Role in the pharmaceutical industry - secondary metabolites
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Introduction
Although they do not have a panoply as extensive as prokaryotes in terms of metabolism, eukaryotes are nevertheless capable of synthesizing a wide variety of metabolites which have a multitude of biological activities: antibiotics, vasodilators, narcotics, immunosuppressants… but also mutagens, carcinogens, teratogens… or some that are simply toxic. Indeed, the positivity or negativity of their effects depends on the point of view from which one places oneself. Many of these products are used medicinally because of their beneficial effects on physiology, but are fatal in high doses! When absorbed inappropriately, they can cause a variety of syndromes (Box 30 and Box 31). These products are synthesized by specific reactions which are not essential for the life of the organism. Their production is therefore variable depending on the species. They are then said to be products of secondary metabolism, as opposed to those of primary metabolism which are essential for normal cell life and which will be common to a large number of organisms.
Roles of secondary metabolites
The role of secondary metabolism in the physiology of producer species is still debated and two schools of thought are in conflict. For the former, the producing organisms live in environments where the proportions of the various nutrients are not optimal and therefore if a nutrient, such as nitrogen or phosphate, is lacking, potentially toxic intermediate metabolites generated by the primary metabolism can accumulate. They are then transformed into secondary metabolites to remedy their harmful effects. For the latter, the production of secondary metabolites was selected for during evolution because they have generally toxic effects. They therefore intervene in the fight against competitors or in interspecific communications, allowing synergies. Both hypotheses are probably true and play a variable part in the appearance and maintenance of different metabolites. Main producers of secondary metabolites are prokaryotes such as streptomycetes and cyanobacteria, and for eukaryotes, plants, Eumycota (Box 30) and certain algae, e.g. Dinophyceae (Box 31). These organisms have access to carbon in significant quantity through photosynthesis or degradation of plant biomass, but nitrogen availability is often very limited. Secondary metabolites are often produced when external conditions become stressful for the organism, for example when the environment becomes exhausted. But, it is also clear that I do not want to eat an apple that is rotten by a fungus and exhibiting a foul odor. It is therefore likely that the rapid rotting of fruits by fungi is a competitive strategy with respect to other fruit consumers. Secondary metabolites are therefore necessary both for metabolism optimization and luxury products that will confer selective benefits.
The production of a particular metabolite is generally restricted to a given species or even to a few strains, and depends on external conditions that are often difficult to control. For example, Aspergillus nidulans has been shown to produce polyketide-type metabolites when it is in direct physical contact with a strain of Streptomyces hygroscopicus but not with the 57 other strains of actinomycetes that have been tested! Likewise, production requires physical contact between the two organisms. Note that the same metabolites can be synthesized by phylogenetically distant organisms. In most cases, horizontal transfers of the genes involved in their biosynthesis are suspected, for example between plants and their endophytic or parasitic fungi which produce the same products, and more rarely convergent evolution is thought to be the cause.
Biosynthesis follows various modalities in fungi and algae. In a few cases, it would in fact be carried out by endosymbiotic bacteria. However, in many cases the necessary genes are directly encoded by the protist genome. Figure 364 shows a general diagram of the metabolism of a eukaryotic cell and where the majority of the chains of biosynthesis of secondary metabolites start. The starting point can be amino acids or acetate in the form of acetyl-coA. The syntheses most often require a very large number of enzymes. In Eumycota, these are most often encoded by genes grouped in “clusters” (Figure 365). The origin of these clusters is often mysterious but correlates with the fact that they could be transmitted horizontally. It appears that clustering participates in the coordinated regulation of the expression of different enzymes; a transcription factor that positively regulates the expression of genes encoding enzymes is often present in the cluster.
Types of secondary metabolites
Alkaloids
There are currently three major groups of metabolites. First, the alkaloids, which are molecules rich in nitrogen and which often derive from amino acids or nitrogenous bases (Figure 366). A famous example is LSD, well known for its hallucinogenic effects. Its precursor, lysergic acid, is produced by Claviceps purpurea, a Pezizomycotina better known as rye ergot (Figure 366 and Box 30). Ergotamine, another alkaloid produced by Claviceps purpurea has a vasoconstrictor effect and is used to stem bleeding. A third example is the saxitoxin produced by certain Dinoflagellata (Box 31).
Polyketides
The second group of metabolites, polyketides, are synthesized by multifunctional enzymes by adding more or less modified two-carbon molecules, and are derived from acetyl-CoA or malonyl-CoA. The same precursors are used in the synthesis of fatty acids and the polyketide synthases belong to the same family of proteins as the fatty acid synthetases. The molecules can then be circularized. These enzymes work in domains that sequentially add the two-carbon molecules. It seems that their operation is modular and that in the future we can use them to manufacture specific molecules at will. Changes, such as oxidations, methylation, etc., are then often made to the end products by additional enzymes. The best known and most common polyketides are probably the HLD melanins which result from the polymerization of 1,8-dihydroxynaphthalene (Figure 367). They are insoluble molecules, colored and not very reactive but which provide reinforcement of the cell walls and protection against ultraviolet rays, reactive oxygen species and other damage in Ascomycota. On the other hand, there are highly toxic polyketides such as patulin (Figure 367), aflatoxins, and particularly aflatoxin B1 (Figure 367 and Box 30), or okadaic acid (Box 31).
Cyclopeptides
The third important class contains cyclic peptides or cyclopeptides which are assembled by gigantic proteins outside of translation. For example, cyclosporin A (Figure 368) is an immunosuppressant produced from Tolypocladium inflatum, a Sordariomycetes species. Cyclosporin A is a cyclic peptide comprising 11 amino acids, not all of which are used in translation. Its synthesis is carried out by a gigantic enzyme of 15,000 amino acids catalyzing about 40 enzymatic reactions!
Certain cyclic peptides are produced via translation in the form of a precursor and then cyclized by proteases of the prolyl oligopeptidase family (Figure 369). This is the case with α-amanitine which inhibits RNA polymerase II and phallacidin, molecules produced by many Agaricomycotina fungi, including Amanita (Box 30). Precursor peptides contain two conserved regions surrounding a variable domain (Figure 369). It is the amino acids contained in these variable domains that are found cyclized. These genes evolve like normal genes and in different species the bordering regions will be different, while the central peptide is conserved (Figure 369). These cyclic peptides are therefore at the border between secondary metabolites and small peptides which have biological activities, such as those present in the venoms of insects or snakes. Unlike fungal cyclopeptides, the latter small peptides are secreted. Eukaryotic protists also produce this kind of secreted peptides, some of which have bactericidal or fungicidal activities.
There are many other classes of molecules, such as hybrids of polyketides and cyclopeptides, lactones, quinones, oxylipines, terpenoids… Let us mention penicillin G and cephalosporin C which are antibiotics synthesized from condensation without cyclization of three amino acids: L-α-aminoadipic acid, cysteine and valine, the valine having to be converted into its D form before condensation. Protists, particularly Eumycota, are also capable of synthesizing volatile products that derive from primary or secondary metabolites. These volatile compounds give mushrooms their characteristic odors which often make it possible to recognize a species by its smell of anise, earth or bleach! These compounds can, however, also have practical applications. This is the case for those manufactured by Muscodor albus, an Ascomycota which synthesizes a mixture of volatile products, each of which, taken separately, has a low activity but which when mixed has a lethal action on many fungi and other microorganisms. Treatments based on such cocktails of volatile molecules could open up new perspectives for medicine in the search of new antibiotics.
Even if the first antibiotic discovered, penicillin, comes from an Eumycota, the Eurotiomycetes Penicillium notatum/chrysogenum, our Western pharmacopoeia mainly uses antibiotics of bacterial origin and more specifically from streptomycetes. It seems, however, that if the secondary metabolism of plants and streptomycetes has been well explored, that of Eumycota is still very under-exploited. It is probable that these will therefore provide new avenues for antibiotic molecules in the future. The Eumycota nevertheless continue to actively contribute to our catalog of medicines. Some have essential roles in modern medicine. Currently, of the twenty most prescribed molecules, six have a fungal origin. Penicillin or its derivatives are still the most prescribed antibiotics. Lovastatin is an anti-cholesterolemic polyketide widely prescribed for the elderly. Cyclosporin A (Figure 368) is a very effective immunosuppressant with little side effects and is therefore widely used during organ transplants.
The Chinese pharmacopoeia also uses many Eumycota in the form of ground powder. The two most widely used species are Ganoderma lucidum and Cordyceps militaris (Figure 370). They produce numerous molecules with putative anticancer, anti-inflammatory, anti-oxidant, anti-viral, anti-microbial, anti-diabetic, anti-aging activities… In the West, microalgae, such as Chlorella, are used as food supplements for various purposes.
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