taxonomic diversity in their collections [59]. and only few experts are still acquainted with them. There is good reason to assume that the wealth of information on molecular data on the internet. [68] studied 15 isolates of Fusarium compactum for metabolite profiles. Talbot et al. The number of sequences deposited in GenBank (http://www. 1. including minimization of human capital. and ultrastructural investigations.edu/general/software/packages/genbank/genbank.66] general overviews on these topics are readily available. along with further information relating to biodiversity [61]. PCR Fingerprinting: A Valuable Alternative PCR fingerprinting and DNA sequencing have become state-of-the-art methodology in many disciplines of mycology and biotechnology. Another remarkable early application of RAPD and PCR primers for the cyclosporin synthetase gene was published by Moller et al. However. Other groups with high potential as untapped sources for novel bioactive agents (e. They used parsimony analyses of both the data on molecular characters and metabolite profiles and concluded that genetic features would be suited well to optimize the chances of identifying a wide range of metabolites from a given species. it should be kept in mind that among other reasons.g.58]. ecological groups like endophytes of seed plants and insectassociated fungi do not show many discriminative characters in culture.psc. HTS was invented to reduce the costs of the screening process. viride were able to produce the characteristic antibiotics. and by restriction fragment-length polymorphism (RFLP) methodology. They found a broad diversity among the isolates of this species from different habitats and localities and suggested . Even unculturable species such as obligate parasites can now be studied for molecular taxonomy as specific PCR-based methods allow for their in situ characterization using minimal amounts of material [62]. harzianum and T. and Xylariaceae are difficult to discriminate by morphological characters. [69]. Hence. Predominantly. Helotiales [57. from which manifold unique compounds have already been reported) are strangely being neglected by taxonomists.. Trichocomaceae.

alba to the Clavicipitaceae have only recently become evident: C. this has already been accomplished with Fusarium (see below).76. 4) and structurally related aromatically substituted drimane terpenes. While none of the latter studies included chemotaxonomic parameters. [32. The other aforementioned examples of RAPD studies in conjunction with morphology and secondary metabolites dealt with anamorphic Hypocreaceae. Recent evidence gained from molecular data suggests that the genus Trichoderma and the taxa studied by Fujimori and Okuda [67] will soon be segregated into several further species [72–74]. the taxonomy of this fungus did at first not become clear from morphological data but was ultimately deduced from a comparison of metabolite profiles and DNA sequences. The characteristic metabolites of Stachybotrys and Memnoniella are satratoxin and other trichothecenes in addition to numerous spiro-drimane indene terpene alkaloids (Fig. pustulata. a new species of this genus. In this context. These chemotypes of drimane producers and trichothecene producers. whose taxonomy is about to change considerably. chartatum [75]. respectively. The distribution of secondary metabolites in these fungi was recently investigated in isolates from waterdamaged buildings. were exam- Figure 4 Some characteristic metabolites of Stachybotrys spp.77]. The affinities of C. Interestingly. [69]. This work resulted in the discovery of an undescribed species. employing the phylogenetic species concept [71]. which produced a new bioactive alkaloid. and molecular data [70]. chemotaxonomic. as well as in the recognition of two different ‘‘chemotypes’’ within St. alba would be likely to be split into ¨ several species.284 Stadler and Hellwig the broader use of RAPD in selection for screening isolates as this method reflected high interspecific diversity. was characterized from a comparison of morphological. They stated that C. the authors discussed the aforementioned results by Moller et al. .

This methodology facilitated the recognition of species that are otherwise difficult to identify. The role of cryptic species in St. should qualify for general characterization of screening strains prior to HTS. In the following. while the fungus known as ‘‘D. pyrenaica). D. and PCR fingerprinting methods were used.e. loculata. No relationship as to the correspondence of these chemotypes and cryptic species has so far been established. In recent years. childiae and D.. Correlations between molecular and chemotaxonomic features were also noted in Alternaria species from South Africa. D. the latter of which also constitutes a key enzyme of secondary metabolite biogenesis [79]. Thus. several species and strains of Daldinia were compared by ARDRA (Amplified Ribosomal DNA Restriction Analysis) of the 18S nrDNA. and trichodiene synthase. The results so far published mainly involved the xylariaceous genus Daldinia and its allies. morphological studies. high ‘‘metabolic creativity’’ as revealed from the rather large number of previously reported metabolites. some characteristic pigments of Daldinia spp. were identified and their distribution was evaluated in some 500 specimens and cultures from around the world. in contrast. For their characterization. The most suitable PCR method.) was recognized as an European taxon preferably associated with Fraxinus.84]. concentrica’’ by American and Asian mycologists was reclassified as D. these terpenoids are not mycotoxins. Fragments obtained using three different restriction enzymes were compared on their similarity. [Connors]). Another striking example of the usefulness of molecular data in conjunction with HPLC profiling techniques is the identification of the pneumocandin-producing anamorphic fungus as Glarea lozoyensis [81] (see Chap. In this case. DNA sequences were employed. the type species. colors of stromatal pigments (i. and frequent occurrence in strain contingencies that are usually provided for NP screening. Both species are otherwise quite similar but drastically differ in their secondary metabolite profiles (Fig. Only in 1999. their secondary metabolite profiles were studied [80]. chartarum was also recently discussed in a study involving molecular phylogenetic analyses of chitin synthase. Characteristic and specific patterns were only obtained with those species containing large introns in their 18S nrDNA (e. and. Ces & de Not. different PCR fingerprinting methods were compared on their discriminatory power [40. some of them were reported to possess apparently selective bioactivities [77. 5). The data in Antibase and DNP suggest that the production of the spiro-drimanes in Nature be restricted to Stachybotrys and Memnoniella. and in addition to PCR fingerprinting. reflecting metabolic diversity. a key biosynthetic enzyme of trichothecene biosynthesis [76].:Fr. In the last world monograph [82]. Concurrently. In Fig. 6. These were subjected to ITS sequencing. chartarum ‘‘chemotypes’’ to be in agreement with the PCR-based characterization. and a group consisting of D. Obviously.78]. fissa.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 285 ined in concert employing RAPD and a specific PCR primer to detect Tri 5. a basis for a polyphasic approach to their taxonomy had been provided. Daldinia concentrica (Bolt. characters relating to secondary metabolism) were first employed for segregation of species in addition to anamorphic and teleomorphic morphology. we have therefore attempted to find indirect methods to determine the metabolic diversity in particular groups of ascomycetes that have in common strong morphological similarity. HPLC profiling. along with unsolved problems as to their taxonomy. (Table 5) These examples show that in many groups of fungi the chemical diversity to be expected from their evaluation in screening programs can be deduced indirectly from their morphology or molecular taxonomy. Several other species did not even differ in their ARDRA patterns from . even a new genus was erected for the producer of a pharmaceutically important product. beta-tubulin. The results revealed St. concurrently.. and. childiae [83].g.

286 Stadler and Hellwig Table 4 Occurrence of Some Characteristic Terpenes in Basidiomycete Cultures (C) and Fruitbodies (F) Genus Cyathus (C. F)a— 14 of 15 records examined Chemical Type Cyathane diterpenes Structure of Characteristic Compound (usually one of several congeners produced) Armillaria (C)— 11 of 11 records examined Protoilludane orsellinates (sesquiterpenes) Albatrellus (F)— 17 of 18 records examined Farnesyl phenols Hebeloma (F)—14 of 15 records examined Hebelomic acids (triterpenes) Hypholoma (C)a— 17 of 21 records examined Caryophyllane sesquiterpenes a Confirmation of previous investigation cited by Erkel and Anke [30]. .

from culture.84. The HPLC-UV and HPLC–mass spectra of 1 to 4 are also depicted [83. Morphological characters are quite similar in both species. anamorphs (Nodulisporium. childiae (right).88. B) and ascospore ornamentation by SEM (10. while they differ in their ascospore ornamentation by SEM.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 287 Figure 5 HPLC-UV chromatograms of stromatal MeOH extracts (210 nm. C) in Daldinia concentrica (left) and D.89]. A). concentricol (2).000 . daldinin C (4). and daldinol (5). 1000 . Their secondary metabolite profiles strongly differ. daldinal A (3). as demonstrated here by the occurrence of BNT (1). .

and.90]. can be divided into several groups that strongly differ in their stromatal metabolite profiles. childiae and D. especially when records from different continents are compared.87]. respectively. A comprehensive SEM study later confirmed these results [88]. and TaqI. One example is shown for each profile type. Species-specific data confirmed that D. it was concluded that morphologically alike Daldinia spp. the metabolite patterns observed in stromata were not closely correlated with the anamorphic branching patterns as defined by Ju and Rogers [91]. childiae. childiae is indeed cosmopolitan and occurs besides D. the upper example is derived from D. In Xylariaceae. as exemplified by D. HaeIII. of the three morphologically similar Daldinia spp. Moreover. childiae. D. concentrica. some of which were meanwhile formally described [86. pyrenaica. about 50 different compounds were obtained.288 Stadler and Hellwig Figure 6 Diversity within Daldinia and allies as deduced from ARDRA profile types A through M (Table 5). care should be taken when assuming the conspecificity of these fungi. or the results were not species-consistent as deduced from morphological and chemotaxonomic results. concentrica is common only in Western and Northern Europe on Fraxinus but absent outside Europe and quite rare on other hosts. concentrica in Europe. while D. The central part shows the profiles obtained after partial digestion of PCR products with HpaII. (Modified from Ref. and the Editor of Mycotaxon. From the data gathered so far. concentrica [89. 40. Only four of those are present in more than one of the three examined species. Here. they also pointed toward the existence of several new species that had been erroneously deposited under the names of known species. and D. thus far examined extensively on their stromatal constituents (D. In summary. and the other from D.). Recently. vernicosa’’ ATCC 36660 is an isolate of D. strain ‘‘D. eschscholzii). with kind permission of Mycotaxon Ltd. The close relationship between both species is reflected by their identical ARDRA patterns (which are otherwise unique within Daldinia spp. except for type C.) the representatives of other genera. 7. further secondary metabolites were identified as minor or major components in the stromata of D. allowing for . The MIS-PCR data also revealed the identity of some species of uncertain correspondence (e. A better resolution was obtained by using Minisatellite (MIS) PCR [85] as shown in Fig. caldariorum). concentrica.g.. their metabolite profiles in culture differ from those of closely related genera such as Hypoxylon. and 80% of them constituted novel NPs at the time of their first discovery. stromatal metabolite profiles and SEM will be helpful to establish the identity of newly collected materials with previously described taxa.

PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 289 Table 5 ARDRA Pattern Types (corresponding to Fig. (Cuba) Biscogniauxia nummularia D. polymorpha D.2 1. decipiens Hypoxylon fragiforme. (Canary Islands).8 1. did not show similarities to any of several examined teleomorphic species with Nodulisporium-like anamorphs.2 Taxa highlighted in bold showed specific patterns because of large introns contained in their rDNA.p.8 1. caldariorum ATCC 36660 D. concentrica. there are several examples for secondary metabolites or profiles of such compounds . D.93].2 1. UK). “Russian Far East” D. grandis. [40] with kind permission of Mycotaxon Ltd. D.p. USA) D. petriniae. D.8 1. H. albofibrosa. D. D. and the Editor of Mycotaxon.2 1. clavata Daldinia sp. Such complementary investigations also provide a good basis for studies on molecular phylogeny and may even be necessary to ensure concordance in this morphologically similar group of fungi. 4) and Fragment Sizes of PCR Productsa Profile Type A Species Daldinia bambusicola. loculata Entoleuca mammata Kretzschmaria deusta Nemania serpens Rosellinia corticium Size of Amplified Fragment (kb) 1. multiforme. the extensive use of secondary metabolites in fungal classification is also strongly suggested. if not a genus of their own. which had recently been segregated from the paraphyletic genus Verticillium [94].8S nrDNA) in relation to their ability to produce the characteristic metabolite [95]. Recent findings also point toward the feasibility of this methodology for species discrimination in anamorphic Clavicipitaceae of the genus Pochonia [92.8 1. D. caldariorum (Mexico. X. (Asia. D. fissa D. In this family.4 2. Source: Modified from Ref. childiae D.2 1. were also evaluated by a combination of various PCR-based methods (AP-PCR and sequencing of the ITS/5.8 1. As inferred from these molecular data. longipes. pyrenaica D. Anamorphic Xylariaceae of the genus Nodulisporium. From the accumulating data gained on trichocomaceous Eurotiales. H. howeianum Xylaria hypoxylon. eschscholzii p.2 A B C D E F G H I K L M 1. eschscholzii p. undigested 18s rDNA segments delimited by the primers NS1 and RDR116.6 1. They appeared monophyletic and may therefore deserve the rank of a separate species. X. D. steglichii. which are present in several tropical countries. comparison of type materials. including producers of the important antiparasitic agent. the producers of this compound. nodulisporic acid.p. eschscholzii p. a Estimated from the electrophoretic mobility of the amplified.

84.290 Stadler and Hellwig Figure 7 Agarose gel electrophoresis of amplified MIS regions of Daldinia childiae. D. (Modified from Ref. with kind permission of the Editor-in-chief of Mycological Research. D. concentrica.eschscholzii and D.) . Rather specific fingerprints were obtained with D. eschscholzii and further taxa of the genus using primer M13 core. concentrica. childiae and D. fissa isolates. while a higher degree of variability was found among the D.

As many secondary metabolite genes in fungi are organized in clusters. Even though these mainly dealt with economically important mycotoxins and their producers. PCR-fingerprinting data are rather easy to obtain and can serve as prerequisite for the selection of ‘‘uniques’’ prior to more cost-and time intensive chemotaxonomic. less frequently. and taste) have been traditionally used in ascomycete and basidiomycete taxonomy and are also important cultural characters in conidial fungi [41]. including the production of secondary metabolites. The biosynthesis of secondary metabolites requires several specific genes. albeit some are cited below and in the next chapter. DNA sequences are now widely used not only to complement morphological and other data but even to establish phylogenetic relationships. and the rather frequent occurrence of template nrDNA copies. the same applies to the morphogenetic genes that encode for particular morphological phenotypes. mitochondrial ribosomal DNA sequences (nrDNA and mrDNA.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 291 that easily help to discriminate certain species from their morphologically similar allies [41. morphological. secondary metabolite genes have so far been playing a secondary role as surrogate parameters for verification of molecular data. specific primers. Especially nuclear or. Not even the most significant key publications in this field can all be discussed here in detail. In time. and chemotaxonomic data in concert [99.g. and DNA sequencing studies if representative individuals among a large number of range of closely related strains shall be studied. odor. In conclusion. morphological and chemotaxonomic characters remain valuable. Taken together. ‘‘Conventional’’ molecular data were even .96–98]. In recent years. It will be a long way to go until such correlations are fully understood (e. Chemotaxonomy and Molecular Phylogeny Throughout the last decade. Evidently. Fusarium species are already being examined. especially for segregation of fungi at generic and subgeneric levels. the genetic background for secondary metabolism has been subject of several intensive studies. However. the endowment of molecular biology helped to solve various problems relating to fungal taxonomy. This especially applies to those fungi that are studied by nontaxonomists because of their value for biotechnological applications. On the contrary. For the time being. from the wider application of functional genomics). for example.100]. 1. many fungal taxa now still regarded as ‘‘strains’’ or ‘‘chemotypes’’ will be recognized as good species from a combination of these methods. Secondary metabolites or indirect characters based on their presence or absence (color.4. respectively) were widely used for practical reasons such as the availability of universal primers. using morphological. There have been many discussions as to whether a phylogenetic species concept would stand in contradiction with phenetic or morphological data. which remain to be recognized and studied in most cases.. However. it is the strong opinion of the authors that the final goal of fungal taxonomy and phylogeny should rather be a polythetic species concept. the methodology available appears ready to find application for basic phylogenetic research on other fungi. straightforward PCRbased methods can be developed to compare large numbers of individuals on their genetic properties by using. facilitating their PCR-based amplification. the value of chemotaxonomy alone or in conjunction with PCR-fingerprinting techniques for strain dereplication in screening is indisputable because it has been demonstrated in most of the fungal families that are well-known to contain unique chemical entities. additional genes encoding for proteins are already being widely considered and studied on their significance. molecular.

the data sets in combination showed that morphological and secondary metabolite characters used in taxonomy were not strongly correlated with phylogeny. This was interpreted as ‘‘a transpecific polymorphism within the virulence-associated secondary metabolite genes. including at least two major groups of strains that show evidence for a long history of geographic isolation’’. Here. the functions of some genes involved in trichothecene biosynthesis have only recently been clarified. the results ‘‘further demonstrated that A.292 Stadler and Hellwig compared with the distribution of genes encoding for secondary metabolite biosynthesis. graminearum complex. while it is generally accepted that the possession of trichothecene biosynthesis genes significantly contributes to the fitness and virulence of Fusarium spp. In Aspergillus flavus. which has been maintained during evolutionary processes by balancing selection acting on chemotype differences that originated in the ancestors. graminearum. For molecular taxonomy of the economically important F. All are derived from a rather complicated carbon skeleton as a common biogenetic precursor. a species widely used in food technology. Such functional but nonessential genes may easily be altered by mutation. omt12 [111]. A.. where the evaluation of secondary metabolite genes concentrates on those encoding for the fumonisins [108] and the fungal phytohormones of the gibberellin type. morphological and chemotaxonomic data were compared with the sequences of partial -tubulin and hydrophobin [110]. leading to either of the main trichothecene types was described as the activated stage of a single cytochrome P-450 [107]. As stated by the authors. the 5. flavus in the current sense constitutes a nonmonophyletic assemblage. appears to have arisen from convergent evolutionary processes. and allies. the production of trichothecenes should be regarded as specific for certain lineages instead of species in Fusarium. resulting in an analogous organization of gene clusters [109]. the phylogenetics of mycotoxin and sclerotium production were studied by using partial sequences of a gene encoding aflatoxin biosynthesis gene. trichothecene production and genes encoding for those compounds were also taken into consideration. While evolutionary relationships between homothallic and heterothallic isolates were recognized. Multiple gene loci genealogies were found useful for segregation of species complexes such as Gibberella fujikuroi and F. Also. Neither the strain-specific differences in metabolite profiles (chemotypes) nor the sequences obtained from the trichothecene gene cluster were wellcorrelated with the concurrent results of the aforementioned genealogical concordancez [105]. Fumigati and corresponding Neosartorya teleomorphs. pointing toward a convergent evolution of secondary metabolism in both genera. oryzae.8S/ITS nrDNA region (widely used for species delineation in other fungi) appeared less informative than other genes. Evidently. For instance. respectively [101–104].’’ It should be considered that the differences in the chemical structures and biosynthesis of the main Fusarium trichothecenes are fairly small. the biosynthesis of the latter compounds in plants and fungi. oryzae. Here. In Aspergillus sect. a main determinant of trichothecene diversity in Fusarium species. Later. respectively. Interestingly. six nuclear genes were finally considered in a genealogical concordance. Similar investigations are under way in the Gibberella fujikuroi complex. protein-encoding genes were employed because of insufficient phylogenetically informative characters as inferred from the large subunit of the nrDNA. the structure and organization of biogenetic genes appears to differ considerably in Fusarium and the macrocyclic trichothecene-producing Myrothecium species [106]. Lately. They concluded that taxonomic changes in this paraphyletic group be necessary and found evidence that the nonaflatoxigenic A. As many as 59 out of 81 secondary metabolites were only present in one out of 18 species in the data set and therefore excluded from the phylogenetic analyses. .

These can subsequently be checked for related compounds or overproducers of a desired NP lead. All such genes may be subjected to the design of specific PCR-primers. which may afford a dilution or purification step before employing standardized PCR protocols. FASTA or related software tools to those of metabolite producing strains found in the screening. However. Main hindrances are PCR inhibitors and DNAases. even allowing for production of new analogues by altering their chemical structures by site-directed mutagenesis. the elucidation of the molecular genetic background of the biosynthesis of marketed drugs such as lovastatin [113] and cyclosporin [114] also made considerable progress. 1.. The results obtained in the course of these studies may eventually facilitate screening approaches aimed at the discovery of additional producer strains for a given lead. for sequencing or PCR fingerprinting in the course of the above described dereplication procedure). . As discussed above. However. Soon. Hopefully. Naturally. Remarkable efforts were also made with the elucidation of the molecular background of biosynthesis and expression of loline alkaloids in Neotyphodium uncinatum. the knowledge on the genetic background of fungal secondary metabolites is soon going to increase even in the case of those compounds that neither constitute mycotoxins nor developmental candidates of the pharmaceutical and agrochemical industry. several important groups of fungi known to produce unique secondary metabolites are still underrepresented in these Internet databases. the remainder may be stored until further use. more basic research on these matters is needed. followed by a ‘‘genetic screening. Once the DNA has been extracted and purified adequately (e. Hence. the endophytic anamorph of Epichloe species [112]. this allows NP researchers to verify taxonomic relationships and to search for the most likely candidates among fungi showing similar sequences. oryzae appears safe once again from being included as an infraspecific taxon in the toxigenic and pathogenic A. the integration of such approaches into NP screening has been facilitated. such techniques will also be used to characterize large strain contingents on the presence of particular biosynthetic gene clusters. As a result of the introduction of capillary electrophoresis and other powerful sequencing equipment.and time-intensive step in PCRbased characterization of fungi is usually the extraction of DNA. It should be pointed out that the most work. they may not even be informative enough for phylogenetic purposes. as suggested by some authors from the interpretation of nrDNA sequence data and other characters. Finally. The Boletales Example The Boletales are a good example to demonstrate that fungal pigments and other secondary metabolites are relevant to the recognition of phylogenetic relationships and thus to the identification of chemical redundancies and of chemical diversity within a fungal order. Moreover. Needless to say. Thus. it should be kept in mind that these internet databases may occasionally contain sequences of misidentified strains. flavus.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 293 is genetically distinct from A. the correlations between the prevailing (rDNA) sequences and secondary metabolite production in fungi are still poorly understood. differing from the latter in possessing a divergent class of non-functional alleles in the aflatoxin gene cluster (and in the general absence of aflatoxins). A. the sequence data stored in the Internet can be compared by BLAST.’’ in which DNA preparations from large strain collection libraries may be employed.g.5. not only PCR fingerprinting but even DNA sequencing nowadays can be done with a high throughput and at lower costs. flavus. In principle.

as exemplified below. A second group of relevant pigments are the terphenylquinones.. such as the yellow pigments variegatic acid and xerocomic acid (Fig. these substances are more likely than the aromatic pigments to exhibit selective pharmacological activities because of their unique chemical structures.g. Representatives are atromentin and the flavomentins and spiromentins from Paxillus atrotomentosus. which are responsible for the bluing reaction of many representatives of the Boletales [28]. host-specificity of mycoparasites such as Sepedonium).g.e. i. such as being formed during injury of the basidiocarps or causing bitter or pungent sensations. pungent. (Figs. distribution and biosynthesis of pigments and colorless metabolites). . On the other hand. which are responsible for the bluing reaction in these species. 9. the taxonomic classification of species now assigned to the Boletales had been dependent on the importance associated with several criteria: morphology (e. it was essential to have a closer look at the other secondary metabolites of the various species. the correlation of results from chemotaxonomy and from molecular phylogeny can now be evaluated. while the Boletales nowadays comprise various fungi whose fruitbodies bear little or no morphological resemblance to the typical bolete. In recent years. 10). and chemotaxonomic aspects (e.. including pigments and colorless substances. These results served as a basis for interdisciplinary taxonomic research. 8). However. In addition. Tylopilus. several colorless metabolites (e. ( )-form from Chamonixia caespitosa and ( )-form from Paxillus involutus [115]. bitter substances) were isolated from their toadstools (Fig. This implicated that the common pigments like the pulvinic acids are useful to confirm the affiliation of controversially discussed taxa to the Boletales.. or even toxic principles. macroscopic characteristics). ecology (e. at that time TLC with reference standards were used as simple and efficient tool. Suillus. The pigments of the Boletales are well-investigated. their pigmentation pattern was considered to be homogenous.9 [28]). While many of them are considered edible and are traditionally collected as food throughout the world. Some genera of the Boletales are characterized by the presence of cyclopentenones [28]. In addition. These include chamonixin.294 Stadler and Hellwig The traditional core group of these fungi comprises mycorrhizal species with fleshy fruitbodies and poroid hymenophore. 8. soon other groups of substances were found in selected Boletaceae (Figs. These compounds often have a definite property. another cyclopentenone pigment from Paxillus involutus [28] induces a brown discoloration upon bruising of the fruitbodies. as well as the biosynthetically closely related grevillins from the genus Suillus [28]. which are now classified in the type genus Boletus and other genera with mostly or exclusively boletoid habit (Leccinum. Species of various subtaxa included in the Boletales have motivated research on their chemistry and biology for many decades.g. mainly due to the efforts of Steglich et al. and gyrocyanin from Gyroporus cyanescens. Involutin... not only the chemistry of their fruitbodies (and to some extent also of the corresponding cultures) but also the molecular phylogeny of the Boletales have been studied extensively. mycorrhizal association. Traditionally. Therefore. Such metabolites turned out to be important markers for the taxonomic relationships within particular Boletaceae subtaxa.g. For investigations of the pigments. and Xerocomus). Most important in this respect are the pulvinic acids. At an earlier stage of the chemical investigation of the Boletaceae. which are derived from the dimerization of 4-hydroxyphenylpyruvic acid. 10). HPLC and LC hyphenated spectral techniques allow for the fast detection and identification not only of pigments but also of colorless compounds with various characteristics. Most. if not all of these boletoid fungi are still included in the Boletaceae. Nowadays. others bear close morphological resemblance to their edible counterparts but contain bitter.

and (c) cyclopentenones. .PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 295 Figure 8 Some characteristic pigments of Boletales: (a) pulvinic acids. (b) terphenylquinones and grevillins.

and Gastroboletus.296 Stadler and Hellwig Figure 9 Some characteristic pigments of Suillus and related species.. rhizopogone. These metabolites were found to differ in the prenylation position of 3. are gasteroid. Chamonixia.g. 9) [28]. Suillus Among the Boletales. 1. Gomphidius and Chroogomphus are characterized by the occurrence of polyprenylated phenols and quinones as additional pigments. Such pigments are also found in Rhizopogon. Interestingly. terphenylquinones and cyclopentenones. while Gomphidius and Chroogomphus are agaricoid and Rhizopogon. the recent taxonomic investigations of species within the genus Suillus and related genera were one of the first examples in which extensive chemotaxonomic investigations were compiled with molecular data. Characteristic pigments of various members of the Boletales are pulvinic acids. Independent from these chemotaxonomic findings. the boviquinones and tridentoquinone (Fig.5. Fig.. molecular studies came to congruent results. DNA analyses confirmed the relationship between the genera Suillus and . 9) [116. (e. Suillus and Gastroboletus. Examples are e.117].g. also containing the Gomphidiaceae and Rhizopogonaceae. suillin.4-dihydroxybenzoic acid during the biosynthesis. recognized that the structurally homologous fungal ¨ metabolites boviquinone-3 and boviquinone-4 are produced by two different pathways in the agaricoid Chroogomphus rutilus and the boletoid Suillus bovinus [118]. These genera mainly differ in the habit and anatomy of their basidiocarps. which is a member of the new suborder Suillenae.1. Besl and Bresinsky [117] therefore erected the new family Suillaceae with the genera Boletinus. Suillus is boletoid. Suillus and Gastrosuillus. Muhlbauer et al.

Sclerodermaceae Due to chemotaxonomic investigations. Rhizopogon [119] and demonstrated the close link of Gomphidius and Chroogomphus with Suillus [120].4-di-O-methylatromentate. confirming its relationship to Scleroderma [123]. broomeianus can be infected by Sepedonium. a mycoparasite genus generally known to parasitise Boletaceae and Paxillaceae.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 297 Figure 10 Some characteristic metabolites of selected Boletales: (a) retipolides and (b) (cyclo)calopins. Chlorinated pulvinic acids had already been known before from the boletoid Pulveroboletus auriflammeus [124] and Xerocomus chrysenteron [125]. 1. which was further confirmed by the fact that M. In the fruitbodies of the hypogaeous Melanogaster broomeianus. chemotaxonomic relations were reflected by . Thus. the cyclopentanoids chamonixin and involutin were detected.5′-dichloro-4.5.4′-di-O-methylatromentate). This pointed out a close relationship between the Melanogastraceae and the Boletales. were isolated from the tropical species Scleroderma sinnamariense [122]. Three pigments – representing methylated and chlorinated derivatives of atromentic acid (methyl 4. methyl per-O-methylatromentate and methyl 2′. the gasteroid genera Scleroderma and Pisolithus already were shown to have affinities to the Boletales. From Pisolithus arhizus the naphthalenoid pulvinic acid derivative pisoquinone was isolated.2. The status of the Suillaceae as a sister family of the Boletaceae had not been recognized prior to these studies [121].

paxilloid habitat) members of the newly established Omphalotaceae were until recently included in the Paxillaceae [128]. which were congruent to the three groups distinguished in the chemotaxo- . and their characteristic illudane type sesquiterpenoids (Table 3) were named after the producing fungus. Another clear chemotaxonomic character to discriminate Omphalotaceae from Paxillaceae besides the occurrence of sesquiterpenoids is the presence of white rot (as opposed to brown rot in Paxillus and Tapinella. which may be derived biosynthetically from three molecules of 4-hydroxyphenylpyruvic acid [47. but the biosynthesis of these compounds may well have arisen independently several times in the course of the evolution of homobasidiomycetes. retipes it remained uncertain how to differentiate between these two species or whether they are distinct species at all. Only in some species of the first group traces of xerocomic acid were detected. which is not known from Boletales. 1. the detection of atromentin and pulvinic acids in cultures of Omphalotus and Lampteromyces had pointed toward their affinities to the Boletaceae [129]. Because the protoilludane and illudane carbon skeleton is frequently encountered in certain groups of Agaricales such as Armillaria and Agrocybe. both genera have been included in Clitocybe and Pleurotus. Recently. 1. while the sequence data clearly differ from the Boletales [135]. This was confirmed when using a wider range of taxa [136]. A concurrent study of the 25S rDNA data of the same collections resulted in three major clades. Omphalotaceae Due to morphological features (e.g. the retipolides A-E (Fig.4. This family forms a distinct clade within the Agaricales. [134]. 10). A recent chemotaxonomic study of several collections from North America and Japan created three separate groups according to the occurrence of characteristic metabolites [138].3. the peptidic omphalotins and the terpenoid illudins may constitute determinants of a more advanced evolution of secondary metabolism. In contrast. lamellae. Investigations of their secondary metabolites guided by the bitter taste of these toadstools led Steglich et al. Moreover. the nematicidal omphalotins were isolated from O.. ‘‘Clitocybe illudens ( syn. respectively.5. the species of the first group have a novel yellow pigment in common in contrast to the second group. These sesquiterpenoids are neither found in the Paxillaceae nor in other Boletales. Retiboletus Since the first description of Boletus ornatipes and B. Binder et al. the Omphalotaceae may be closely related to other gilled mushrooms.137]. As inferred from phylogenetic studies of 28S nrDNA sequences. The first and the second group contain retipolide A and C.5.298 Stadler and Hellwig host-parasite relationships [126] and later proved by comparison of the 28S nrDNA sequences [127]. the Coniophoraceae and all other wood-destroying Boletales) and the occurrence of bioluminescence. it was revealed that the pleurotoid Nothopanus is closely related to the Omphalotaceae [135]. olearius [131–133]. placed the two genera in a separate family Omphalotaceae. Due to a comparison of 28S nrDNA data. Before.48. In addition. The third group was found to contain the retipolides A-D. Omphalotus olearius’’ [130]). which lacks this pigment. Their production appears to be a rather constant feature of Lampteromyces and Omphalotus spp. to the discovery of a unique group of macrocyclic butenolides. The occurrence of pulvinic acids is unparalleled in agaricoid taxa.

xerocomic acid was detected as side metabolite.PCR-Based Data and Secondary Metabolites as Chemotaxonomic Markers 299 nomic investigations [138]. as well as in the North American species B. Boletus calopus Boletus calopus and closely related mushrooms of Boletus sect. coniferarum. R. are found in Boletus calopus and B. Important for the process of drug discovery are those compounds which exhibit selective bioactivities. This will facilitate rendering the search for new chemical entities from nature less complicated and less time. 10) [140]. molecular data were actually used to validate these findings in retrospective [141] and chemotaxonomy probably helped to explain the molecular data. Calopodes. called calopins and cyclocalopins. These findings can easily interpreted in view of the chemotaxonomic preselection described above: In contrast to the more ubiquitous metabolites the species-specific compounds often possess unique chemical structures probably more likely to exhibit selective bioactivities. the retipolides constitute the major pigments of this complex. These examples suggest that also metabolites other than pigments may play an important role in chemotaxonomy within the Boletales. which were not always in clear accordance to morphological features. Not only concerning the Boletales. Calopodes are characterized by their bitter taste. B. Sequence data of the 28S nrDNA indicated that B. Up to now. ornatipes complex. peckii (Fig. ornatipes. Again. they may disturb the HTS workflow by generating false positive hits and are probably not suitable for drug development. The example of R.5.and cost-intensive. which is caused by O-acetylcyclocalopin A. but also in many other areas of fungal taxonomy there is a lot of knowledge based on classical taxonomy and on chemotaxonomy. but not species-specific metabolites may also show nonselective reactions. since only in one group of the B. Thus. Besides this bitter compound. It is the challenge of NP research within the search for new lead structures to exploit and to extend this knowledge.. In Boletales. a careful preselection within the strain collections and the screening samples and hits is necessary. the new genus Retiboletus with R. while some of the common pigments may be biosynthesized in a more convergent way and often represent precursors for more specialized metabolites. rubripes and B. Instead. while other interesting. retipes from R. ornatipes. while other ‘‘typical’’ Bolete pigments were not found at all. Therefore.g. ornatipes are distinct from other groups in the Boletaceae. However. including Boletus itself. are usually derived by a more complex biogenetic pathway and often are characteristic of one or only a few species. retipes/ornatipes reveals the importance of chemotaxonomic and PCR data to identify morphological apparently similar or identical biological species producing different chemical substances. e. This was in agreement with the chemotaxonomic investigations. confirmed their relationship. To find these metabolites while avoiding handling of the unwelcome compounds. the Boletales example gives an idea of how to identify the producing strains of these desired compounds and how to avoid duplicates of other species. retipes and R. there are still no morphological or anatomical characteristics available to distinguish R. molecular data agreed well with these results [139]. retipes/B. the calopins with a new carbon skeleton seem to be produced only by species of sect. terpenoids. several structurally related analogues. 1. The common occurrence of the bitter principles in all species of the section. radicans. HPLC profiling at an early stage of the process will be rewarding not only in drug discov- . In conclusion.5. These compounds. retipes/B. independent from their geographical distribution. flavoniger [139] was erected.

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such as luciferase [9]. and smaller assay volumes.7]. cholesterol statins. in vitro assays were developed to detect inhibitors of critical enzymes and binding to crucial receptors. advances in assay technology. such as mevinolin and simvistatin [4]. and even higher-density well formats allow hundreds of thousands of samples to be screened in a day. include antibiotics. These therapeutic agents. McCALLUM Merck Research Laboratories. to modern screening techniques is a continuing challenge. immunosuppressants.A. and robotics have provided more diversity in detection techniques. and glutathione transferase [11]. In vivo assays were then expanded to include multicellular organisms. As knowledge of the biochemistry of the targets advanced. such as penicillins [1–4]. such as free-living adult and larval insects and adult and larval parasitic worms both in and outside their hosts. Molecular biology has provided techniques for creating designer cells with increased permeability that over-express or under-express the desired target (receptor or enzyme) and contain genes for reporter reactions. and vasoconstrictors. which are either the natural metabolite per se or derivatives of the natural products. Rahway. 1. Mammalian cell cultures were employed to find anticancer and antiviral agents. Adaption of fungal and other natural product extracts. Initially. such as syntomsetrine [8]. These cells are more sensitive to inhibitors in low concentration and produce an easily measured signal.10 Screening for Biological Activity in Fungal Extracts MARVIN SCHULMAN and CHRISTINE D. instrumentation. New Jersey.S. INTRODUCTION Many clinically important pharmaceuticals have been found by screening fungal extracts. In addition. fungal extracts were tested for antimicrobial activity using whole-cell assays on agar plates. and griseofulvin [5]. greater sensitivity. U. which contain chromophores and fluorophores. cephalosporins [1–4]. Sample prepa309 . to search for insecticides and anthelminths. such as the cyclosporins [6. 1536-. Assays in 384-. -galactosidase [10].

It should not be undertaken unless the assay has a high throughput and low cost. The common practice is to store the extracts at 20 C to 80 C or to dry the extracts at low temperature and store the dried sample at 20 C to 80 C. This supply of sample can then be aliquoted for screening. one has to achieve an acceptable signal-to-noise ratio for the positive control in the presence of inactive extracts and demonstrate a proportional increase in activity with increasing concentration of the positive control in the presence . however. In experiments conducted at Merck Research Laboratories. such as glass beads or vermiculite. Storage of extracts and samples for screening is an important issue. In this chapter. Assay validation. screening of fractionated samples did not lead to discovery of any actives not found in crude extracts. whole broth extracts of fungi grown in suspended aqueous culture contain all of the potentially active metabolites. Since fungal metabolites are both intracellular and extracellular [12]. and assay support for isolating the active components are all crucial to the success of a program. and to decrease the amount of interference in assays (as discussed later in this chapter). An acceptable assay will have a signal-to-noise ratio of at least four-fold. extracts of the mycelia must suffice. Screening samples are kept frozen at 80 C. screening concentration. Fractionation prior to screening dramatically increases both the number of samples to be screened and the cost. reported one such discovery at the SIM meeting in 2000 [13]. we discuss a variety of these assays and related issues and also suggest some techniques and approaches that can be successfully employed. and for isolation of active compounds. and assay support for isolation are related issues. confirmation of activity. and then refrozen. 2. GENERAL CONSIDERATIONS The type of fungal extract has a significant impact on the screening program. Both polar (methanol:water) and nonpolar (2-butanone) extracts are needed for a broad screening program because most of the antimicrobials found to date have been polar compounds whereas most of the therapeutic agents have been nonpolar compounds. Samples for screening are generally prepared by dissolving a known amount of dried extracts in DMSO. Testing extracts periodically in a fixed number of assays would provide a better measure of component stability but is time-consuming and expensive. for fractionation prior to screening. Quantification can be based either on the dry weight of extract in the screening sample or on whole broth equivalents. ScheringPlough. confirmation of activity. therefore. This. which might otherwise escape detection. detection of activity. If cells are grown on solid media. does not ensure that the assay will perform well with fungal extracts. thawed for use. To validate the assay for use with fungal extracts. which often contain significant amounts of natural pigments including chromophores and fluorophores. Quantification of the screening samples is imperative because it provides the basis for determining the specific activity of an extract in a given assay and of the active components during isolation. however. general rules for ensuring chemical stability should be applied.310 Schulman and McCallum ration. Both methods have been successfully employed at Merck. Fresh samples should be prepared after several freeze-thaw cycles and at least every year. Fungal samples have been fractionated prior to screening in an attempt to increase the concentration of minor components. interference. Comparison of HPLC patterns of fresh and stored extracts can provide a gross measure of change but is a poor indicator of the stability of individual components. Assay development is generally done using a pure active compound as the positive control and DMSO as the blank. There is a paucity of experimental evidence concerning the stability of extracts. assay validation.

Screening for Biological Activity in Fungal Extracts 311 of the inactive extracts. These parameters are a matter of choice and are determined by the importance of the program and the support available for validation and isolation of actives. a 40% inhibition may be selected statistically if the assay is not noisy. These are often highly pigmented and/or contain long-chain fatty acids.e. At Merck. the standard deviation of the mean of all samples in a plate (not just the controls)] and they are not subject to an arbitrary cutoff. Many of the hits will be false positives due to interference and will not confirm. receptor) is omitted. activity jumps from the 0% to 10% range directly to the 90% to 100% range. which act as detergents. it is important to cast a wide net because the active compound may only be a minor component in the mixture. This provided not only a repeat of the original assay but also a titration curve and a measure of potency. It is important that this titration of activity yield incremental increases in activity with increasing concentration because a common type of interference encountered with fungal extracts results in an all or none response—that is. the threshold of activity is set and all samples displaying activity above this threshold (% inhibition or activation) are considered active.. depending on the assay detection methodology. In screening fungal extracts that are crude mixtures. we found this was best achieved by titration of the extract to determine an IC50 or EC50. It will therefore yield a different IC50 value (concentration of sample that yields 50% inhibition or activation. The desired hit rate is a function of the screening concentration and the threshold chosen for either activity or statistical outliers. (Inactive extracts are found by limited screening of samples in the assay prior to this validation. Hits generally are defined either by using a fixed cutoff activity level or by statistical analysis. The inactive extracts chosen should include those containing chromophores and fluorophores. or in mock assays in which the target (enzyme.) The titration curve of the positive control in the presence of the extract may be displaced from that obtained with the positive control plus DMSO. we typically aimed for a 50% confirmation rate because it provided sensitivity and a manageable number of samples to pursue. which show up as actives in several unrelated assays. Figure 1 presents an analysis of a screen and indicates the top 1% actives by either percentage of activity or the number of standard deviations from the mean (score) [14]. After validation of the assay. An EC50 value (concentration of sample that yields activation or inhibition equal to 50% of the range of activity observed) can be used when the titration curves do not cover a full 0% to 100% range. This will eliminate extracts that appear . The statistically selected hits differ because selection is a function of the noise in the assay [i. the screening concentration is determined empirically by testing several plates of samples at different concentrations and observing the frequency of actives (hit rate). In the statistical method. usually determined via nonlinear regression plots) but the shape of the curve should be similar. each plate is analyzed and outliers (generally 2 standard deviations) from the plate mean or median are considered hits. Databases can be employed to compile lists of interfering extracts. those by percentage of inhibition are in the lower right quadrant and those chosen statistically are in the upper left quadrant. it is important to check the titrated actives in assays with a different order of addition of reactants. In the Natural Products program. a different format (nonhomogeneous). Statistical packages such as ‘‘SAS’’ and visualization programs such as ‘‘Spotfire’’ are often used to facilitate this approach. The number of inactive extracts to use is a matter of choice. In the fixed cutoff method. Actives selected by both methods are in the lower left quadrant. Thus. but we found 10 to 20 to be a reasonable number to ensure a reliable assay. Because some interfering compounds (false positives) may titrate nicely. Confirmation of actives is critical since it determines which extracts are to be pursued and which will be dropped.

Counterscreens can be used to prioritize but not to eliminate actives since lack of specificity of the extract does not predict a lack of specificity of the purified component. there could be a detergent-like molecule that prevents antibody binding in an ELISA assay or a high concentration of biotin in a SPA assay using streptavidin-coated beads.312 Schulman and McCallum Figure 1 Statistical selection of actives. active because they are interfering with the detection system employed. Quantitation of activity during isolation is critical because it permits monitoring of the total activity applied and recovered in each step of the isolation procedure. ‘‘Scores’’ is a statistical value related to the number of standard deviations from the mean. This entails additional work but actually saves resources by limiting the number of samples selected for isolation. This information must be available in a timely fashion (within 2 to 3 days) to avoid potential loss of activity during the isolation due to instability of the active components. Broad counter-screening to establish . Assay support for isolation of the active component requires the largest commitment of time and resources because it entails titration (10 points) of each fraction produced in each step of the isolation procedure. For example. This provides a measure of the specific activity of each component in the mixture and prevents the loss of potent minors.

ASSAY FORMATS There are a wide variety of assay formats currently in use. Some rely on conventional technologies and are more labor intensive.or 384-well microtiter plates. Centrifugation Assay A centrifugation assay successfully used to screen for inhibitors of the Hepatitis C NS3 protease is outlined in Figure 3 [17]. The filters are then transferred to scintillation vials. filtered.1. The stopped reaction mixture is then filtered to separate unbound ligand from ligand. the reaction mixture is transferred to a millipore filter system. A brief description and diagram of the commonly used assay formats is presented for the convenience of readers inexperienced in screening assays. 3. 3.or 384-well plates. A synthetic substrate containing the CYS-ALA scissile bond and radiolabel Figure 2 Filtration assay in microtiter format.16]. More complete information on a given assay can be obtained from the references. The reaction is then stopped by the addition of excess cold ligand and rapid filtration. which are expensive. Comparison of the radioactivity detected in the control (no fungal extract added) with the samples (fungal extract present) is used to indicate binding inhibition or stimulation. and then washed. This reaction can be performed in small tubes or in 96-well plates. The assay steps include incubation of cells with radiolabeled ligand to allow binding. This type of assay has been used for both receptors and enzymes [15.receptor complex. and the amount of bound radioactivity is determined. If the incubation is in 96. . 3. and others rely on automated systems. The latter often require special reagents and detectors.Screening for Biological Activity in Fungal Extracts 313 specificity of the active component is important once a high degree of purity (90%) is obtained. Filtration Assay Figure 2 outlines a filtration assay for the binding of ligand to a generic cell-surface receptor. If the incubation is run in tubes. fluid is added.2. samples can be filtered manually using microtiter filtration plates and a filtration manifold or automatically using a cell harvester. This can be done in small tubes or in 96.

A second antibody raised against another portion of the peptide substrate and coupled to a reporter enzyme (in this case. but for clarity. horseradish peroxidase) is added and allowed to react.18].3. The reaction is stopped by addition of Toyo-Pearl-DEAE beads. The reaction is centrifuged and an aliquot of the supernatant containing the cleaved C-terminal fragment of the substrate is transferred to a microtiter plate designed for scintillation counting or to a scintillation vial and counted. which bind the glutamate-rich region of the intact substrate and the N-terminal portion of the cleaved substrate. The reaction is terminated and the liquid is removed from the well. Enzyme-linked immuosobant assays (ELISAs) are generally run in microtiter plates with adherent surfaces. and the reaction components are added. let us assume that this is a tyrosine kinase assay and the primary antibody recognizes the phosphotyrosine product. the primary antibody is added to the wells and allowed to adhere. 3. The excess antibody is then removed. on the C-terminus is incubated with the active protease in the presence or absence of extract. Finally. Before the assay is begun. which is converted to a fluorescent . leaving the newly formed phosphotyrosine bound to the primary antibody. A generic ELISA is depicted in Figure 4. A comparison between the control and the sample indicates if the sample is an inhibitor (decrease in soluble radioactivity). Enzyme-Linked Immunosorbant Assay This assay has very broad application and is limited only by the availability of specific antibodies against the reaction product [14.314 Schulman and McCallum Figure 3 Centrifugation assay. Hepatitis C NS3 protease. a nonfluorescent substrate. The liquid is removed and the plate is again washed to remove excess detection antibody.

emission at 620 nm). in which the second antibody is conjugated to Europium rather than a reporter enzyme. is both time-dependent and proportional to enzyme concentration. Absorbance Assay This assay measures a change in absorbance (generally an increase). . shown in the inset. is added and the amount of fluorescent product is determined in a fluorometer. After the plate is washed. The substrate contains a chromophore that is liberated during the enzymatic reaction. All assays that use antibodies for detection are subject to interference by compounds that interfere with antibody binding. causing a time-dependent increase in absorbance.Screening for Biological Activity in Fungal Extracts 315 Figure 4 ELISA (enzyme linked immunsorbant assay). The format is simple and is often used for proteases. and it is important to validate them for use with fungal extracts. Figure 5 provides an example of an absorbance assay for the protease papain [21]. peptidases. lipases. 3. and esterases [20]. This assay is very sensitive but is more qualitative than quantitative because the amount of phosphorylated product is amplified by the horseradish peroxidase reaction. This type of assay is subject to interference by chromophores and fluorophores. product by horseradish peroxidase. The increase in absorbance. A quantitative assay can be obtained using the Delfia technique [19].4. which interfere with absorbance measurements. Europium fluorescence is then detected directly in a fluorometer (excitation at 337 nm.

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Figure 5 Absorbance assay.

3.5. Fluorescence Assay This assay is similar to the absorbance assay except that a change (generally an increase) in fluorescence is measured. It is also often used for proteases, peptidases, lipases, esterases, and glycosidases. It is approximately 10 to 1000 times more sensitive than an equivalent absorbance assay because it is much easier to measure increased light emitted than to measure decreased light transmitted. In the example of a -galactosidase assay depicted in Figure 6, the substrate contains the fluorophore 7-hydroxycoumarin-3-carboxylic acid bound to the -D-galactose via a glycosidic bond [22]. Cleavage of the glycosidic bond liberates the fluorophore 7-hydroxycoumarin-3-carboxylic acid, which when excited at

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Figure 6 Fluorescence assay for -galactosidase.

390 nm fluoresces at 460 nm. The increase in fluorescence is therefore a measure of substrate cleaved and will increase with both time and enzyme concentration. 3.6. Scintillation Proximity Assay Scintillation proximity assay (SPA) provides a homogeneous format for conducting assays with radiolabeled ligands or substrates that eliminates the need to wash and remove bound ligand from free and eliminates the use of scintillation fluid. The basis for this assay is that the scintillant is locked into the SPA bead and is only excited by radioligands that are in proximity—that is, bound to the bead. Assays therefore are designed to allow the product but not the substrate to bind to the bead and be detected. Figures 7 and 8 outline two different SPA assays—one for a nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR ) [23] and the other for a tyrosine kinase [24]. In the PPAR assay, the cloned PPAR receptor has been engineered as a fusion protein with glutathione transferase (GST). The added tritiated ligand (H) will either be free in solution or bound to the PPAR receptor. The bound ligand is detected using an anti-GST antibody and a

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Figure 7 Scintillation proximity assay for peroxisome proliferator-activated receptor . Scintillation proximity bead coated with protein A (A), anti-GST Ab, GST-hPPAR fusion protein, tritiated ligand (3H).

protein A-coated SPA bead. As depicted, binding of the protein A bead to anti-GST antibody that is bound to receptor brings the receptor-bound ligand in sufficient proximity to the bead to allow excitation of the scintillant and detection of bound ligand. Free ligand is not detected. In the tyrosine kinase assay [24], the peptide substrate is biotinylated and the SPA beads are coated with streptavidin. Radioactive -labeled ATP is used and detection of the reaction product, phosphotyrosine peptide, occurs via binding of the biotinylated, phosphorylated peptide to the streptavidin-coated bead. Beads with a wide variety of coatings are currently available, and more are under development to allow a wide variety of assays to be run in this format [24]. 3.7. Flash Plate Assay Flash plates are another means of conducting assays that use radiolabeled ligands without the addition of scintillation fluid [25]. In this case, special assay plates are used in which the scintillant is coated onto the walls of the wells. The walls are also coated with gelatin or other substances that allow receptors and proteins to adhere. Figure 9 depicts a generic flash plate assay in which a receptor is shown adhering to the wall of a well coated with scintillation fluid. After addition of radiolabeled ligand and incubation to allow binding, the well is washed and filled with appropriate buffer. Bound radioligand is detected because it is close enough to the scintillant to be detected. It is not imperative that the plates be washed to remove unbound ligand, but this generally is preferred since it lowers the background. A variety of flash plates are available [25], but this format is used less frequently than SPA beads. 3.8. Fluoresence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) is a homogeneous assay technique based on energy transfer between two fluorophores within the same molecule [26]. Cleavage of

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Figure 8 Scintillation proximity assay for tyrosine kinase. Biotin (B), tyrosine (Y), streptavidin
(SA), scintillation proximity assay bead (SPA).

Figure 9 Flash plate assay.

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the molecule separates the fluorophores and disrupts the energy transfer, thereby altering the wavelength of the emitted light. In the -lactamase assay depicted in Figure 10, the cephalosporin has coumarin bound to the -lactam ring and fluorescein bound to the dihydrothiazine ring. Excitation of this molecule at the coumarin absorption maximum (395 nm) yields the green fluorescence of fluorescein (535 nm) rather than the blue fluorescence of coumarin (460 nm) due to energy transfer from the coumarin to fluorescein. After cleavage of the molecule by -lactamase, there is no longer any energy transfer, and excitation at 395 nm yields the blue fluorescence of coumarin. The ease of readout allows this assay to be run with either enzyme preparations or whole cells to detect and determine -lactamase activity or to find inhibitors of -lactamase [27]. This assay is most often used to detect -lactamase when it is used as a reporter gene for activation or

Figure 10 FRET (fluorescence resonance energy transfer).

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inhibition of receptors. Because energy transfer requires close proximity between donor and acceptor dyes, FRET has also been used to measure protein--protein or protein--ligand interactions, by conjugating dyes directly to the proteins or to antibodies that recognize the proteins. 3.9. Homogeneous Time-Resolved Fluorescence Homogeneous time-resolved fluorescence (HTRF) also involves energy transfer between fluorophores in close proximity, but in this case, the fluorescence emitted is long-lived thereby allowing measurements to be delayed sufficiently after excitation to avoid interference from normal (short-lived) background fluorescence [28,29]. This is achieved by excitation of lanthanides and energy transfer to XL-665 (allophycocyanin), a 105-kd protein that functions as an accessory pigment in cyanobacteria and red algae [30]. Figure 11 outlines the concepts of an HTRF assay [31]. Line 1 indicates a complete reaction in which there is energy transfer from the donor lanthanide (europium) excited at 337 nm to the acceptor XL-665, which then emits light at 665 nm. Lines 2 and 3 indicate events where there is no energy transfer. Europium absorbs light at 337 nm and emits long-lived fluorescence at 620 nm. XL-665 also absorbs at 337 nm and emits normal (short-lived) fluorescence at 665 nm. In a typical assay, long-lived fluorescence at both 665 nm (channel A) and 620 nm (channel B) are measured and the ratio of A/B is calculated. High-channel B readings are often an indication of interference [31]. Figure 12 is an HTRF tyrosine kinase assay in which the substrate is biotinylated, streptavidin is labeled with the acceptor

Figure 11 HTRF concepts.

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Figure 12 Tyrosine kinase assay in HTRF format.

dye XL-665, and an antibody against phosphotyrosine is labeled with europium cryptate [Eu(K)]. The XL-665-streptavidin binds to the biotinylated substrate and after phosphorylation, the europium-labeled anti-phosphotyrosine antibody binds to the phosphotyrosine enabling energy transfer, and an increase in fluorescence at 665 nm is measured. 3.10. Luminescence Luminescence, a long-lived light emission produced by any one of a variety of luciferases acting on their corresponding substrate luciferins, is commonly used as readout in cellbased receptor assays. The luciferase gene is cloned into the cellular DNA downstream of the promoter of interest, and is used as a reporter gene. Figure 13 illustrates such an assay for a generic nuclear receptor. After binding of the ligand to the receptor’s ligandbinding domain (LBD), the receptor–ligand complex enters the nucleus where the receptor’s DNA-binding domain (DBD) binds to the DNA response element. After complexing with transcription factors, the promoter is activated and transcription of the luciferase reporter occurs. The cells are then lysed, luciferin is added, and resulting luminescence provides a measure of receptor activity. A comparison of luminescence in the control (no fungal extract) with those containing sample indicates antagonism or agonism. Also, luminescence is commonly used in assays for cytotoxicity in which luciferase and excess luciferin are added to measure levels of ATP [32,61].

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Figure 13 Nuclear receptor assay using luminescence as the reporter.

4. ASSAYS 4.1. Enzyme assays Enzyme inhibition assays are probably the most common type of in vitro assay. They are rapid, require small amounts of material, and are easily adapted for high-throughput screening. The types of enzymes screened include proteases, peptidases, topoisomerases, kinases, oxidases, reductases, dehydrogenases, beta lactamases, RNA and DNA synthetases, and enzymes of bacterial and fungal cell-wall synthesis (e.g., transpeptidases, glucan synthetases). Enzymes have been successfully used as targets in a wide variety of therapeutic areas such as anti-microbials (i.e., anti-bacterials, anti-fungals and anti-virals), anticancer treatments, anti-inflammatories, anti-hypertensives, anti-obesity therapies, anti-parasitics, and cholesterol-lowering drugs. Because there are numerous assay types, this discussion focuses more narrowly on various assay formats and their applicability to screening of fungal extracts. Nonhomogeneous formats are an older technology and rely on separation of unreacted tagged-reagents and macromolecular products via filtration, centrifugation, or binding to a surface. Protein kinases and polysaccharide synthetases (glucan, chitin) are examples of assays using filtration. In the kinase assays, -labeled ATP is separated from the phosphorylated protein product; in the glucan synthase or chitin synthetase assays, the radiolabeled glucose or N-acetylglucosamine is separated from the respective oligosaccharide products. Separation via centrifugation has been successfully employed in protease assays. In this case, the tagged unreacted protein substrate is either precipitated (by acid) or bound to ion exchange resin and then separated from the released radiolabeled amino acid or peptide by centrifugation. ELISA has been used for a wide variety of enzymes and is limited only by the availability of specific primary and secondary antibodies. In this technique, the immobilized primary antibody traps the reaction product, and detection occurs via binding of the secondary enzyme-linked antibody. In all nonhomogeneous assays, almost complete separation of reactants (including the screening sample) and products can be achieved by washing (filtration, ELISA) or centrifugation. This can be advantageous in screening fungal extracts because they contain fluorophores and chromophores, which may interfere with the signal detection. The extracts can quench a liquid scintillation assay and can quench or enhance (via autofluoresence) a fluorescence or absorbance assay signal. Assays employing ion exchange resins and ELISA assays are also subject to interference by compounds that inhibit binding of the ligand to the resin or antibody, respectively. If not employed in primary screening, nonhomogeneous assays are very useful for confirmation and should be used to verify all actives prior to beginning isolation.

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Homogeneous assay formats are available for virtually all detection techniques and are commonly used in moderate- and high-throughput screening because they simplify the assay and decrease the time required. These assays, however, tend to be expensive due to the need to purchase specialized reagents and equipment, and they are more prone to interference because products are not separated from the reactants. Common types of homogeneous formats include measurement of increase in absorbsance, increase/decrease in fluorescence, SPA, flash plate, FRET, HTRF, and luminescence. As mentioned earlier, the measurement of changes in absorbance and fluorescence is subject to interference by chromophores and fluorophores present in the fungal extract. Increases in absorbance can be masked by a high intrinsic absorbance of an extract, which can elevate the background sufficiently to decrease the sensitivity of the assay and lower the signal to noise ratio. False positives can result from a sample that fluoresces at the measuring wavelength. These difficulties have limited the use of absorbance assays with crude extracts. Increases in fluorescence can be masked by chromophores, which either absorb the excitation light or quench the emitted fluorescent light. Alternatively, a sample with autofluoresence would be a false positive. These difficulties in fluorescence assays can be overcome by use of time-resolved fluorescent assays employing lanthanides. These compounds emit a longlived fluorescence that can be resolved from the background fluorescence produced by the extract. In these assays it is important to measure both the donor (lanthanide) and acceptor (XL-665) emissions and to use the ratio of these measurements to monitor changes in the donor emission due to changes in association between the donor and acceptor. SPA beads are a popular format for conducting radiolabeled assays in a homogeneous format. This technique relies on specific binding of the radioligand to the bead to allow detection by the scintillant embedded in the bead, and it is subject to interference by compounds that inhibit this binding, such as long-chain fatty acids, which are commonly found in fungal extracts. Once radioligand is bound to the bead, detection of radioactivity in SPAbased assays ultimately relies on light emission by the scintillant in the SPA bead and is therefore subject to the same types of interference as normal scintillation counting—i.e. color quench, chemical quench, and interference by fluorescence or luminescence. The scintillation counter must therefore be set to correct for luminescence, and quench curves must be prepared and used to correct the measured counts per minute (cpm) to disintegrations per minute (dpm). Luminescence assays using luciferase are also subject to interference by chromophores that may quench the emitted light, and an appropriate blank (containing the active extracts) must be used to confirm all actives. Interference from fluorescence is not significant because of the long life of the luminescent signal. 4.2. Receptor-Based Assays Receptor–ligand binding is an important aspect of biology. There are two main types of receptors—cell-surface receptors and intracellular (nuclear) receptors [33,34]. The cellsurface receptors are divided into three families based on their mechanism of action: ligand-gated ion channel receptors, G-protein–coupled receptors, and enzyme-linked receptors [34]. Ligand-gated ion channel receptors open or close in response to ligand binding (acetylcholine, glutamate) and either allow or restrict passage of specific ions [35]. These receptors are commonly found on postsynaptic membranes. G-protein–coupled receptors undergo a conformational change upon ligand binding (epinephrine, glucagon). This in turn causes dissociation of the coupled G-protein, which induces downstream signaling [36]. Enzyme-linked receptors either have intrinsic enzyme activity (insulin receptor) or

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can recruit proteins with catalytic function (tyrosine or serine-threonine kinases), which are activated by the conformational change elicited by ligand binding (insulin, interleukins, integrins) [37]. Nuclear receptors are transcription factors that reside in the cytoplasm or nucleus, and their ligands (testosterone, cortisol) consequently must pass through the membranes to reach these receptors. Ligand binding induces a conformational change that allows DNA binding and subsequent regulation of transcription [23,38]. Assays of ligand-receptor interactions are similar regardless of the receptor type. Binding to receptors can be measured in whole cells, cell membrane preparations, detergent-solubilized receptors, or with purified soluble receptor. In each case, the principle is the same: to measure the amount of specifically bound ligand. Irrespective of the assay format, the binding must be well-characterized prior to setting up a screen. Several textbooks provide a detailed discussion of receptor biochemistry [39–41], which is beyond the scope of this chapter. Briefly, conditions for saturable binding of ligand to receptor must first be determined, and the KD calculated. The extent of nonspecific binding is measured by including an excess of unlabeled ligand to effectively compete for all the specific (reversible) binding sites in the preparation. The assay should then be designed to measure competition for binding of labeled ligand under equilibrium, at a concentration of labeled ligand below the KD. Ideally, a ligand concentration of 10% to 20% of the KD value should be used to obtain maximum sensitivity and a low ratio of nonspecific/specific binding. Binding assays are most commonly used for primary screening, and wholecell–based assays using reporter genes are generally used to confirm actives and demonstrate whole-cell functionality. Whole-cell functional assays have been used for primary screening. However, this is generally more time-consuming. A variety of assay types are available for measuring ligand-receptor interactions. Traditionally, this is achieved by using radiolabeled ligand and removing any unbound ligand by filtration and washing [16,42]. Centrifugation binding assays are popular for measuring binding to receptors in membrane preparations. Here, the membranes bearing receptors and the bound radiolabeled ligand are sedimented, allowing unbound radioligand to be removed by aspirating the supernatant [15,43]. Charcoal adsorption and gel filtration are other methods that can be used to separate free radioligand from that bound to soluble or detergent-solubilized receptors [15,44]. Filtration, centrifugation assays, and gel filtration assays can be done in high-throughput format but are less desirable than homogeneous assays due to the radiolabel use and necessity for washing steps. Homogeneous assays for ligand-receptor interactions include: SPA assay (PPAR receptor) in which receptor is immobilized on the bead and radioactive ligand interaction is detected (Fig. 7) [45]; fluorescence-based assays in which cells, membranes, or receptors are immobilized and bound fluorescent ligand is detected [46]; and HTRF in which receptor and ligand are in solution, each tagged with fluorophore (or fluorophore-linked antibody) and ligand binding is detected by long lived fluorescence at 655 nm [47]. Fluorescence polarization can be used for competitive assays to measure the capacity of a competitor compound to displace a high-affinity fluorescent ligand from either crude membrane preparations or purified receptors [48]. Because smaller molecules rotate faster than larger molecules in solution, fluorophores attached to smaller molecules yield a smaller polarization signal. The binding of fluorophore-labeled ligand to receptor results in an increase in the polarization signal compared to that of the labeled ligand alone. The polarization signal is linearly dependent on the extent of binding. In the presence of unlabeled competitor ligands, the polarization signal is reduced if the unlabeled ligand replaces the fluoro-

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phore-labeled ligand at the receptor binding site. This technique requires a fluorescence polarization detector, which is available for microplate format. In addition to potential interference by fluorophores in the assay samples, polarization measurements are particularly sensitive to viscosity. Enzyme fragment complementation of -galactosidase is a new technique for measuring displacement of ligands from solubilized receptors [49]. This technique uses -galactosidase, genetically engineered into two inactive fragments that can recombine in solution to form active enzyme and cleave fluorescent or chemiluminescent substrate (see 3.5). To measure receptor binding, one enzyme fragment is conjugated to the ligand; the other is free in solution. Binding of the -galactosidase-ligand-conjugate to receptor prevents recombination with the other fragment and thus results in reduced enzyme activity. Displacement of the conjugated ligand from the receptor due to competition from a component in the screening sample is detected by an increase in enzyme activity because the conjugated fragment is now free to recombine with the other fragment to form active -galactosidase. Receptor–ligand interactions can be assayed in whole cells genetically engineered to overexpress the receptor of interest and to contain reporter genes to provide convenient means of measurement. Whole-cell assays have been used for G-protein–coupled receptors and nuclear receptors. Such an assay for nuclear receptors using luciferase as a reporter was described earlier as an example of a luminescence assay (Fig. 13). -galactosidase is another commonly used reporter gene that can be detected using the fluorescent substrates as described earlier (Fig. 6). In functional assays for the enzyme-linked receptors, the activity of the linked enzyme is measured directly following cell lysis. Because these are virtually all kinases, any of the assay formats described can be used. Ligand-gated ion channels are conveniently assayed using ionspecific fluorophores whose fluorescence intensity yield significantly increases (100-fold) on ion binding [50,51]. In a typical assay, cells are preloaded with the fluorophore and following stimulation, the influx of the requisite ion is monitored by measuring the increase in fluorescence. 5. SCREENING FOR ANTIMICROBIAL ACTIVITY Natural products, including fungal metabolites, have yielded most of the clinically useful antimicrobial agents to date. Examples of useful antimicrobial drugs produced by fungi include penicillin, made by Penicillium chrysogenum, cephalosporin by Acremonium, and griseofulvin by Penicillium griseofulvin. In addition, active fungal metabolites that are unattractive as drugs themselves due to toxicity or low potency have been useful in identifying novel chemical classes, which have served as scaffolds for synthesis of successful antibiotics. Structural classes of natural product antimicrobial drugs have been reviewed by Silver and Bostian [52]. Identification of novel antibiotics can be achieved by screening fungal metabolites with various assay methodologies, including target-based screening, empiric growth assays, and the use of microbial genomics for discovery of novel antimicrobial targets. In target-based screening, enzymatic and receptor-binding assays are used to find inhibitors of specific biochemical targets. This approach requires extensive knowledge of the microbe and the difference in its biochemistry and physiology compared to mammalian cells in order to validate a given target. Current antimicrobial agents target members of macromolecular metabolism pathways, cell division, and nutrient transport. Tetracycline, streptomycin, chloramphenicol, and others inhibit key enzymes involved in bacterial protein synthesis [53]. Beta-lactams and glycopeptides are examples of antibiotics that target cell-wall

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synthesis in bacteria [54], and echinocandin and nikkomycin target fungal cell-wall synthesis [55]. Bacterial fatty acid biosynthesis is inhibited by the hand soap additive triclosan, and RNA and DNA synthesis is inhibited by rifamycin and novobiocin, respectfully [52,53]. Therefore, once a target enzyme or pathway is validated, specific in vitro assays may be designed using purified enzymes and assay formats as discussed earlier in this chapter. Cell-based assays may also be used to identify novel antimicrobials. Growth inhibition assays are advantageous in that they identify compounds that must be physically able to interact with their target protein in the cell (i.e., must be cell permeable if target is intracellular). Demonstration of cell-permeability at the initial hit identification stage eliminates the disappointment associated with the discovery of potent enzyme inhibitors that are later found to lack whole-cell activity because they cannot physically interact with their target in the cell. However, an enzymatic assay may identify a chemical class of potent inhibitors that can then be modified and made permeable, whereas a whole-cell assay would preclude finding such inhibitors. Another advantage of the cell-based growth screen is that it does not require knowledge of a particular cellular target. Such an empiric approach should allow for discovery of new antibiotics that act against novel targets. Simple growth assays that measure the growth of bacteria or fungi in liquid culture are an inexpensive and straightforward means of screening for antimicrobial activity. Growth inhibition can be monitored in liquid culture by turbidity measurements (light absorbance at 600 nm) or by commercially available homogeneous chromogenic and fluorogenic assays. For example, metabolic activity can be measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] or Alamar Blue reduction. The MTT assay involves incubation of the bacterial culture with MTT, a soluble tetrazolium salt. The MTT is generally added after incubation of the culture with chemical compounds or fungal extract for several hours. In metabolically active viable cells, MTT is bioreduced by dehydrogenases into a colored formazan product. The quantity of formazan product produced is directly proportional to the number of living cells, and it is measured by light absorbance at 490 nm [56]. The MTT assay is highly reproducible and sensitive. Less is understood about the action of Alamar Blue (resazurin) although it has been widely used with success in high-throughput assays for antimicrobials [57,58]. A mitochondrial dehydrogenase is thought to reduce Alamar Blue to a pink product, which can be detected by absorbance or, for increased sensitivity, by fluorescence [59]. Other means of monitoring cell growth are commercially available. The Oxygen Biosensor is a fluorescence-based method that detects the amount of oxygen consumed over time, where oxygen consumption is proportional to the rate of cell respiration [60]. No chemicals are required for the oxygen detection because the system for measuring oxygen consumption is contained within the Oxygen Biosensor assay plate. In addition, bioluminescent detection of cellular ATP can be used to monitor cell toxicity [61]. Unlike the homogeneous assays described, ATP measurement requires the cells to be lysed at the end of the test period prior to the addition of a luciferase reporter to quantify cellular ATP. Since only viable cells at the time of lysis will contain ATP, and because the turnover of luciferin by firefly luciferase requires ATP, the luminescent intensity is directly proportional to the number of viable cells. These assays are all readily amenable to miniaturization to at least 384-well format. Because natural products contain chromophores and fluorophores, the same considerations mentioned earlier for enzyme assays relying on fluorescence and absorbance must be addressed. Bioluminescent assays are less prone to interference from the chromophores; fluorophores and turbidity commonly present in natural product extracts. For all liquid assays of growth

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inhibition, confirmation of activity is generally achieved by demonstrating a dose-dependent inhibition of cell growth by the fungal extract. This confirmation of activity requires titration and retesting of the active extract and is used to determine the minimum inhibitory concentration. Agar-based growth assays are also useful for screening for antimicrobial agents, but they require more sophisticated optical imaging systems and software for quantitation in high-density screening formats. To perform an agar diffusion assay, agar seeded with bacteria or fungi are poured into plates. Samples of interest are then spotted on the plates. Following incubation for sufficient time to establish a lawn of growth, zones of growth inhibition are measured as indication of antibiotic activity [62]. The size of the zone of inhibition is proportional to the concentration and potency of the drug. The clear zones of inhibition on an opaque lawn of growth can be measured manually. For higher density screening formats digital images of screening plates can be analyzed by software that recognizes pixel density differences and calculates the radii of the zones. Monitoring growth inhibition in agar is advantageous in that it allows a titration of drug to be achieved from a single spot due to the radial diffusion of the drug in agar. Thus, a dose titration is achieved at the same time as primary screening for active metabolites. Of course, different compounds in a complex mixture such as a fungal extract will have differing rates of diffusion. A zone of inhibition around a fungal extract may result from multiple weak antibiotics in the mixture or could be due to a single potent antibiotic present at very low concentration in the mixture. Either way, fractionation of extracts with confirmed activity will allow identification of the active component(s). Thus, an agar diffusion assay should facilitate discovery of antibiotics present as minor components in fungal extracts. Examples of antimicrobial agar diffusion screening assays are plentiful in the literature [63–67]. Caution must be taken when applying samples to agar. In particular, compatible solvents must be used to maintain the agar in a gel state and to maintain the metabolites in a soluble format to allow diffusion from the point of application. Samples may be spotted on top of agar plates in very small volumes, or a well may be formed in the agar to allow larger volumes of samples to be applied. Whether monitoring growth in liquid or in agar, screening of fungal extracts is challenging because it is possible that a small concentration of a potent antibiotic could go undetected in an extract mixture because the presence of interfering compounds limits the concentration at which these natural product mixtures can be tested. Prefractionating the fungal extracts is one means of potentially enriching for the active metabolite. Fractionating the extracts may also help to identify and eliminate all the known antibiotic activities in the extracts, saving time and reagents by avoiding discovery of the same antibiotics repeatedly. Prefractionation of samples may not always be practical, however, because it drastically increases the number of samples to be screened. In an alternative approach, the cells can be rendered more sensitive to inhibitors. For example, gram-negative bacteria such as Eschericia coli are more resistant to antibiotics than gram positive bacteria due to limited entry of the drugs to the site of action. E. coli strains deficient in the envA gene product render the cells more permeable and therefore more sensitive to all antibiotics [68,69]. This will increase the chances of finding an antibiotic present at low concentration in an extract but would likely also increase the chances of finding nonspecific toxins. More specifically, microbes can be rendered more sensitive to inhibitors of particular pathways or enzymes, increasing the chances of finding a rare antibiotic. Microbial genomics provide the tools for engineering such particularly susceptible fungi and bacteria. In general, target specificity may be obtained by using genetically engineered strains, where

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expression of particular genes are altered and sensitivity to fungal extracts is compared with the parent wildtype strain [70,71]. Here, the gene of interest is placed under the control of an inducible promoter to allow overexpression or underexpression of the protein. When putting a gene under the control of an inducible promoter, it is essential that the gene and promoter are chromosomally located for the construct to be inherited through the rapid divisions these cells undergo throughout the course of a growth assay. One could theoretically screen several targets in a single operon if it is behind an inducible promoter. This would allow screening for inhibitors of any stage of a given pathway, for example, rather than searching for inhibitors of one particular enzyme. This can be done both in liquid or agar but requires twice the screening (i.e., two-plate assays for comparisons of the overproducer or underproducer with the wildtype) [63]. With this strategy, it is possible to find leads that are active not only against whole cells but that are also specific for a biochemical target. Similar to the enzymatic and receptor-based screens, this approach requires extensive knowledge and validation of the biochemical targets. Microbial genomics is useful to help guide target selection. With more than 60 complete genomic sequences of prokaryotes currently available, new strategies for discovering novel antibiotics are emerging [72]. Essential genes can be identified by highthroughput gene disruption analysis, in which genes are disrupted by various strategies and the ability of the strain to grow in the absence of the gene product is assessed [73–75]. Entire arrays of target-specific screening strains can be constructed in which each strain in the array has lowered expression of a single, essential gene product [76]. Inhibition of gene expression can be obtained through the use of antisense RNA. Elitra Pharmaceuticals has identified a large number of essential genes in Staphylococcus aureus by cloning DNA fragments coding for antisense RNA under control of a xylose-inducible promoter [77]. Expression of an antisense RNA complementary to the mRNA of an essential gene results in RNA–RNA duplexes that effectively prevent translation of that specific mRNA. This results in a reduction in the level of target protein and thereby confers specific sensitivity to compounds targeting that gene product. For example, an S. aureus clone expressing an antisense RNA to the gene encoding -ketoacyl-acyl carrier protein synthase was found to have 12-fold increased sensitivity to the inhibitor cerulenin when the antisense RNA to the gene was induced with xylose, compared with the same clone grown in the absence of xylose. Such hypersensitivity to antibiotics specific to the gene product of interest should allow identification of antibiotics in complex mixtures. In Elitra’s shotgun antisense approach, essential genes were identified after conditionally expressing random genomic fragments and screening the fragments for growth inhibition. Clones showing strong growth inhibition upon antisense induction were sequenced and the essential genes identified by BLAST analysis [77]. Conditional lethal mutants can also be used to identify essential genes as potential antibiotic targets. A conditional lethal mutation allows the gene product to function normally under one condition while being rendered inactive under another. Temperature-sensitive mutants are one example of conditional lethal mutants. Conditional mutants can also be generated by positioning a complementary copy of the essential gene of interest under the control of an inducible promoter, such as by positioning the gene at the ara locus of E. coli. This renders the expression of the gene and the growth of the E. coli dependent on arabinose. In the absence of arabinose, the gene product will be depleted and the organism will be more sensitive to inhibitors of that protein [75]. Parallel screening of multiple essential drug targets can be achieved using these methodologies. Pools or arrays of mutants can be screened together with each mutant distinguishable

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by a molecular tag. The susceptible mutant can then be identified from the pool, indicating the target of the fungal metabolites [76]. With emerging technology for identifying novel targets, the chemical diversity of fungal metabolites can be exploited for discovery of novel antibiotics.

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Madrid. we will focus on the molecular mechanisms underlying sordarins’ mode of action in the fungal cell with the aim of understanding the basis for their selectivity. The AIDS pandemic as well as side effects of cancer chemotherapies have been responsible for the renewed emergence of these life-threatening pathologies. many pharmaceutical companies initiated programs devoted to the discovery of new drugs that may help to improve their current antifungal arsenal. These molecules exert their antifungal effect by selectively impairing fungal. is a key intermediate in the synthesis 335 . we will summarize the more relevant features of their biological profile in order to envision their potential clinical applications. This chapter provides a general perspective on the current knowledge about this family of compounds. Consequently. A. a tetracyclic diterpene glycoside that was originally isolated by workers at Sandoz in 1971 as a metabolite of Sordaria araneosa [1].11 Sordarins: Inhibitors of Fungal Elongation Factor-2 ´ ´ JUAN MANUEL DOMINGUEZ and JULIO J. Finally. MARTIN GlaxoSmithKline Research and Development. INTRODUCTION During the past two decades. Spain 1. touching upon their chemical tractability including biotransformation procedures.. It is in this context that a family of natural products known as sordarins appeared in the antifungal arena in the mid-1990s. protein synthesis. 2. This exquisite selectivity appeared as a remarkable and surprising property of sordarins given the high similarity among translational machineries in all living organisms. This became even more noticeable once it was shown that elongation factor2 (EF-2) was the molecular target for sordarins. After describing their chemical features. S. and not mammalian or bacterial. SORDARINS AS A CHEMICAL FAMILY Sordarin. the impact of systemic fungal infections associated with immunocompromised conditions has increased overwhelmingly.

Nevertheless. Rosellinia subiculata (sordarins by Merck) [4]. and its aglycone. All natural sordarin analogues contain a common aglycone moiety known as sordaricin. Graphium putredinis (producer of GR135402 by GlaxoSmithKline) [5] and the marine fungus Hypoxylon croceum (producer of hypoxisordarin) [6]. it shows only weak activity against whole yeast cells. such as Zopfiella marina (producer of Zofimarin by Sankyo) [2]. the new analogues still having a remarkable antifungal activity [7]. Banyu. sordaricin shows no activity at all [7. The general liability of the aldehyde groups as a possible drawback for the development of sordarins as drugs has given rise to the search for more stable bioisosters. In fact. Merck. Although sordarin itself is considerably potent as a protein translation inhibitor in fungal cell – free systems. other producing microorganisms have been identified. which bear as key groups in the putative pharmacophore a carboxylic function vicinal to a formyl group. not forming a cyclic hydroxylactone because of their rigid conformation in a high dihedral angle (Fig. acylated sordarin derivatives such as Zofimarin and GR135402 exhibit a potent profile as inhibitors of the protein synthesis in yeast and fungi. . Since sordarin’s isolation. Sordaricin aglycone and sordarose sugar moieties are shown.8].e. It is important to note that whereas sordarin derivatives containing nonsubstituted sordarose show a relatively modest antifungal activity. Most chemical modifications of the aldehyde and carboxylic groups in these analogues abolished the activity of sordarins. both in cell-free Figure 1 Chemical structure of sordarin. The sugar moiety (i. attempts to make a more flexible aglycone by cleaving ring bonds have led to compounds devoid of activity. Over the last few years. Penicillium mineoluteum (producer of BE-31405 by Banyu) [3].336 Domınguez and Martın ´ ´ of novel antifungal agents known as sordarins. sordarose) seems to play a role in enhancing the binding of sordarins to the active site. and Schering-Plough—have reported the discovery of new members of this family as a result of their screening campaigns. several pharmaceutical companies—such as GlaxoSmithKline.. it has been possible to replace the aldehyde by nitrile group. especially to chemical or biological oxidation. 1).

Hence. and hydroxisordarin. and . acylations. the antifungal activity is markedly enhanced over that of sordarin and sordaricin [7]. or morpholines (azasordarins by GlaxoSmithKline) [12]. In brief. and reduction of aldehyde to alcohol by Mortierella. the biosynthesis of sordarins has not yet been studied in depth. we could distinguish the following approaches to new sordarin derivatives: 1. Unfortunately. it has not been possible to perform rational genetic engineering for new producing microorganisms. GSK).16. This has been the most productive approach in terms of the number of new products generated. access to new sordarin derivatives has mostly been carried out by using natural product intermediates. therefore. new semisynthetic sordaricin derivatives with potent antifungal activity have been obtained in which sordarose has been replaced by alkyl-ethers (by Merck) [7]. 3. Semisynthetic derivatives starting from demethylsordarins.19]. oxidation of aldehyde to carboxylic acid by Xylaria. Sordarin derivatives have also been made by using solid-phase chemistry in a combinatorial fashion (personal communication from Dr. as well as in terms of the potency and pharmacological properties of the compounds made. this approach has enabled access to other intermediates that expand the chemical tractability of sordarins. Zofimarin. neosordarin. De novo chemical synthesis of sordaricin derivatives has been reported [13]. the biotransformation products exhibited reduced antifungal activity compared with either sordarin. In general.. sordarin. esterifications. SCH57404 [9]. Some of the compounds directly isolated are Sordarin. there are also sordaricin derivatives bearing unusual sugar moieites that retain antifungal activity. GR135402. xylarin. It is beyond the scope of the current chapter to present a comprehensive review of all the synthetic efforts made by the different groups involved in the chemistry of sordarins. it seems that the lipophilicity of such an appendage is critical for the effective modulation of the cell uptake. or GR135402. Chemical synthesis of new derivatives from natural product intermediates as starting materials. such as BE-31405 [3]. zofimarin. Although de novo total chemical synthesis of the methyl-ester of sordaricin has been described [13]. such as tetrahydrofurans and azasordarins. Although most of the natural sordarins isolated so far only differ in the type of sordarose substitution. demethylation of sordarose in sordarin by Streptomyces [15]. oximes (by Bristol-Myers Squibb) [11]. An interesting development in the exploration of this natural product series was the discovery that when sordaricin is functionalized with lipophilic side chains. hypoxysordarin. and 4′-O-demethyl sordarin have been extensively derivatized at the sordarose moiety (e. S. 2. However. substitutions of hydroxyles by nitrogen or carbon. including its replacement by nonsugar arrangements.). 4′-O-demethyl sordarin. Moreover. Sordaricin. and simplified aglycone analogues that resembled the pharmacophore of sordaricin have also been pursued [18. Biotransformation of natural product intermediates with microbial organisms. have undergone profuse preclinical investigations by GlaxoSmithKline [9. and Xylarin [10]. Isolation of microbial metabolites. the chemical synthesis of sordarin is too complex to make it exploitable in drug discovery programs. SCH57404. Unfortunately. Garcia-Ochoa. Some of the sodarin derivatisations accomplished by microbial transformations are hydroxylation of zofimarin aglycone at position 6 by Streptomyces [14]. etc. esterification of sordaricin and sordarin by Pithomyces.5 Sordarins: Inhibitors of Fungal EF-2 337 and whole-cell assays.g. 4.17].

g. including clinical isolates of yeasts (Candida sp. toxicity. Over the past 10 years.1. Blastomyces. a considerable effort has been made in the pharmaceutical industry to come up with new chemical classes of antifungals through biased screening against novel molecular targets. such as resistance. less common mold . the target selectivity profile against fungal EF-2 is retained. The azoles and allylamines inhibit its biosynthesis. dermatophytes (e. Coccidioides). Hence. GlaxoSmithKline has conducted advanced preclinical investigations on sordarins. 3. Trichophyton. As a result. fungal infections caused by opportunistic microorganisms have emerged in recent decades as an important reason of morbidity and mortality in immunocompromised patients. They all interfere with the functionality on ergosterol in the fungal plasma membrane. polyenes. have been more recently identified and have the additional advantage of an easier chemical synthesis. generically named azasordarins. Present antifungal drugs on the market suffer from several drawbacks. For more than 15 years. Histoplasma. Microsporon). there is still an unmet need for new antifungal agents having a different mode of action from those currently used. A summary of the antifungal profile of the four selected sordarin derivatives to inhibit the growth of key fungal microorganisms in culture is displayed in Table 1.. Thus.338 Domınguez and Martın ´ ´ none of the simpler and more synthetically amenable structures has retained an interesting biological profile. SORDARINS AS AN ANTIFUNGAL FAMILY As mentioned. GM193663 contains a dioxolane ring.3)-glucan synthetase inhibitors. Merck recently launched (caspofungin acetate). whereas GM237354 contains a tetrahydrofurane ring. azoles. whereas no activity at all was exhibited at 100 g/mL against a similar counterpart system from rabbit reticulocyte [8]. and Cryptococcus neoformans). These derivatives. dimorphic endemic fungal pathogens (e. The second series includes compounds in which the sordarose sugar moeity in sordarin is replaced by a 6-methylmorpholin-2-yl group with different substitutions at N-4′position [9]. and other emerging. filamentous fungi (Aspergillus sp. The in vitro activity of sordarins has been extensively characterized by several workers. and limited spectrum of action.). The first series includes compounds presenting different types of fused rings at positions C3′ to C4′ of the 4′-O-demethylsordarin [17]. On the other hand. GW570009 and GW587270 may be considered as the best balanced and promising leads within this series in terms of antifungal potency. 3. This is the first lipo-depsipeptide antifungal in the market that belongs to the also known echinocandins or pneumocandins series of -(1. spectrum. The most advanced of this company’s sordarin derivatives can be grouped into two main chemical series represented by the four compounds included in Table 1.g. and allylamines have been the only three chemical classes of antifungals for the treatment of systemic fungal infections. and polyenes physically interact with ergosterol and form pores that impair fungal membrane permeability. Pneumocystis carinii. In Vitro Antifungal Profile All the compounds showed low nanomolar potency to inhibit a cell-free translational system from Candida albicans. Epidermophyton. and safety profile.. such as those individuals affected by AIDS or receiving immunosuppresive therapy upon organ transplantation or oncological treatment.

015 0. fluconazole-resistant strain 339 .008 <0.004 0. flavus Other fungal pathogens Pneumocystis carinii Histoplasma capsulatum MIC.03 0.03 0. krusei Cryptococcus neoformans Filamentous fungi Aspergillus fumigatus A. parapsilosis C.008 0. GM 193663 H O O H O H O O OH MIC ( g/mL) 0. minimum inhibitory concentration.001 >32 0. glabrata C.06 8 >32 0.008 <0.Table 1 In Vitro Antifungal Activity of Sordarin Derivatives. Flur.25 0.25 0. albicans Flur C.001 0.008 0.25 >32 >32 4 >64 >64 <0.06 2 >16 >16 >16 >16 NT NT 0.12 0. not tested.001 64 4 0.001 <0.001 1 0.5 >16 >16 >16 >16 NT NT Chiral O H H O H O H O O OH H O O OH GM 237354 H O H H O H O H Chiral Chiral H O O H O /9 #%' GM 193663 Chiral O H N H Sordarins: Inhibitors of Fungal EF-2 H O O OH Microorganism Yeasts Candida albicans C. NT. tropicalis C.

In terms of sensitivity to sordarins. Poorly sensitive or resistant (micromolar MIC or inactive): Interestingly. parapsilosis and C.. and monkeys [16. Likewise. 3. GM237354 has shown therapeutic efficacy against systemic or oral candidiasis after subcutaneous or oral administration. tropicalis and C. Sordarins have also turned out to be more effective than fluconazole.21].g.25–28]. sordarin derivatives (e. albicans in fungal opportunistic infections. Given the high prevalence of C. kefir. parapsilosis. However. 4. glabrata and Cryptococcus neoformans.or submicromolar MIC): Although less potent. 3. the gold standard antifungal agent. Within this group of microorganisms we can also include Aspergillus sp. Some sordarins exhibit a marked slowness of Aspergillus growth. which is the standard treatment for PCP. carinii and Histoplasma capsulatum.22–25].g.17. Irregular responses are found for sordarin derivatives. sordarins are not intrinsically cidal drugs according to their primary effect. which points to the possibility of more effective sordarins.20].16.. some compounds such as GM237354 have shown some activity against C. Moreover. in the treatment of mice with disseminated histoplasmosis and coccidioidomycosis. krusei are two Candida sp. dogs. we can distinguish the following groups of fungal microorganisms: 1. with MIC values in the low.3. and particular structure – activity relationships are found within members of the different chemical classes.17. subnanomolar MICs have been reported for P.340 Domınguez and Martın ´ ´ pathogens [9. Short lifetimes and high clearance of sordarins in mice and rats have obligated to aggressive administration regimes in murine animal models. rats. As expected for protein synthesis inhibitors. C. Therapeutic Efficacy in Animal Models Sordarin derivatives have generally shown good activities in animal models for a wide range of fungal infections [16.e. GM211676.or subnanomolar range. Nevertheless. and they were not clastogenic in cultured human lymphocytes. Pharmacokinetics and Pharmacodynamics Pharmacokinetics of sordarin derivatives has been studied in mice. 48 hours) of the yeast cell to the compound. albicans. Moderately sensitive (low. GM193663. including fluconazole-resistant ones. murine candidiasis models have been the most extensively studied. and slight trailing growth is observed even in the highest concentrations. they are also active against C. Remarkably. sordarins showed superior activity over cotrimoxazole. though the end-points were less sharp than those obtained with yeasts. 2. However. Likewise. Highly sensitive (low-nanomolar MIC): Sordarins are also active against C. Sordarin derivatives show negligible activity with respect to inhibiting the protein synthesis in intact mammalian cell lines cultured in vitro. reluctant to the action of sordarins.17. and GM237354) were effective in protecting immunosuppressed rats to develop Pneumocystis carinii pneumonia (PCP).. albicans in the event of long exposure times (e.2. allometric relationships have been found among the species studied. all the compounds tested showed no apparent evidence of genotoxicity in the Ames test. 3. it has been shown that some sordarins produce a fungicidal effect (i. It has been shown that resistance resides in the nature of EF-2 rather than in the permeability or cellular stability of the compound [8. Extremely sensitive (subnanomolar MIC): Sordarins are extremely potent compounds against all strains of C. which predict more favorable . 3-log reduction in the number of viable cells) against C.

4. polyuridylic acid) or the use of high ionic strength to force initiation. However. Hence. 150 mg/kg) turned out to be lethal when intravenously infused in a rapid bolus due to cardiovascular effects.e.1... in vivo levels in serum were reproduced in vitro [27].9 hours) [28]. both using a double pronged approach with molecular biology and biochemistry tools. and tracheal inflation pressure when the lower doses of compound are infused at a slower rate (i. albicans) it has been possible to correlate the efficacy of GM237354 only when the free. 4. albicans whole cells [8] and comparing .. those artificial components demand more evidence to support the notion that protein synthesis was the real target of sordarin derivatives within the fungal cell. the physiological significance of such an assay is rather questionable because of the presence of ‘‘artificial’’ components such as nonnatural mRNA (i. The task was accomplished simultaneously by two different teams at Merck and GlaxoSmithKline.17]. lead II ECG. Although there was good correlation among the arrest of cell growth and inhibition of the cell-free assay. but not the total. preliminary studies point to phase II conjugation via glucuronidation as significantly responsible for the metabolic elimination of sordarins. Sordarins are well-tolerated when orally administered [16.. Sordarins Are Real Protein Synthesis Inhibitors Even though they were first described in 1971 [1]. Although ADME has not been profusely investigated yet. No significant subchronic toxicological effects were observed in clinical pathology and histopathology after 7 days of administration with GM193663 or GM237354. Toxicology and Safety Pharmacology Information on potential toxicological effects is still scarce. Although high doses of GM237354 (i.e. adequate administration regimes are foreseen for man (predicted t1/2 for GM237354 in human is 8. no significant effects were observed in arterial blood pressure heart rate.Sordarins: Inhibitors of Fungal EF-2 341 pharmacokinetics as long as the body weight increases and the liver blood flow decreases. This was achieved by testing the effect of a sordarin derivative in both protein and RNA synthesis de novo in C.e. sordarins were rediscovered when screening for fungal protein synthesis inhibitors [5] using a radioactive cell-free protein synthesis assay as the one described by Tuite and Plesset [29]. by simulating different PK profiles in an in vitro dynamic system (i. whereas neither Cmax (maximum peak of compound concentration in serum) nor overall time over in vitro microbiological MIC were reliable PK predictors [26]. 4. One of the most challenging problems in chemotherapy is how to ascertain the relationships between in vitro and in vivo activities that allow the prospective analysis of the therapeutic efficacy of compounds. What follows is a summary of how these investigations evolved and how a nicely detailed picture describing the molecular mechanism by which sordarins exert their antifungal action could be devised.e. GM237354 activity in a murine model of lethal candidiasis over a range of dosing intervals has been predicted by establishing a pharmacokinetics–pharmacodynamics (PK-PD) model in which both kidney burden and AUSTC (area under the survival time curve) correlate well with AUC (area under the curve). MODE OF ACTION The discovery of the precise mode of action of sordarins is an interesting story. Moreover. bioreactor with growing C. 57 mg/kg in 2 hours). 3.

Identification of the Molecular Target of Sordarins Once protein synthesis was confirmed as the cellular process targeted by these drugs. is omitted in the interest of greater picture clarity. . which disappeared at longer incubation times. therefore. it was totally dissimilar to that of phenantroline (a known RNA synthesis inhibitor). While the effect of sordarins was almost identical to that of verrucarin (a known protein synthesis inhibitor). Indeed. the results of further investigations were surprisingly different. Given the exquisite selectivity of sordarins toward fungi. However. ribo- Figure 2 Overall scheme of elongation cycle in fungi. adjacent to the Psite but involving exclusively the large ribosomal subunit. Protein synthesis comprises three sequential steps: initiation. depending on whether or not they constitute the ribosome particle and therefore precipitate or remain in the supernatant after ultracentrifugation. As a first approach to inquire into the target’s nature. the machinery of which is well conserved among the eukaryotic kingdom. elongation. The biomolecules involved can be roughly divided into either ribosomal or soluble factors. The ribosomal E-site. and termination. which is depicted in Fig. Translation is a complex process. 2. this sordarin derivative caused a short-term stimulatory effect in RNA synthesis at low concentrations. elongation factor-3 (EF-3) is the only protein known to be different among fungal and mammalian translational systems. the next step was to identify the biomolecules involved. it seemed plausible to deduce that the sordarin target might be one involved in that part of the translational process.2. The in vitro assay inhibited by sordarins includes only the elongation phase. Moreover. had already been described for specific protein synthesis inhibitors added to cultures of amino acid – starved yeast cells [30].342 Domınguez and Martın ´ ´