Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites

Fungal Biology

Biosynthesis

and Molecular

Genetics of

Fungal Secondary

Metabolites

Juan-Francisco Martín

Carlos García-Estrada

Susanne Zeilinger Editors

Fungal Biology

Series Editors:

Vijai Kumar Gupta, PhD

Molecular Glycobiotechnology Group, Department of Biochemistry,

School of Natural Sciences, National University of Ireland Galway,

Galway, Ireland

Maria G. Tuohy, PhD

Molecular Glycobiotechnology Group, Department of Biochemistry,

School of Natural Sciences, National University of Ireland Galway,

Galway, Ireland

For further volumes:

http://www.springer.com/series/11224

Juan-Francisco Martín • Carlos García-Estrada

Susanne Zeilinger

Editors

Biosynthesis and Molecular

Genetics of Fungal

Secondary Metabolites

ISSN 2198-7777 ISSN 2198-7785 (electronic)

ISBN 978-1-4939-1190-5 ISBN 978-1-4939-1191-2 (eBook)

DOI 10.1007/978-1-4939-1191-2

Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014946216

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Editors

Juan-Francisco Martín, Ph.D.

Department of Molecular Biology

University of León

León, Spain

Susanne Zeilinger

Institute of Chemical Engineering

Vienna University of Technology

Vienna, Austria

Carlos García-Estrada, D.V.M., Ph.D.

Parque Científico de León

Instituto de Biotecnología de León

(INBIOTEC)

León, Spain

v

Preface

The Wonderful World of Fungal Secondary Metabolites

There are thousands of fungal species in nature but only a handful of them, most of

them ascomycetes, have been studied in detail. Studies on the model fungi

Neurospora crassa, Aspergillus nidulans, Aspergillus niger, Penicillium chrysogenum,

and others, in comparison with the yeast Saccharomyces cerevisiae, have provided

the basic core of scientific knowledge on the vegetative metabolism and morpho￾logical differentiation of filamentous fungi. However, the biochemistry and molec￾ular genetics of fungal secondary metabolites are less known due to their large

diversity.

Some fungal products are extremely beneficial to combat tumors or bacterial and

fungal infections, and others contribute to control cholesterol metabolism to improve

human health. A large number of fungal metabolites, the mycotoxins, are highly

toxic for humans and for the livestock. They also affect soil-dwelling worms or

other organisms and, therefore, have a profound ecological interest. Finally other

fungal metabolites provide the vivid colors (e.g., β[beta]-carotene, astaxanthin) of

some fungi.

During the last decades, there has been an intense effort to elucidate the biosyn￾thesis pathways of fungal secondary metabolites to characterize the genes that encode

the biosynthetic enzymes and the regulatory mechanisms that control their expres -

sion. One interesting finding is that genes encoding fungal secondary metabolites are

clustered together, as occurs also with the bacterial genes for secondary metabolites.

This is in contrast to fungal primary metabolism genes, which are frequently scat￾tered in the genome. However, in contrast to the bacterial gene clusters, most of the

fungal secondary metabolite genes are expressed as monocistronic transcripts from

individual promoters. This raises the question of possible unbalanced levels of the

different mRNAs of the genes in a pathway and the need of temporal and spatial

coordination of their expression. Furthermore, expression of the secondary metabo -

lites in fungi is correlated with differentiation and with the formation of either sexual

or asexual spores, including cleistothecia and other types of differentiated cells.

vi

Fungal secondary metabolites are complex chemical molecules that are formed by

a few basic mechanisms with multiple late modifications of their chemical structures.

The basic mechanisms include enzymes such as non-ribosomal peptide synthetases

(NRPSs), polyketide synthases (PKSs), terpene synthases and cyclases, and less known

“condensing” enzymes that use as substrates a variety of activated precursors.

In this book we bring together 15 review articles by expert scientists on the best

known secondary metabolites that serve as model of the different biosynthetic types

of fungal secondary metabolites. Each chapter presents an updated review of the

medical, agricultural, food and feed applications, and the ecological relevance of

each compound.

Furthermore, we provide descriptions of the present status of knowledge on the

molecular genetics and biosynthesis of each of these compounds. All together

the expertise of the authors of those chapters provides an impressive overview of the

actual knowledge of the world of fungal secondary metabolites.

León, Spain Juan-Francisco Martín

Preface

vii

1 Valuable Secondary Metabolites from Fungi ....................................... 1

Arnold L. Demain

2 Penicillins................................................................................................. 17

Carlos García-Estrada and Juan-Francisco Martín

3 Cephalosporins........................................................................................ 43

Sandra Bloemendal and Ulrich Kück

4 Cyclosporines: Biosynthesis and Beyond.............................................. 65

Tony Velkov and Alfons Lawen

5 Aflatoxin Biosynthesis: Regulation and Subcellular Localization...... 89

John E. Linz, Josephine M. Wee, and Ludmila V. Roze

6 Roquefortine C and Related Prenylated Indole Alkaloids.................. 111

Juan-Francisco Martín, Paloma Liras, and Carlos García-Estrada

7 Ochratoxin A and Related Mycotoxins................................................. 129

Massimo Reverberi, Anna Adele Fabbri, and Corrado Fanelli

8 Carotenoids.............................................................................................. 149

Javier Ávalos, Violeta Díaz-Sánchez, Jorge García-Martínez,

Marta Castrillo, Macarena Ruger-Herreros, and M. Carmen Limón

9 Astaxanthin and Related Xanthophylls................................................ 187

Jennifer Alcaino, Marcelo Baeza, and Victor Cifuentes

10 Gibberellins and the Red Pigments Bikaverin and Fusarubin ........... 209

Lena Studt and Bettina Tudzynski

11 Fusarins and Fusaric Acid in Fusaria................................................... 239

Eva-Maria Niehaus, Violeta Díaz-Sánchez,

Katharina Walburga von Bargen, Karin Kleigrewe,

Hans-Ulrich Humpf, M. Carmen Limón, and Bettina Tudzynski

Contents

viii

12 Lovastatin, Compactin, and Related Anticholesterolemic Agents..... 263

David Dietrich and John C. Vederas

13 Meroterpenoids ....................................................................................... 289

Yudai Matsuda and Ikuro Abe

14 Ergot Alkaloids........................................................................................ 303

Paul Tudzynski and Lisa Neubauer

15 Fungal NRPS-Dependent Siderophores:

From Function to Prediction.................................................................. 317

Jens Laurids Sørensen, Michael Knudsen, Frederik Teilfeldt Hansen,

Claus Olesen, Patricia Romans Fuertes, T. Verne Lee,

Teis Esben Sondergaard, Christian Nørgaard Storm Pedersen,

Ditlev Egeskov Brodersen, and Henriette Giese

Index................................................................................................................. 341

Contents

ix

Contributors

Ikuro Abe, Ph.D. Graduate School of Pharmaceutical Sciences, The University of

Tokyo, Tokyo, Japan

Jennifer Alcaino, Sc.D. Departamento de Ciencias Ecológicas, Facultad de

Ciencias, Universidad de Chile, Santaigo, Chile

Javier Ávalos, Ph.D. Department of Genetics, Faculty of Biology, University of

Seville, Sevilla, Spain

Katharina Walburga von Bargen, Dr. rer. nat. University Münster, Institute of

Food Chemistry, Münster, Germany

Sandra Bloemendal, Ph.D. Christian Doppler Laboratory for Fungal

Biotechnology, Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität

Bochum, Bochum, Germany

Ditlev Egeskov Brodersen, Ph.D. Department of Molecular Biology and Genetics,

Aarhus University, Aarhus C, Denmark

Marcelo Baeza, Ph.D. Departamento de Ciencias Ecológicas, Facultad de

Ciencias, Universidad de Chile, Santiago, Chile

Marta Castrillo Department of Genetics, Faculty of Biology, University of Seville,

Sevilla, Spain

Victor Cifuentes, Sc.D. Departamento de Ciencias Ecológicas, Facultad de

Ciencias, Universidad de Chile, Santiago, Chile

Arnold L. Demain, Ph.D., M.S., B.S. Research Institute for Scientists Emeriti

(R.I.S.E.), Drew University, Madison, NJ, USA

Violeta Díaz-Sánchez, Ph.D. Department of Genetics, Faculty of Biology,

University of Seville, Sevilla, Spain

David Dietrich, B.Sc., Ph.D. Department of Chemistry, University of Alberta,

Edmonton, AB, Canada

x

Anna Adele Fabbri, Ph.D. Department of Environmental Biology, Università

Sapienza, Roma, Italy

Corrado Fanelli, Ph.D. Department of Environmental Biology, Università

Sapienza, Roma, Italy

Patricia Romans Fuertes, M.Sc. Department of Biotechnology, Chemistry and

Environmental Engineering, Aalborg University, Aalborg, Denmark

Carlos García-Estrada, D.V.M., Ph.D. INBIOTEC (Institute of Biotechnology of

León), Parque Científico de León, León, Spain

Jorge García-Martínez Department of Genetics, Faculty of Biology, University

of Seville, Sevilla, Spain

Henriette Giese, Ph.D. Department of Biotechnology, Chemistry and

Environmental Engineering, Aalborg University, Aalborg, Denmark

Frederik Teilfeldt Hansen, Ph.D. Department of Molecular Biology and Genetics,

Aarhus University, Aarhus C, Denmark

Hans-Ulrich Humpf, Dr. rer. nat. University of Münster, Institute of Food

Chemistry, Münster, Germany

Karin Kleigrewe, Dr. rer. nat. University Münster, Institute of Food Chemistry,

Münster, Germany

Michael Knudsen, Ph.D. Bioinformatics Research Center, Aarhus University,

Aarhus, Denmark

Ulrich Kück, Ph.D. Christian Doppler Laboratory for Fungal Biotechnology,

Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,

Bochum, Germany

Alfons Lawen, Dipl.-Chem., Dr. rer. nat. Department of Biochemistry and

Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne,

VIC, Australia

T. Verne Lee, Ph.D. AgResearch Structural Biology Laboratory, School of

Biological Sciences, University of Auckland, Auckland, New Zealand

M. Carmen Limón, Ph.D. Department of Genetics, Faculty of Biology, University

of Seville, Sevilla, Spain

John E. Linz, M.S., Ph.D. Department of Food Science and Human Nutrition,

Microbiology and Molecular Genetics, Michigan State University, East Lansing,

MI, USA

Paloma Liras, Ph.D. Department of Molecular Biology, Microbiology Section,

University of León, León, Spain

Juan-Francisco Martín, Ph.D. Department of Molecular Biology, Microbiology

Section, University of León, León, Spain

Contributors

xi

Yudai Matsuda, M.Sc. Graduate School of Pharmaceutical Sciences, The

University of Tokyo, Tokyo, Japan

Lisa Neubauer Institut für Biologie und Biotechnologie der Pflanzen, Westfälische

Wilhelms Universität Münster, Münster, Germany

Eva-Maria Niehaus University of Münster, Institute of Biology und Biotechnology

of Plants, Münster, Germany

Claus Olesen, M.Sc. Department of Molecular Biology and Genetics, Aarhus

University, Aarhus C, Denmark

Christian Nørgaard Storm Pedersen, Ph.D. Bioinformatics Research Center,

Aarhus University, Aarhus C, Denmark

Massimo Reverberi, Ph.D. Department of Environmental Biology, Università

Sapienza, Roma, Italy

Ludmila V. Roze, Ph.D. Department of Plant Biology, Michigan State University,

East Lansing, MI, USA

Macarena Ruger-Herreros, Pharm.D. Department of Genetics, Faculty of

Biology, University of Seville, Sevilla, Spain

Teis Esben Sondergaard, Ph.D. Department of Biotechnology, Chemistry and

Environmental Engineering, Aalborg University, Aalborg, Denmark

Jens Laurids Sørensen, Ph.D. Department of Biotechnology, Chemistry and

Environmental Engineering, Aalborg University, Aalborg, Denmark

Lena Studt, Ph.D., Dr. rer. nat. Institute for Biology and Biotechnology of

Plants, University of Münster, Münster, Germany

Bettina Tudzynski, Ph.D., Dr. habil. rer. nat. University of Münster, Institute of

Biology and Biotechnology of Plants, Münster, NRW, Germany

Paul Tudzynski, Dr. rer. nat. Institut für Biologie und Biotechnologie der

Pflanzen, Westfälische Wilhelms Universität Münster, Münster, Germany

John C. Vederas, B.Sc., Ph.D. Department of Chemistry, University of Alberta,

Edmonton, AB, Canada

Tony Velkov, Ph.D. Department of Pharmaceutics, Monash University, Parkville,

VIC, Australia

Josephine M. Wee, B.Sc. Department of Food Science and Human Nutrition,

Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA

Contributors

J.-F. Martín et al. (eds.), Biosynthesis and Molecular Genetics of Fungal 1

Secondary Metabolites, Fungal Biology, DOI 10.1007/978-1-4939-1191-2_1,

© Springer Science+Business Media New York 2014

Introduction

A major contribution of microbes to the health and well-being of people began back

in 1928, when Alexander Fleming discovered in a Petri dish seeded with

Staphylococcus aureus that a compound produced by a mold killed the bacterium.

The mold, Penicillium notatum , produced an active agent, which was named peni￾cillin. Fleming’s discovery began the microbial drug era. By using the same method,

other naturally occurring substances, like chloramphenicol and streptomycin, were

later isolated from bacterial fermentations. Naturally occurring antibiotics are pro￾duced by fermentation, an old technique that can be traced back almost 8,000 years,

initially for beer and wine production, and recorded in the written history of ancient

Egypt and Mesopotamia. During the last 4,000 years, Penicillium roqueforti has

been utilized for cheese production and for the past 3,000 years, soy sauce in Asia

and bread in Egypt represented examples of traditional fermentations [ 1 ].

Natural products (NPs) with high commercial value can be produced via primary

or secondary metabolism. The present review deals with secondary metabolites.

Due to technical improvements in screening programs and separation and isolation

techniques, the number of natural compounds discovered exceeds one million [ 2 ].

Among them, 50–60 % are produced by plants (alkaloids, fl avonoids, terpenoids,

steroids, carbohydrates, etc.) and 5 % of these plant products have a microbial ori￾gin. From all the reported natural products, about 20–25 % show biological activity

and of these, approximately 10 % have been obtained from microbes. Microorganisms

produce many compounds with biological activity. From the 22,500 biologically

active compounds so far obtained from microbes, about 40 % are produced by fungi

[ 2 , 3 ]. The role of fungi in the production of antibiotics and other drugs for treatment

of noninfective diseases has been dramatic [ 4 ].

Chapter 1

Valuable Secondary Metabolites from Fungi

Arnold L. Demain

A. L. Demain , Ph.D., M.S., B.S. (*)

Research Institute for Scientists Emeriti (R.I.S.E.), Drew University , Madison , NJ , USA

e-mail: [email protected]

2

Biosynthetic genes are present in clusters coding for large, multidomain, and

multi-modular enzymes such as polyketide synthases, prenyltransferases, non￾ribosomal peptide synthases, and terpene cyclases. Genes adjacent to the biosyn￾thetic gene clusters encode regulatory proteins, oxidases, hydroxylases, and

transporters. Aspergilli usually contain 30–40 secondary metabolite gene clusters.

Strategies to activate silent genes have been reviewed by Brakhage and Schroekh [ 3 ].

Currently, with less than 1 % of the microbial world having been cultured, there

have been signifi cant advances in microbial techniques for growth of uncultured

organisms as a potential source of new chemicals [ 5 ]. Furthermore, metagenom￾ics—i.e., the extraction of DNA from soil, plants, and marine habitats and its incor￾poration into known organisms—is allowing access to a vast untapped reservoir of

genetic and metabolic diversity [ 6 , 7 ]. The potential for discovery of new secondary

metabolites with benefi cial use for humans is great. A method to predict secondary

metabolite gene clusters in fi lamentous fungi has recently been devised [ 8 ].

Microbes normally produce secondary metabolites in only tiny amounts due to

the evolution of regulatory mechanisms that limit production to a low level. Such a

level is probably enough to allow the organism to compete with other organisms

and/or coexist with other living species in nature. The industrial microbiologist,

however, desires a strain that will overproduce the molecule of interest. Development

of higher-producing strains involves mutagenesis and, more recently, recombinant

DNA technologies [ 9 ]. Although some metabolites of interest can be made by plants

or animals, or by chemical synthesis, the recombinant microbe is usually the “crea￾ture of choice.” Thousandfold increases in production of small molecules have been

obtained by mutagenesis and/or genetic engineering. Other important parts of

industrial production include creating a proper nutritional environment for the

organism to grow and produce its product, and the avoidance of negative effects

such as inhibition and/or repression by carbon sources, nitrogen sources, phospho￾rus sources, metals, and the fi nal product itself. Avoidance of enzyme decay is also

desired [ 4 , 10 ].

Applications of Microbial Natural Products

Over the years, the pharmaceutical industry extended their antibiotic screening pro￾grams to other areas [ 11 , 12 ]. Since microorganisms are such a prolifi c source of

structurally diverse bioactive metabolites, the industry extended their screening pro￾grams in order to look for microbes with activity in other disease areas. As a result of

this move, some of the most important products of the pharmaceutical industry were

obtained. For example, the immunosuppressants have revolutionized medicine by

facilitating organ transplantation [ 13 ]. Other products include antitumor drugs,

hypocholesterolemic drugs, enzyme inhibitors, gastrointestinal motor stimulator

agents, ruminant growth stimulants, insecticides, herbicides, antiparasitics versus

coccidia and helminths, and other pharmacological activities. Catalyzed by the use of

simple enzyme assays for screening prior to testing in intact animals or in the fi eld,

further applications are emerging in various areas of pharmacology and agriculture.

A.L. Demain

3

Antibiotics

Of the 12,000 antibiotics known in 1955, fi lamentous fungi produced 22 % [ 14 , 15 ].

The beta-lactams are the most important class of antibiotics in terms of use. They

constitute a major part of the antibiotic market. Included are the penicillins, cephalo￾sporins, clavulanic acid, and the carbapenems. Of these, fungi are responsible for

production of penicillins and cephalosporins. The natural penicillin G and the biosyn￾thetic penicillin V had a market of $4.4 billion by the late 1990s. Major markets also

included semisynthetic penicillins and cephalosporins with a market of $11 billion.

In 2006, the market for cephalosporins amounted to $9.4 billion and that for penicil￾lins was $6.7 billion. By 2003, production of all beta-lactams had reached over

60,000 t. The titer of penicillin is over 100 g L −1 and that for cephalosporin C is about

35 g L −1 [ 16 , 17 ]. Recovery yields are more than 90 %. There have been more than

15,000 molecules based on penicillin that have been made by semisynthesis or by

total synthesis. By the mid 1990s, 160 antibiotics and their derivatives were already

on the market [ 15 , 18 ]. The market in 2000 was $35 billion. Despite these impressive

fi gures, more antibiotics are needed to combat evolving pathogens, naturally resistant

microbes, and bacteria and fungi that have developed resistance to current antibiotics.

A new and approved cephalosporin is ceftobiprole, which is active against methicil￾lin-resistant S. aureus (MRSA) and is not hydrolyzed by a number of beta-lactamases

from Gram-positive bacteria [ 19 ]. Another antibiotic of note is cerulenin, an antifun￾gal agent produced by Acremonium caerelens . It was the fi rst inhibitor of fatty acid

biosynthesis discovered [ 20 ]. It alkylates and inactivates the active-site nucleophylic

cysteine of the ketosynthase enzyme of fatty acid synthetase by epoxide ring opening.

Other properties that are desired in new antibiotics are improved pharmacological

properties, ability to combat viruses and parasites, and improved potency and safety.

Pharmacological Agents

Years ago, noninfectious diseases were mainly treated with synthetic compounds.

Despite testing thousands of synthetic chemicals, only a handful of promising struc￾tures was obtained. As new synthetic lead compounds became extremely diffi cult to

fi nd, microbial products came into play. Poor or toxic antibiotics produced by fungi

such as cyclosporin A or mycotoxins such as ergot alkaloids, gibberellins, zearela￾none were then successfully applied in medicine and agriculture. This led to the use

of fungal products as immunosuppressive agents, hypocholesterolemic drugs, anti￾tumor agents, and for other applications.

Hypocholesterolemic Agents

Only about 30 % of cholesterol in humans comes from the diet. The rest is synthe￾sized by the body, predominantly in the liver. Many people cannot control their level

of cholesterol at a healthy level by diet alone and require hypocholesterolemic

1 Valuable Secondary Metabolites from Fungi