Professor Hanson is Emeritus Professor of Chemistry at the University of Sussex.

The Chemistry of Fungi

By James R. Hanson

The Royal Society of Chemistry

Copyright © 2008 James R. Hanson
All rights reserved.
ISBN: 978-0-85404-136-7

Contents

Chapter 1 Fungi and the Development of Microbiological Chemistry,
Chapter 2 The Chemistry of Growing Fungi,
Chapter 3 Fungal Metabolites Derived from Amino Acids,
Chapter 4 Polyketides from Fungi,
Chapter 5 Terpenoid Fungal Metabolites,
Chapter 6 Fungal Metabolites Derived from the Citric Acid Cycle,
Chapter 7 Pigments and Odours of Fungi,
Chapter 8 The Chemistry of Some Fungal Diseases of Plants,
Chapter 9 Mycotoxins,
Chapter 10 Fungi as Reagents,
Epilogue, 188,
Further Reading and Bibliography, 190,
Glossary, 204,
Subject Index, 209,

CHAPTER 1

Fungi and the Development of Microbiological Chemistry

1.1 Introduction

Fungi are widespread, non-photosynthetic microorganisms that play a vital role in the environment, particularly in the biodegradation of organic material. The study of their metabolites and metabolism has made many contributions to the overall development of chemistry. Although the biosynthetic pathways that fungi utilize to construct their metabolites have general features in common with those found in bacteria, plants and mammals, they differ in detail and the structures of the resultant natural products are often different. This book is restricted to fungal metabolites but the reader should not lose sight of other natural products produced elsewhere in the living world.

Since fungi do not contain chlorophyll and are not photosynthetic organisms, they gain their energy and many of the nutrients to supply their biosynthetic pathways through the degradation of plant and other matter. Their environmental role is that of recycling. Their widespread provenance is often illustrated in one of the first practical exercises of many microbiology courses. A Petri dish containing a nutrient agar is exposed to the atmosphere for a few minutes. It is then incubated to reveal the range of organisms, both bacteria and fungi, whose spores are present in the atmosphere and which fell onto the plate in a relatively short time. It was a chance contaminant of an agar plate that led to the isolation of penicillin and changed the face of medicinal chemistry.

Fungi are eukaryotic organisms with a distinct nucleus, unlike bacteria which are prokaryotes. This also distinguishes them from another wide family of soil microorganisms, the Actinomycetes (e.g. Streptomycetes), which are often considered along with the bacteria. Yeasts, however, are regarded as a unicellular form of a fungus. Some fungi grow in a symbiotic relationship with photosynthetic algae or cyanobacteria in the form of lichens.

Fungi do not grow in isolation. Some attack plants, insects and mammals as pathogens whilst others are saprophytic and grow on dead material. Some live in a positive symbiotic relationship with a host organism. Thus, there are mycorrhizal fungi that are associated with the roots of plants and facilitate the uptake of nutrients by the plant. Others are endophytic organisms that grow within the vascular system of the plant. Throughout the natural world there is a chemical language between the fungus and its host which determines the nature of this relationship. We are beginning to understand the role of fungal and plant metabolites in this ecological communication.

The chemistry of fungi impinges on many aspects of our daily life whether it be in the role of yeasts in the production of bread and wine, the edible mushrooms or the manufacture of antibiotics such as the penicillins. The fungal diseases of crops, ornamental plants and trees and the spoilage of stored foodstuffs are serious economic problems. The control of the phytopathogenic organisms and the detection of their toxic metabolites in the food chain provide further chemical problems.

The microbiological chemist is interested in the structure, chemistry and biological activity of fungal metabolites. The biosynthesis of these metabolites, the sequences, stereochemistry and mechanism of the individual steps, together with the structure and regulation of the enzymes involved, is a major area of enquiry. The ecological chemistry of fungal interactions with plants and insects has provided another area of chemical investigation. An understanding of the chemical basis of fungal bio-control agents may have useful agrochemical applications.

As biodegradative organisms, fungi can carry out microbiological transformations of extraneous chemical substances. They can behave as self-replicating, environmentally friendly, chiral reagents. Their ability to carry out transformations that are chemically difficult, e.g. hydroxylations at sites that are remote from other reactive centres, has been exploited commercially. The scope of these biotransformations and the development of predictive models so that the use of an organism can be built into a synthetic strategy is yet another area of investigation. The use of the biodegradative ability of fungi in the bioremediation of contaminated land is a further application of chemical interest.

There are various estimates of the number of species of fungi. These range from 100 000 to 250 000. What is clear is that only a relatively small number, of the order of a few thousand, have been thoroughly investigated by microbiological chemists. Furthermore, there are often different strains of the same species. Whilst these may be morphologically similar, their metabolites can be quite diverse. Some metabolites may be produced consistently by all the strains of a particular species whilst other metabolites may be variable. The chemistry of an organism can also vary with the conditions under which it is grown. Un-surprisingly, therefore, some species of economic importance, e.g. Penicillium chrysogenum, have generated immense chemical interest.

1.2 Structure of Fungi

At first sight the structures of fungi appear quite diverse. The fruiting body of the common edible mushroom, Agaricus bisporus, is very different from the green Penicillium species growing on the surface of some cheese. However, there are some common features. The basic structural units of most fungi are the filaments known as the hyphae. Collectively, hyphae can aggregate to form a felt known as the mycelium. In some of the higher fungi, the hyphae can aggregate to form long strands and even differentiate to create a structure almost like a boot-lace, which is known as a rhizomorph. Another name for the honey-fungus, Armillaria mellea, which does considerable damage to trees, is the ‘boot-lace fungus’, which aptly describes the rhizomorphs by which it spreads underground.

The higher fungi, the mushrooms and toadstools, develop complex and readily observable structures known as fruiting bodies. These sprout from their mycelium, particularly in the autumn, and produce spores. At the other extreme some unicellular micro-fungi, such as the yeasts, produce small globular or ellipsoid cells that are only visible under the microscope.

The hyphae may be long single multi-nucleate aseptate (undivided) cells through which the cellular cytoplasmic fluids may flow. Other hyphae are septate and have distinct divisions. In these much of the chemical activity takes place at the growing tip. The lower micro-fungi only become septate as the culture ages whilst the higher macro-fungi become septate at an early stage and, as rhizomorphs are formed, their function may differentiate.

The form a fungus takes can depend on the culture conditions. Some fungi will have a yeast-like form under one set of conditions and a filamentous form under others. Under inhospitable conditions, often exploited in the storage of cultures, an organism can develop a ‘resting’ stage. In the wild this can allow spores to over-winter in the soil. In the laboratory, fungal cultures are often stored at low temperatures on agar under oil or in sealed vials on sand.

When a fungus is grown in suspension in a nutrient medium contained within a conical flask, the mycelium will sometimes clump together whilst at other times a well-dispersed mycelial suspension or even a mycelial mat is formed. The aeration and hence the metabolic capabilities of these forms can differ. The aeration can be quite poor within tightly formed clumps and this can affect the metabolism of the fungus. It is often difficult to get higher fungi to produce fruiting bodies in laboratory culture and again this can affect their metabolite production. Some rapidly growing fungi such as Rhizopus species produce fine long hyphae that spread rapidly across the agar in a Petri dish. They may produce a covering of aerial mycelium with the appearance of household dust. Indeed, quite a lot of household dust is fungal mycelium.

Fungi usually reproduce by spores although they can also develop vegetatively from mycelial fragments. The spores may be pigmented and some may have a gelatinous polysaccharide coating to facilitate their dissemination by a carrier and their attachment to a host. They are often borne on a specific thallus or germ tube. Hyphae that carry these are known as conidiophores. A culture such as that of Botrytis cinerea may appear light grey as the mycelium spreads across a Petri dish and then it develops a ring of green-black sclerotial mycelium bearing spores.

1.3 Classification of Fungi

In the general taxonomic classification, fungi are grouped in terms of the following ranks: division, class, order, family, tribe, genus, section, and species. In the binomial description of a fungus, the first name is that of the genus and the second name is the species. The name (not italicized) that follows this may be that of the author who first described the species. There are often varieties and strains of particular species. The accession number in a culture collection can be important in defining the organism used to isolate a particular metabolite. Although some metabolites may be specific to particular strains, others may be more common and are found in a section of a genus. The structure of the reproductive organs and the mechanisms of reproduction form the basis of the classification of fungi. These organisms may be broadly grouped in the following way. There are the Phycomycetes or lower fungi, which have a simple thallus bearing the spores. They possess unicellular aseptate hyphae. In some classifications this class name is treated as a trivial name for the Mastigomycotina and Zygomycotina. Typical examples are the Peronosporales, which include plant pathogens such as Pythium and Phytophthora species and the Mucorales, which include the common Mucor, Rhizopus and Phycomyces species. The ‘damping-off’ fungus Pythium ultimum, found growing across over-zealously watered germinating seeds, is an example.

A second group are the higher fungi which have septate hyphae, and these can be divided into the Ascomycetes and the Basidiomycetes. In the Ascomycetes the spores are borne in a sac-like structure known as an ascus. This type of fruiting body or ascocarp is found in Monascus species. The fungus M. ruber, which produces the red colour on Chinese red rice, is an example of these. The genera Penicillium and Aspergillus belong to the class of Ascomycetes known as the Plectomycetes. The spores are held in a pear-shaped perithecium in another class known as the Pyrenomycetes. The saprophytic plant parasites of the Hypocreales are also members of this group. Some of the best known of the higher fungi are Basidiomycetes. Here the spores are borne in special distinctive fruiting bodies. The edible part of the common mushroom, Agaricus bisporus, is a typical example.

The final large group are the Fungi Imperfecti or Deuteromycetes. In these organisms, the perfect stage of reproduction is rare or unknown and for the most part they are cultured vegetatively. The Fusaria are the best known of these. This classification can be confusing because some fungi originally classified within the Fungi Imperfecti do have both an asexual imperfect stage and a perfect stage. Thus the fungus that produces the gibberellin plant hormones, Gibberella fujikuroi, is the perfect stage of Fusarium monoliforme.

The naming of fungi has undergone many changes over the years and this can be a source of confusion. For example, Ophiobolus graminis was the name given to a serious pathogen of wheat. This name was incorporated into that given to a family of terpenoid metabolites, the ophiobolanes, which were isolated from the fungus. However, the fungus is now known as Gaeumannomyces graminis. Ophiobolanes are also produced by a rice pathogen that was at one time known as Helminthosporium oryzae or Drechslera oryzae and is now described as Bipolaris oryzae. Many of the Polyporus species, which gave their names to the triterpenoid polyporenic acids, have also been renamed as Piptopterus. When attempting to re-isolate a fungal metabolite, particularly from a culture that has been deposited in one of the culture collections, it is helpful to trace the provenance and naming of a particular isolate. When describing the isolation of a fungal metabolite, it is important to record the accession number of the culture in one of the major culture collections. If the strain of the organism is a new isolate it should be deposited in an accessible culture collection. Much valuable time has been wasted in unsuccessful attempts to re-isolate a fungal metabolite when the original culture has been lost.

1.4 The Fungal Cell Wall

The chemistry of the fungal cell wall contains some useful taxonomic markers. The cell wall is also a very important target for anti-fungal agents. The fungal cell wall differs in its structural components both from the bacterial cell wall and mammalian cell membranes. The fungal cell wall is a complex of chitin [a polymer of N-acetylglucosamine (1.1)], various mannoproteins together with α- and β-linked 1,3-D-glucans. Electron microscopy of the cell walls of the yeast Candida albicans shows that they are in layers attached to a plasma membrane. The major sterol in these is ergosterol (1.2) rather than cholesterol which is found in mammalian systems. Inhibitors of the biosynthesis of these components can, therefore, be selectively fungicidal.

The development of novel anti-fungal agents is a continuing area of research. Furthermore, opportunistic fungal infections, particularly caused by Candida and Aspergillus species, are emerging as a source of morbidity and mortality amongst immunocompromised patients. Polyene antibiotics such as nystatin and amphotericin B bind to ergosterol much more avidly than to cholesterol and hence disrupt the fungal cell membrane. Ergosterol biosynthesis inhibitors such as the azole fungicides target a key stage in the biosynthesis of ergosterol, the C-14α demethylation of lanosterol. Melanins are dark brown or black pigments that are present in fungal cell walls and are formed by the oxidation of phenolic precursors such as tyrosine (1.3), 3,4-dihydroxyphenylalanine (1.4) and 1,8-dihydroxynaphthalene (1.5). Some anti-fungal agents such as tricyclazole produce a weakening of cell walls by inhibiting melanin biosynthesis. More recently, several compounds that target the β-(l,3)-D-glucan and chitin synthases have been developed.

Part of the antagonistic interaction between fungi, such as that between Trichoderma and other organisms, includes the production of a chitinase. This allows the Trichoderma to attack the cell wall of its target organism. The hyphae of the Trichoderma can then penetrate the target fungus and sequester its nutrients.

1.5 History of Fungal Metabolites

The chemical activities of fungi have a long history. Many fungi, because of the competitive environment in which they live, produce antibiotics of varying efficiency. The Greek physician, Dioscorides described the use of an infusion that he called Agaricium, which was obtained from the larch polypore, Fomitopsis (Polyporus) officinalis, and was used for the treatment of consumption (tuberculosis). This biological activity has been attributed to the presence of agaricic or laricic acid [α-cetylcitric acid (1.6)]. The ‘ice-man’, whose 5300 year old body was discovered some years ago in the ice in the Alps between Italy and Austria, had the birch polypore, Piptoporus betulinus, with him. This fungus is active against wound bacteria such as Staphylococcus aureus. There are records of the use of other fungi, particularly Ganoderma lucidum, in ancient Chinese medicine. The identification of moulds growing on cloth by their pigmentation and their treatment is described in the Old Testament of the Bible in Leviticus Chapter 13, Verse 47.

The hallucinogenic properties of fungi such as Amanita muscari were known to several peoples. It is possibly the Soma which was used in parts of Asia and Scandinavia. There are records from travellers in the 18th century of its use.

The toxicity of ergot was apparently known to the Syrians in 600 BC. The metabolites of the ergot fungus, Claviceps purpurea which grows on rye, contaminated rye bread and brought about the disease known in the Middle Ages as St Anthony’s Fire. Ergotism involved damage to the nervous system and vascular constriction, leading to death of the affected parts of the body. Subsequently, medicinal uses of ergot were developed. In the early nineteenth century ergot was used to induce childbirth and to prevent post-natal haemorrhage.

(Continues…)Excerpted from The Chemistry of Fungi by James R. Hanson. Copyright © 2008 James R. Hanson. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.