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Secondary Metabolites

Introduction

Secondary metabolites are compounds produced by an organism that are not required for primary metabolic processes. Fungi produce an enormous array of secondary metabolites, some of which are important in industry. Many fungi express secondary metabolites that influence competitive outcomes (see above). LINK The compounds are expressed along with enzymes necessary for extracellular digestion. LINK The precise function of many of these compounds in the natural environment, however, is unclear.

Some of the compounds released by fungi influence the organisms that interact with fungi leading to an anthropocentric interpretation of function in the fungus. Some metabolites, referred to as toxins, are compounds that have the potential to kill an organism at concentrations we might use. The activity of the metabolites in biological conditions may differ. This page examines the concept of secondary metabolites with a view to elucidate their importance to the fungi. It also examines their importance to humans.

Primary and secondary metabolites may be confused. Primary metabolites may accumulate as staling products slow growth of the fungus, or as essential nutrients are removed from the growth medium. LINK Regulatory compounds may also appear as growth slows, and they can be confused with secondary metabolites.

Secondary metabolites are generally produced following active growth, and many have an unusual chemical structure. Some metabolites are found in a range of related fungi, while others are only found in one or a few species. The restricted distribution implies a lack of general function of secondary metabolites in fungi.

Clear reasons exist for studying secondary metabolites. Many have been found to have use in industry and medicine. LINK Indeed, six of the twenty most commonly prescribed medications for humans are of fungal origin. These metabolites have been subjected to combinatorial chemistry following growth in selective media. Some metabolites are toxic to humans and other animals. Yet others can modify the growth and metabolism of plants. Interestingly, the most important secondary metabolites seem to be synthesised from one or a combination of three biosynthetic pathways: polyketides arising from Acetyl Coenzyme A, mevalonate pathway that also arises from Acetyl Coenzyme A, and from amino acids. In addition, genes for the synthesis of some important secondary metabolites are found clustered together, and expression of the cluster appears to be induced by one or a few global regulators.

Some of the 'global regulators' are also involved with sporulation and hyphal elongation. Thus the expression of secondary metabolites may be a normal part of and occur at a predictable point in the life cycle of some fungi.

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Polyketide Metabolites

Polymerisation of acetate may result in the formation of a fatty acid or a polyketide. Polyketides result when a primer other than acetate is included, and processing during chain elongation results in the inclusion of various other compounds. The chain may be further processed by cyclisation, lactonisation, or formation of thioesters or amides. The result is a staggering number of possible structures built from the simple primer units.

Included among the polyketide secondary metabolites are orsellinic acid, tetrahydroxynaphthalene (precursor for melanin), sterigmatocyctin, aflatoxins, statins, and fumonisin.

Aflotoxins are produced in members of the Aspergillus parasiticus group via the polyketide pathway. The pathway has around 20 steps, and the end products include a diversity of related compounds (bisfuranocoumarins) that can be readily converted one to another.

Aflatoxin B1 is one of the most toxic compounds known. The toxin is formed commonly in plant materials held at relatively high moisture and temperature for long periods (ie growth in tropics and sub-tropics). Peanuts, corn and cotton are readily contaminated in the field. The Aspergillus parasiticus group of fungi are common in soil. The fungi can colonise roots and spread through the plant. When harvested, contaminated seed will become toxic if not dried immediately and held in a dry form.


Structure of Aflatoxin B1.

Aflatoxins are toxic and carcinogenic. The LSD 50 for ducks is 0.33 mg/kg. At lower levels and following prolonged exposure, the toxins cause liver cancer in humans. Aflatoxin B1 is converted to Aflatoxin M (in milk) on passage through cows. Though less toxic, it does illustrate the potential damage caused by consumption of contaminated product.

Aflatoxicosis first came to note in the west when contaminated peanut meal was fed to turkeys in the UK. Since then, chickens, pigs and cattle have also been severely affected by consumption of contaminated feed. All peanuts are now screened for the presence of the toxins.

The function of aflatoxins in fungi is unknown. Toxicosis only takes place after consumption of the contaminated plant material. Animals tend to avoid contaminated feed, but as B1 is so highly toxic, even large animals can be killed by small, almost undetectable quantities.


Structure of Patulin.

Patulin is a polyketide antibiotic synthesised on an acetate/malonate pathway. Its biosynthetic pathway is still unclear, but it appears that several alternate pathways may result in the same end product. Further, several related compounds may be produced from minor variations of the cultural conditions.

Patulin was first thought to be a potential antibiotic. However, it is now also known to be a compound produced by Penicillium expansum in contaminated apples. Patulin associated with storage rot of apples, and it is toxic to mammals. Patulin illustrates the close relationship between anthropogenic benefits and detriments.

The role of patulin in rotting apples seems unclear, but it may be a general inhibitor of competitors, including mammals and microbes. LINK One speculative explanation indicates coevolution between the plant and fungus, that is colonised apples have a greater chance of producing seed that germinates. However, the life cycle of apples tends to be decades, that of the fungus much shorter. The disparity in the length of the life cycle between organisms associated with fungi and the fungus is common. The disparity makes it difficult to argue for the co-evolution of the associated organisms.

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Aromatic Compounds

Cyclic compounds can be synthesised via the polyketide or shikimic acid pathways. Zearalenone is one interesting example from this group. The compound regulates perithecium formation in the fungus. It also has an oestrogenic effect in mammals.


Structure of Zearalenone.

The reproductive behaviour of pigs consuming feed contaminated with Fusarium graminearum may be modified. Consumption of high doses of zearalenone is commonly detrimental to pigs, but the compound has also been used deliberately at low doses as a growth supplement in sheep and cattle.

F. graminearum is a common hyphomycete found in soil. Some isolates are widely recognised as important plant pathogens in temperate and warm temperate climates. Thus contamination of feeds is likely to be widespread where there is poor control over humidity during storage. The main function of the compound appears to be its role in perithecium formation. The effect on mammals appears to be entirely coincidental.

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Amino Acid Pathway

Penicillin and cephalosporin are β lactam antibiotics.  β lactam antibiotics are produced by a few Ascomycota and several bacteria. LINK The precursors of these antibiotics are amino acids. Synthesis of active antibiotics is directed by the inclusion in the growth medium of different organic and fatty acids resulting in different side chains on the compound.

A second group of antibiotics derived from the amino acid pathway are the defensins. Defensins are peptides that act against bacteria. They are found in animals where they function to protect organs such as the gingiva where bacterial densities are very high. The first defensin found in fungi has been called plectasin. The role of plectasin in its host Pseudoplectania nigrella is unknown.

Toxins derived from amino acid synthesis include psilocybin (Psilocybe) and Bufotenine (Amanita). LINK These compounds act on nerve impulses, resulting in hallucinations. The result is thought to be due to the similarities between the compounds and serotonin.

Amatoxins (Amanita) are cyclic peptides that act on RNA synthesis in all eukaryotic organisms. They are extremely toxic. LINK They are particularly dangerous because their effect does not become evident until 24 to 48 hours after ingestion.

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Combination of Pathways

Ergot alkaloids are synthesised from several pathways. Trypotophan from the shikimic acid pathway is attached to an isoprenoid moiety from the mevalonate pathway, and several amino acids from primary metabolism are added depending on the final product. Ergot alkaloids are produced as a complex mixture of related compounds from a branched pathway.

The activity of the alkaloids is as varied as the compounds. LINK In essence, the compounds may function as vasodilators, hormone regulators, and feeding deterrents. They can be active in mammals and insects. Their function in the fungus is unclear, but theories about coevolution of the fungi and their grass hosts are based on the benefit of the toxins in selecting grasses that deter herbivory. The role of the toxins in the fungi are, otherwise, almost inexplicable.

Given the importance of amino acids in the synthesis of alkaloids, it follows that nitrogen nutrition of the fungi is important. Carbon/nitrogen balance, carbon source, availability of precursors, and repression by excess of regulators such as ammonium are important both in industry (ergotamine) and in colonised grasses. However, the regulation of other secondary metabolites is as varied as their structures.

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Plant Growth Regulators

Many pathogenic and benign fungi produce auxin, cytokinins, gibberellins and abscisic acid. In fact the gibberlellins were first found in the fungus Gibberella fujikuroi, a pathogen that causes tall, straggly growth of rice. The gibberellins are diterpenes produced by the mevalonate/isoprenoid pathway. The function of these compounds in fungi that colonise plants seem clear. Modification of host tissue enhances colonisation, releases nutrients for fungal metabolism and regulates host reproduction. The function of plant growth regulation in fungi found outside plants is unclear.

Some plant fungi appear to modify host production of growth regulators, resulting in alterations of host metabolism. For instance, initiation of AM in roots results in a slowing of root tip elongation and increase in lateral formation. The cause may be associated with a change in concentrations of auxins and/or cytokinins from the fungus or induced in the host, an increase in local concentration of phosphate due to the fungi, or a factor influenced by either. LINK Increases in expression of plant hormones may be direct or indirect. Resolution of the identity and importance of the fungal compounds remains.

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Toxins

In general, toxins associated with fruiting bodies are important because consumption of the fruiting body can result in poisoning. LINK Toxins associated with microfungi are important because they become evident after consumption of contaminated food. The principles of toxicosis are the same, though the topics of toxin fungi and toxic foods are commonly separated. In addition, toxins are produced from a myriad of pathways, and have enormously diverse effects. That they may be produced at a different point in the life cycle of a fungus is simply another aspect of the complex subject.

structure of sporodesmin
Structure of Sporodesmin.

The function of toxins to fungi has been subject of much speculation. Colonisation (contamination) of organic materials is a prelude to the digestion of the material by the fungus. The production and expression of toxins is one mechanism the microbe has to protect the food, provided competitors detect the presence of the microbe and toxin. The conditioned response to the fungus thus reduces the consumption of the fungal substrate. The “detection” molecule may be other than the toxin. Objectionable flavours and smells may thus be warnings to competitors. Overall, the resultant reduction of feeding increases the chances of the fungi surviving.

structure of vomitoxin
Structure of Vomitoxin.

This hypothesis fits neatly into the Plant Defence theory. The theory predicts that plants allocate defences only if they provide improved fitness. The presence of fungi in plants has been suggested to be due to their production of defence and deterrence compounds. Colonised plants would thus be relatively protected from herbivory and produce more offspring in the next generation. The fungi would be provided with a home and nutrients, and in a position to colonise the subsequent generation. Further, fungi are able to evolve new and more appropriate deterrents more rapidly than plants being under greater selection pressure, and having shorter generation times. The result is suggested to explain the relationship between grasses, which have few defences, and the Balansioid fungi. LINK

Needless to say, those animals that have evolved the capacity to detect the toxins or to detoxify them, will have a selective advantage. This has reached its most advanced state in the insects. Some species require toxins in their diet, or feed only from plants containing toxins. Further, some microbes in the GIT of animals have evolved the capacity to detoxify metabolites. LINK The relationship between microbes and their hosts, and the organisms with which they interact is complex and yet to be explained. The role of fungi in this process of coevolution has not been clarified.

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Conclusion

Fungi produce a diversity of metabolites, many of which appear unnecessary for the primary function of the host. However, many metabolites appear indirectly important, influencing fungal growth and survival. The structure of secondary metabolites varies enormously. Some are produced quite widely, and others are specific to a single fungus. Marked differences in expression can even be found even within a single species, and the environment plays an important role in expression.

The function of secondary metabolites is as varied as their structure. Some appear to directly or indirectly benefit the fungus, while the function of many others remains obscure. Secondary metabolites play an extremely important role in the functioning of fungi, providing one crucial element of their diversity.

Secondary metabolites are exploited in medicine and industry. Screening for different activities remains the mainstay of much of traditional biotechnology. The potential for secondary metabolites to explain behaviour needs to form part of any investigation of new and different fungi.

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References

Cox RJ (2007) The biosynthesis of polyketides, acyl tetramic acids and pyridones by filamentous fungi. In: Exploitation of Fungi Eds Robson GD, van West P & Gadd GM CUP.

Griffin DH (1994) Fungal Physiology. Wiley, New York.

Hocking AD & Pitt JI (1996) Fungi and mycotoxins in food. In: Fungi of Australia. 1B, 315 – 342.

Mann J. (1986) Secondary Metabolism.OUP, Oxford.

Vining LC (1990) Functions of secondary metabolites. Annual review of Microbiology 44, 395 – 427.

Yu J-H & Keller N. (2005) Regulation of secondary metabolism in filamentous fungi. Annual Review of Phytopathology 43: 437-458.

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