Biochemistry of Secondary Metabolism of Fungi

Abstract

Fungi, eukaryotic organisms with a kingdom of their own, include microorganisms from moulds and yeasts to the most known and appreciated mushrooms. The incredible biodiversity of these organisms is not limited to their morphology but is reflected in their chemistry, namely in the variety of compounds they produce. Therefore, like other living beings, fungi can be an excellent source of bioactive compounds.

Although they may be primary metabolites, fungal bioactive compounds are mainly produced through secondary metabolism. These compounds have an essential role in the fungal survival and adaptation to almost all habitats on earth. Besides, they can also exert beneficial effects on human health, such as antioxidant, antimicrobial, anti-UV radiation, or even anti-inflammatory or antitumor activity. Given the wide bioactivity of the molecules produced, fungi have become, over time, an exciting source of compounds with possible application in various industries, including the food, pharmaceutical, or cosmetics industries.

Fungal secondary metabolites are mainly produced via acetyl-CoA and via the shikimate pathway. Even though it is possible to find in the literature some different classifications regarding secondary metabolites of fungi, in this manuscript, we define polyketides, non-ribosomal peptides, terpenoids, and indole alkaloids as the main structural classes.

The present chapter will present a brief introduction to fungal secondary metabolism, including some examples of the most well-known compounds and their principal functions in ecosystems. The biosynthetic pathways of the main classes of fungal secondary metabolites will also be depicted.

1 Fungal Secondary Metabolites: An Overview

Fungi are widely known to produce a variety of secondary metabolites that include antibiotics, vitamins, pigments, amino acids, and other organic compounds, which in turn have recognized biological activities (Devi et al. 2020). Penicillins and other β-lactam antibiotics, the cholesterol-lowering lovastatin, or the immunosuppressant cyclosporin are some of the most well-known fungal metabolites used as medicines worldwide.

Albrecht Kossel first introduced the term “secondary” metabolite in 1891 (Devi et al. 2020; Hartmann 2007). Unlike primary metabolites, secondary metabolites are low molecular weight molecules, which are not present in every living cell capable of dividing and are not essential for the producing organism’s normal growth, development, reproduction, or energy production (Avalos and Limón 2021; G. F. Bills and Gloer 2016; Thirumurugan et al. 2018). Although the classic definition of secondary metabolism remains in this sense, the truth is that it results from the evolutionary process of the species, and the synthetized compounds have become essential for their existence. In fact, secondary metabolites can confer some adaptative and survival advantages to the producing organisms. Throughout their evolution, fungi conquered a wide range of habitats, ubiquitous in terrestrial and freshwater environments, less common in marine territories, and a cosmopolitan distribution (Webster and Weber 2007). This adaptive success to the most diverse ecosystems is also associated with its developed secondary metabolism, which is complex and capable of originating an enormous diversity of compounds with several functions.

Although they seem very different mechanisms, the line separating primary from secondary metabolism becomes very thin, as secondary metabolites are derived from central metabolic pathways and primary metabolite pools, with acetyl-CoA as the initial building block, leading to the synthesis of polyketides and terpenes, and amino acids being used for the synthesis of non-ribosomal peptides (Keller 2019). This cross-linkage between fungal primary and secondary metabolism is represented in Fig. 13.1.

Fig. 13.1

Schematic diagram representing the cross-linkage between primary and secondary metabolism in fungi. There is main evidence on the central pathways (), leading building blocks (), and main originated compounds ()

As shown in Fig. 13.1, the production of primary and secondary metabolites is a dynamic process, with common biochemical pathways. Indeed, some products of the primary metabolism are often considered secondary metabolites, depending on their need and function. Some examples are amino acids and other organic acids, such as oxalic acid, alcohols, or sugars. This classification of primary/secondary metabolite is influenced by the organism’s growth, cell differentiation and development, combined with the edaphoclimatic conditions in which it develops (G. F. Bills and Gloer 2016; Keller 2019; Thirumurugan et al. 2018).

The main structural classes or chemical families of fungal secondary metabolites are polyketides (PKs), non-ribosomal peptides (NRPs), terpenoids, and indole alkaloids (Daley et al. 2017; Devi et al. 2020). It is important to highlight that there are also hybrid molecules, resulting from the joint action of different classes, namely polyketide–terpene, non-ribosomal peptide–polyketides, and polyketide–fatty acid (Keller 2019). Some authors even consider hybrid non-ribosomal peptide/polyketides the fifth class of fungi secondary metabolites (Avalos and Limón 2021). However, after a critical reading of the information available in the literature, this chapter will consider and address the four chemical classes initially mentioned.

Fungi secondary metabolites are primarily synthesized via the shikimic acid pathway and acetyl-CoA through the malonic and mevalonic pathways (Fig. 13.1). In addition to differences at the function level, primary and secondary metabolism also have their particularities at the genomic level. The genes encoding the synthesis of primary metabolites are dispersed throughout the fungal genome. In contrast, the genes encoding the enzymatic activities to produce secondary metabolites are organized in biosynthetic gene clusters (BGC), ranging from two genes to over twenty genes (Brakhage 2013; Keller 2019). BGCs mainly contain genes that encode one or more enzymes that synthesize the core structure of the compound, the so-called backbone enzymes. Since most secondary metabolites are derived from polyketides (PKs) or non-ribosomal peptides (NRPs), the most common backbone enzymes are polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) (Brakhage 2013; Kjærbølling et al. 2019). Accordingly, secondary metabolites are essentially originated through the polymerization of primary metabolites by backbone enzymes, which will thus determine the chemical class of the generated compounds. For example, polyketide synthases (PKSs) produce polyketides from acyl-CoAs, non-ribosomal peptide synthetases (NRPSs) generate non-ribosomal peptides from amino acids, and terpene synthases and terpene cyclases generate terpenes from activated isoprene units. However, other enzymes can alter the metabolites’ bioactivities (Keller 2019).

The secondary metabolism is mainly developed in fungi of the division Ascomycota and Basidiomycota, while underdeveloped in the unicellular forms of the divisions Ascomycota, Basidiomycota, Zygomycota, Blastocladiomycota, and Chytridiomycota (G. F. Bills and Gloer 2016). The diversity of fungal species, particularly in the Ascomycota and Basidiomycota divisions, along with the diversification and clustering of the biosynthetic genes present, contribute highly to the enormous variety of metabolites originated (G. F. Bills and Gloer 2016; Keller 2019; Keller and Hohn 1997).

As previously mentioned, fungi secondary metabolites display a comprehensive range of biological activities, allowing fungi to survive in the most diverse ecosystems. For example, fungi pigments may confer protection against environmental stress, playing a pivotal role against photo-oxidation effects (carotenoids) or function as UV radiation protectors (melanin) (Dufossé et al. 2014; Gmoser et al. 2017; Kalra et al. 2020). Volatile compounds are released by fungi and used for species communication (Farh and Jeon 2020). Phenolic compounds and organic acids may act as signalling molecules in host-parasite/symbiotic relationships (Gaude et al. 2015), and antibiotics are produced to avoid species competition, limiting bacterial and fungal growth (Fan et al. 2017). In response to warmth, humidity, and moisture, the production of toxins by fungi (mycotoxins) is also well-known, mainly by those growing on crops (Brown et al. 2021). Overall, given the bioactive potential of their secondary metabolites, fungi produce these molecules as a response to biotic and abiotic factors, being involved in both communication and competition processes (Netzker et al. 2015; Shalaby and Horwitz 2015).

Therefore, fungi constitute a great source of attractive compounds for different pharmaceutical, cosmetic, and food sectors. In recent years fungi have become a good source of microbial metabolites, accounting for about 45% of the total production. This percentage includes metabolites produced by Basidiomycetes (mushrooms; 11%) and filamentous fungi such as Penicillium, Aspergillus, or Trichoderma (33%). Other types of fungi, such as yeasts and slime moulds, account for nearly 1.5% of the production of all metabolites (Bérdy 2012). Among the incredible variety of secondary metabolites produced by fungi, we can highlight molecules that have provided benefits that have revolutionized society, such as penicillins, cyclosporin, and statins (Cole et al. 2003; Devi et al. 2020; Keller et al. 2005; Rosazza 1984; Turner and Aldridge 1971). On the other hand, some fungal secondary compounds are also associated with severe problems, like mould-contaminated indoor environments or food and livestock contaminants, including aflatoxins and trichothecenes (Bills and Gloer 2016; Bräse et al. 2009, 2013; Hoffmeister and Keller 2007).

Overall, secondary metabolites synthesized by fungi are organized into four main classes and are extremely important to their environment adaptation and survival. Given their chemical diversity and bioactive potential, they have become attractive to humans from a food and pharmaceutical/cosmetic point of view.

2 Biosynthetic Pathways of Secondary Metabolites in Fungi

Fungal secondary metabolites may be divided into four major chemical classes: polyketides (PKs), non-ribosomal peptides (NRPs), terpenoids, and indole alkaloids. Beneath, some of the most known fungal secondary metabolites will be displayed, describing their biosynthetic pathway, including the enzymes involved in their origin.

2.1 Polyketides

Polyketides are one of the most chemically and functionally diverse compounds, synthesized by fungi (Cox et al. 2018). This chemical class encompasses molecules with an essential ecological function that man uses to develop products with diverse applications in agriculture or pharmacology.

Polyketides are often associated with toxicity, being the precursors of several toxins such as T-toxins (trichothecene mycotoxins), fumonisins, cytochalasin, or the well-known aflatoxins (Fig. 13.2). Although these polyketide mycotoxins are frequently associated with harmful effects on the production of diverse crops, affecting the agricultural sector and animal and human health, in nature, they allow the fungus to perform balanced maintenance of the pathogens that share its environment, competing with the surrounding species (Sakhkhari et al. 2019; Schuemann and Hertweck 2009).

Fig. 13.2

Chemical structure of fungal polyketide-derived compounds with important ecological roles. (a) T-toxin (C41); (b) Fumonisin B1; (c) Cytochalasin B; (d) Aflatoxin B1; and (e) Melanin

The polyketides class also includes pigments associated with fungal protection, as is the case of melanins (Fig. 13.2e). Melanins play essential ecological and biochemical roles in the fungal lifecycle. Their functions are associated with protection against adverse conditions, including UV light or heavy metals toxicity, and are also involved in charge transport and structural stability phenomena (Cordero and Casadevall 2017; Gómez and Nosanchuk 2003). Figure 13.2 displays the chemical structure of some of the described polyketide-derived compounds with a crucial function in fungal survival in ecosystems.

Given their role in ecosystems, polyketides have inherent bioactivities that are interesting to explore. Therefore, the man tries to synthesize them and use them at an industrial level, as is the case of strobilurins and griseofulvins. Strobilurins are a group of natural products and synthetic analogues used as pesticides in agriculture. The first natural strobilurin, strobilurin A, was extracted from the fungus Strobilurus tenacellus. Nowadays, these polyketides are produced at an industrial level through chemical modifications that promote their activity and photostability, and there are more than ten strobilurins available on the market. Indeed, these broad-spectrum fungicides represent an essential component in the agricultural fungicide trade (Cox et al. 2018; Feng et al. 2020). Griseofulvin (Fig. 13.3a), first obtained from the fungus Penicillium griseofulvum, is also an antifungal polyketide but used as medicine for years to treat ringworm in animals and humans (Petersen et al. 2014).

Fig. 13.3

Chemical structure of fungal polyketide-derived compounds with important pharmacological activity. (a) Griseofulvin; (b) Erythromycin A; and (c) Lovastatin

Polyketides also include other valuable and revolutionary drugs used in medicine, such as erythromycin or lovastatin. Erythromycin A (Fig. 13.3b) is an antibiotic (macrolide family of antibiotics) first isolated in 1952 from Saccharopolyspora erythraea (McGuire et al. 1952). It is widely used against several respiratory infections, being the main treatment for many pulmonary infections such as Legionnaire’s disease. It is also used to treat some sexually transmitted infections, such as chlamydia or syphilis (Farzam et al. 2021). Lovastatin (Fig. 13.3c) is a statin, the class of agents mostly used to treat hypercholesterolemia (Mulder et al. 2015). First isolated from the filamentous fungi Aspergillus terreus, this polyketide-derived natural product may also be present in higher fungi, such as Pleurotus ostreatus, Cantharellus cibarius, and Lentinula edodes (Kała et al. 2020). The commercial lovastatin is derived from A. terreus batch fermentation. It acts on the liver, reducing its ability to produce cholesterol by blocking the enzyme HMG-CoA reductase (Casas López et al. 2004; Mulder et al. 2015). Figure 13.3 displays the chemical structure of some of the described polyketide-derived compounds used worldwide due to their pharmacological potential.

Polyketide’s biosynthesis is highly related to fatty acids biosynthesis. Indeed, their carbon backbone is formed by a series of decarboxylative condensation reactions between acetyl-CoA thioesters units and malonate, using enzyme complexes homologous to fatty acid synthases, called polyketide synthases (PKSs). Following the condensation reactions, several chemical reactions occur, justifying the great diversity of this class of compounds (Avalos and Limón 2021; Bhattarai et al. 2021; Cox et al. 2018; Gupta and Rodriguez-Couto 2017; Simpson and Cox 2012). PKS enzymes have been classified into (i) type I, (ii) type II, and (iii) type III PKS. However, fungi polyketides biosynthesis involves mainly single modular iterative type I polyketide synthases (iPKSs), responsible for their carbon backbone construction. Type III PKSs, enzymes with a single keto synthase (KS) domain, may also be present; nevertheless, they are less common. Data on literature reveals that only eleven type III PKSs from fungi have been characterized so far (Avalos and Limón 2021; Bhattarai et al. 2021; Kaneko et al. 2019; Manoharan et al. 2019; Ramakrishnan et al. 2018; Skellam 2022; Yan et al. 2018).

PKSs have a standard set of conserved domains always consisting of ketosynthase (KS), acyltransferase (AT), and an acyl carrier protein (ACP). These three domains are complemented by other catalytic domains such as ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), methyltransferase (MeT) and thioesterase (TE) (Fujii 2010; Fujii et al. 2004; Schuemann and Hertweck 2009; Skellam 2022). Figure 13.4 briefly depicts the biosynthesis of polyketides based on the structure of the PKSs that give rise to them.

Fig. 13.4

Basic schematic representation of the mechanism of polyketide biosynthesis. KS ketoreductase, AT acyltransferase, ACP acyl carrier protein, KR ketoreductase, DH dehydrase, ER enoyl reductase, Enz KS or ACP domain. (Original image based on Crawford and Townsend 2010 and Schuemann and Hertweck 2009)

In polyketides biosynthesis, acyltransferase (AT) recognizes the monomer that will be used in the synthesis and transfers acyl groups from CoA onto the KS and ACP domains. The Claisen condensation is catalyzed by the KS domain to which acetyl-CoA binds, being condensed with malonyl-CoA units that are carried by the ACP domain (Fig. 13.4). In this stage, a ketide unit is added in each catalytic step. During condensation, the acetyl-CoA continuously bonded to malonyl-CoA loses its acidic group, resulting in the β-polyketide chain (Avalos and Limón 2021; Bhattarai et al. 2021; Crawford and Townsend 2010; Javidpour et al. 2011). The ACP, transporting the intermediates through the catalytic cycle, serves as a covalent binding site for the intermediate formed (Avalos and Limón 2021). Therefore, ACP domains serve as anchors for both malonyl-extending units and the acyl chain under construction (Avalos and Limón 2021; Schuemann and Hertweck 2009). The molecules that are being formed move between KS and ACP by thioester transfers, and in the end, a β-ketone is obtained (Fig. 13.4). The complexity of the β-ketone depends on the number of cycles (chain length control) and if it is fully reduced at the β-carbon of the extending chain or not. The keto groups formed in the elongating process can be reduced by introducing different possible groups on the β-carbon during the assembly of the polyketide, by a ketoreductase (KR), dehydrase (DH) or enoyl reductase (ER). This process, also called β-ketone processing, allows the obtention of non-reducing, partially reducing, or highly reducing polyketides (Fig. 13.4) (Avalos and Limón 2021; Crawford and Townsend 2010; Schuemann and Hertweck 2009). In addition, other changes carried out by different domains such as methyltransferase (MT) or condensation/heterocyclization (HC) can be performed, increasing the already enormous variety of polyketides existing in nature (Avalos and Limón 2021).

Overall, fungal polyketides assemblage is carried out mainly by iPKSs, being type III PKSs less frequent. According to the degree of reduction of the β-ketone chain, polyketides and PKSs can be classified as non-reducing, partially reducing, or highly reducing PKs or PKSs.

2.1.1 Non-reducing Polyketides

The most simple and well-known non-reducing polyketide is the phenolic compound orsellinic acid. The first PKS activity was observed in 1968 and was associated with its production; the orsellinic acid synthase present in a cell-free extract of Penicillium madriti (Gaucher and Shepherd 1968; Schuemann and Hertweck 2009).

Non-reducing PKSs (nrPKSs) usually lack domains for β-keto processing. They generally have a conserved set of domains composed of an N-terminal starter unit acyl transferase (SAT) domain, followed by the standard KS, AT, and ACP domains, essential to the chain elongation. Following the general mechanism of polyketides biosynthesis, the SAT domain is responsible for selecting and loading the starter unit, an acetate, a fatty acyl chain or another polyketide. Then, AT loads the malonate extender units, and the KS catalyzes the chain extension of the ACP-bound acyl chain (Cox 2007). What distinguishes these PKSs is a specific structural feature, an additional domain called product template (PT) domain, located between AT and ACP (Cox et al. 2018; Crawford and Townsend 2010; Schuemann and Hertweck 2009; Simpson and Cox 2012). Thus, these enzymes are constituted by an N-terminal loading component (SAT domain), a backbone extension component consisting of KS, AT, PT, and ACP domains, and a C-terminal processing component (Schuemann and Hertweck 2009; Simpson and Cox 2012). The PT domain is responsible for the cyclization of poly-β-ketone intermediates gathered during polyketide biosynthesis, being involved in the chain length determination, and controlling the final product’s structure. This domain can also promote the product release from the enzyme (Barajas et al. 2017; Schuemann and Hertweck 2009; Simpson and Cox 2012; Zheng et al. 2020). Therefore, nr-PKSs are responsible for the synthesis of fungal aromatic polyketides. Some examples are norsolorinic acid and aflatoxin B1 produced by Aspergillus sp. or the fungal red pigment bikaverin produced by Fusarium sp.

Many nrPKSs do not end after the ACP, having a diverse array of domains, including Claisen cyclase/thioesterases (CLC/TE) (Watanabe et al. 1998), C-methyl transferases (C-MeT) (Shimizu et al. 2005), and reductases (R) (Cox 2007), as well as additional ACPs (Fujii et al. 2001). TE domains are the most common C-terminal processing components found in nrPKSs (Schuemann and Hertweck 2009). On the other hand, although few nrPKSs with C-methylation domains are known, many non-reducing polyketides are C-methylated. In this case, the C-methylation (C-MeT) domain after the ACP domain presumably acts during chain extension. This has been particularly studied in the biosynthesis of the mycotoxin citrinin from Monascus ruber or M. purpureus (Cox et al. 2018; Schuemann and Hertweck 2009; Simpson and Cox 2012). Reductases (R) are rare in nrPKSs. This domain is usually used as a mechanism to release an aldehyde or primary alcohol (Simpson and Cox 2012). In the nrPKSs with additional ACP domains, although not all are required for polyketides biosynthesis, all of them are functional (Fujii et al. 2001; Watanabe and Ebizuka 2002).

2.1.2 Partially Reducing Polyketides

Partially reducing PKSs (prPKSs) are much rarer when compared with the nrPKSs, or the hrPKSs. These PKSs (and highly reduced PKSs) have a core domain, named the ketoreductase (KR) domain. So, prPKSs have an N-terminal KS, followed by AT, DH, KR, and a C-terminal ACP. Unlike nrPKSs, they do not possess SAT, PT, or TE domains (Cox et al. 2018; Schuemann and Hertweck 2009).

Although several genes coding for prPKSs are known, only three of these genes are related to the production of secondary metabolites in fungi. One of the best known is associated with the biosynthesis of the fungal metabolite 6-methylsalicylic acid (6-MSA) and has been isolated from P. griseofulvum, A. terreus, and Glearea lozoyensis. During the biosynthetic process of 6-MSA, a single reduction catalyzed by the KR domain occurs (Cox et al. 2018; Schuemann and Hertweck 2009; Simpson and Cox 2012).

2.1.3 Highly Reducing Polyketides

Highly reducing PKSs (hrPKSs), a class of enzymes also very common in fungi, are responsible for the synthesis of highly reduced polyketides, such as the previously mentioned lovastatin, T-toxin or fumonisin B1 (Simpson and Cox 2012).

The N-terminal KS from hrPKSs is followed by AT, DH, ER, and KR domains, ending with a C-terminal ACP. Some hrPKSs lose the ER domain, and instead, they possess an equivalent length sequence without known function. These PKSs are therefore grouped into hrPKSs with functional ER domain and hrPKSs with missing or non-functional ER domain. In addition to the β-keto processing domains, many hrPKSs have a C-MeT domain following the DH (Cox et al. 2018; Simpson and Cox 2012). For example, for the biosynthesis of lovastatin, two enzymes are required, lovastatin nonaketide (LNKS) and lovastatin diketide synthase (LDKS). The first has an inactive ER domain and a C-terminal truncated condensation (C) domain. LDKS encompasses KS, AT, C-MeT, ER, KR and ACP domains (Cox et al. 2018).

In general, fungal polyketides are classified as non-reducing, partially reducing, or highly reducing PKs. This classification is mainly based on the enzymes that originate them and the differences in their structure that induce the formation of different compounds. nrPKSs, one of the most common enzymes, consist of an SAT domain, followed by KS, AT, PT and ACP domains, and a C-terminal varying component. prPKSs have an N-terminal KS followed by AT, DH, KR, and a C-terminal ACP. Finally, hrPKSs have an N-terminal KS followed by AT, DH, ER, and KR domains, ending with a C-terminal ACP.

2.2 Non-ribosomal Peptides

Ribosomes play an essential role in the biosynthesis of proteins that form the building blocks of life. Nonetheless, evidence suggests that some peptides’ formation in soil-inhabiting Actinomycetes and Bacilli, eukaryotic filamentous fungi, and marine microorganisms are based on distinct ribosome-independent mechanisms, resulting in the synthesis of non-ribosomal peptides (NRP) (Martínez-Núñez et al. 2016). These structurally diverse NRPs are synthesized by the mega-enzyme complex referred to as non-ribosomal peptide synthetases (NRPSs), playing specific roles in host protection, stress tolerance and interactions with the environment (Oide and Turgeon 2020). Still, the characterization of these fungal metabolites has led to the development of ground-breaking pharmaceutical formulations, including antimicrobial agents, tumour suppressors, enzyme inhibitors, siderophores, and immunosuppressants in past decades (Guzmán-Chávez et al. 2018; Le Govic et al. 2019; Süssmuth et al. 2011).

Biosynthesis of NRPs begins by a series of repeating steps catalyzed by the three core catalytic domains of NRPS: adenylation (A), thiolation (T), and condensation (C). The multidomain NRPSs act as a molecular assembly line in which an amino acid is incorporated from one module to the next, as shown in Fig. 13.5.

Fig. 13.5

Assembly line of a non-ribosomal peptide synthetase (NRPS)

The A domain starts the NRPS biosynthesis cycle by carrying out an ATP-dependent activation of the amino acid substrates, then loading onto the T domain’s serine residue (Miller et al. 2016). The substrates that the A domain can recognize are proteinogenic and non-proteinogenic amino acids in their d- and l-configurations, fatty acids, α-hydroxy acids, α-keto acids, heterocycles, and other acyl moieties (Iacovelli et al. 2021). The flexibility of the biosynthetic programming pathway of NRPs based on their ability to utilize an extended range of over 500 substrates compared to ribosomal peptides results in the synthesis of structurally diverse NRPs (Izoré et al. 2021). The T domain, also known as the peptidyl carrier protein (PCP), binds the activated substrate to a 4′-phosphopantetheine cofactor (ppan) attached to a serine residue, forming a covalent linkage between the monomer and the enzyme. After adenylation and thiolation, the C domain, usually located at the beginning of each module, condenses the two substrates loaded onto the T domains, producing the peptide bond. After the generation of the complete peptide, the thioesterase (TE) domain, located on the C-terminal of NRPSs by an intra- or intermolecular cyclization event, catalyze peptide release from NRPSs (Guzmán-Chávez et al. 2018). The peptide then goes through a series of modifications, including methylation, glycosylation, acylation, halogenation, heterocyclization, or hydroxylation, generating structurally diverse peptide scaffolds (Le Govic et al. 2019). According to the collinearity rule, the number of modules in the NRPS is expected to correspond to the number of amino acid building blocks incorporated into the peptide metabolite. This implies that a 3 module NRPS will produce a tripeptide, while a 5 module NRPS will produce a pentapeptide (Challis and Naismith 2004). In addition, each module and the active site of each domain are utilized once in the assembly line, but in sporadic cases, these rules of collinearity and module skipping might be violated due to deletion of a specific domain (Gao et al. 2018). NRPs often have cyclic and/or branched structures that are easily recognizable by a peptide backbone, modified side chain on the amino acids, and can carry either methylated, glycosylated, acylated, halogenated, or hydroxylated modifications on the peptide backbone (Fig. 13.6).

Fig. 13.6

Structural backbone of a typical NRP

The discovery of penicillin in 1928 by Alexander Fleming represents a historical milestone in searching for effective antimicrobial agents and is recognized as the earliest advancement in therapeutic medicine. Penicillins and cephalosporins are NRPs that are mainly produced in Penicillium chrysogenum, Aspergillus nidulans, and Acremonium chrysogenum. Structurally, they contain a β-lactam ring formed by cyclisation of the linear tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), made up of l-α-aminoadipic acid, l-cysteine, and l-valine (von Döhren 2004). ACVS is the most well-characterized NRPS and is responsible for conducting the first reaction in the pathway leading to the biosynthesis of penicillin (Iacovelli et al. 2021). As shown in Fig. 13.7, the biosynthesis of penicillin begins with the activation of the three amino acids, followed by them loading onto the PCP domain. The activated substrates are condensed, forming ACV, a reaction catalyzed by ACV synthetase (ACVS). The next step involves the cyclization of ACV by isopenicillin cyclase, an iron-dependent enzyme, forming isopenicinilin N (Niu et al. 2020). In Penicillium chrysogenum, isopenicillin N is converted to penicillin by the enzyme isopenicillin N acyltransferase. In contrast, in Acremonium chrysogenum, isopenicillin N is converted to cephalosporin via a series of distinct enzymatic reactions (Guzmán-Chávez et al. 2018).

Fig. 13.7

Structural backbone of a typical NRP

Cyclic depsipeptides (CDPs) are cyclooligomers containing one or more amino acids being replaced by a hydroxylated carboxylic acid, forming a lactone bond in the core ring. These compounds have been reported in several fungal genera, including Acremonium, Calcarisporium, Fusarium, Phomopsis, and Ramalina (X. Wang et al. 2018). This class of fungal peptides has received increased attention due to their potential biological properties as antibacterial, insecticidal, herbicidal, anti-viral, cytotoxic, and cholesterol-lowering agents (Süssmuth et al. 2011; X. Wang et al. 2018). Besides their promising potential in pharmaceutical formulation development, they also confer several advantages to the producing fungal strain by enhancing uptake of nutrients, protection against other microbes, and more ecological functions (X. Wang et al. 2018). Biosynthesis of cyclic depsipeptides follows a similar module architecture with identical domain arrangement, as shown in Fig. 13.8. The A domain of the first module activates the D-hydroxycarboxylic acid substrate and is covalently bonded onto the PCP of the same module. The L-amino acid substrate is activated at the A domain of the second module and loaded onto the PCP of the corresponding module, which is linked to a terminal C3 domain, where ester bond formation and cyclization take place (Boecker et al. 2018; Süssmuth et al. 2011).

Fig. 13.8

The bimodular architecture in fungal cyclodepsipeptides biosynthesis

Hexadepsipeptides are the largest cyclic fungal depsipeptides that have been well characterized and distributed in the genera Aspergillus, Beauveria, Cordyceps, and Fusarium (Novak et al. 2021). Beauvericins are mycotoxins formed by d-hydroxyisovaleric acid and N-methyl-l-phenylalanine, produced by the soil-inhabiting entomopathogenic fungus Beauveria bassiana, Fusarium proliferatum, Fusarium oxysporum, Aspergillus terreus, Cordyceps cicadae, Peacilomyces tenuipes, and Paecilomyces fumosoroseus (Ulusoy et al. 2022; X. Wang et al. 2018). Increasing scientific research has shown the very promising potential of beauvericin in anti-viral therapy against SARS-CoV-2 and antimicrobial effects in the nematode Caenorhabditis elegans (Al Khoury et al. 2022; Büchter et al. 2020). Enniatins are structurally similar to beauvericin, having the phenylalanine moieties replaced by isoleucine, valine or leucine, forming enniatin A (Fig. 13.9), enniatin B or enniatin C, respectively. These compounds are mainly produced by fungal strains belonging to Alternaria, Fusarium, Halosarpheia, and Verticillium genus and currently, over twenty enniatin analogues have been isolated from fungal cultures (Süssmuth et al. 2011). These highly ionophoric and lipophilic compounds have shown promising potential in several in vitro models of cytotoxicity, oxidative stress, inflammation, and genotoxicity (Novak et al. 2021; Pallarés et al. 2020).

Fig. 13.9

Biosynthetic pathway of beauvericin, bassianolide, and enniatin

Other fungal cyclic hexadepsipeptides include allobeauvericin, aspergillicin, bursaphelocide, cardinalisamide, cordycecin, desmethyldestruxin, desmethylisaridin, destruxin, emericellamide, guangomide, hirsutatin, homodestcardin, isaridin, isoisariin, isariin, oryzamide, pullularin, sporidesmolide, and trichomide, among others (X. Wang et al. 2018). Bassianolide is the most well-characterized fungal cyclic octadepsipeptides isolated from Beauveria bassiana, Verticillium lecanii, and wood-decaying Xylaria spp. Structurally (Fig. 13.9), it is a tetramer containing d-hydroxyisovaleric and N-methyl-leucine monomer units similar to enniatin C (Süssmuth et al. 2011).

Siderophores such as ferrichrome and ferricrocin are also well characterized NRPs (Fig. 13.10). They are cyclic hexapeptides that act as iron chelators and mainly produced in various fungal strains, including Aspergillus fumigatus, Ustilago maydis, Aspergillus nidulans, Omphalotus olearius, Schizosaccharomyces pombe, Magnaporthe grisea, Cochliobolus heterostrophus, Fusarium graminearum, and Alternaria brassicicola (Eisfeld 2009). The siderophore synthetases of ferrichrome combine three N-acylated N-hydroxyornithine residues that form the core heme-binding unit and a ring of glycine, alanine, or serine, forming Ferrichrome A, Ferrichrome C, and ferricrocin, respectively (Bushley et al. 2008).

Fig. 13.10

Structure of ferrichrome-type siderophore

Overall, non-ribosomal peptides represent structurally diverse metabolites in several fungal strains with significant impact and application in pharmaceutical, food, cosmetic, and agricultural industries. Advances in genomic sequencing have ensured the correct identification of most NRPS biosynthetic genes in several fungal strains. This has encouraged re-engineering these NRP metabolites using several combinatorial biosynthetic methods for industrial-scale production of diverse molecular scaffolds of NRPs with improved biological and pharmacological properties (G. Bills et al. 2014).

2.3 Terpenoids/Terpenes

Ascomycota and Basidiomycota are known to produce an array of well-known terpenoid natural products, including mycotoxins, antibiotics, antitumor compounds, and hormones (G. F. Bills and Gloer 2017). However, the studies that have been developed (secondary metabolic pathways at molecular and biochemical levels) focus mainly on Ascomycota, as the Basidiomycota fungi, in general, are difficult to grow under in vitro conditions (G. F. Bills and Gloer 2016).

Fungal terpenoids are derived from five-carbon intermediates of isoprenyl diphosphate intermediates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), synthesized from one of two pathways, the eukaryotic MVA (via mevalonate) pathway or the prokaryotic MEP (methylerythritol phosphate) pathway (G. F. Bills and Gloer 2017; Z.-J. Li et al. 2021). Condensation of IPP and DMAPP monomers results in linear hydrocarbons of varying length: C10 geranyl pyrophosphate (GPP), C15 (2E,6E)-farnesyl pyrophosphate ((2E,6E)-FPP, or FPP), and C20 geranyl geranyl pyrophosphate (GGPP) (Schmidt-Dannert 2015). These linear hydrocarbons undergo a dephosphorylation and cyclization cascade to produce terpenes. Terpene synthases are the enzymes responsible for these highly complex reactions and two distinct classes of terpene synthase exist, defined according to substrate activation mechanism (G. F. Bills and Gloer 2017; Liao et al. 2016; Schmidt-Dannert 2015). Class I terpene synthases catalyze an ionization-dependent cyclization of the substrate, while class II terpene synthases catalyze a protonation-dependent cascade. Depending on the length of the precursor molecule, fungal terpene synthases are known to produce sesquiterpenes (C15), diterpenes (C20) and triterpenes (C30) (Liao et al. 2016).

Terpenoids are classified into two groups based on whether their scaffolds are derived solely from isoprenyl units or mixed biosynthetic origin. The first group includes the carotenoids, rare sesterterpenoids, and it is divided into mono-, sesqui-, di-, or triterpenoids, which contain two to six C-5 isoprene units. The second group includes the meroterpenoids, the indole diterpenoids, and the structurally and biosynthetically diverse group of prenylated aromatic natural products (G. F. Bills and Gloer 2017).

2.3.1 Carotenoids

Carotenoids are terpenoid pigments of yellow, orange, and red colour. Since they are not essential molecules for fungi, they accumulate smaller amounts in these organisms than plants or algae. However, given their antioxidant properties, they can protect fungi from UV radiation, as observed in the parasitic fungi Microbotryum violaceum and the mould Neurospora crassa (Cacciola and Sandmann 2022).

According to their chemical characteristics, microbial carotenoids are classified as carotenes and xanthophylls. Carotenes, such as α-carotene, β-carotene, γ-carotene, δ-carotene, and torulene, are the most well-known, containing carbon and hydrogen atoms in their chemical structure. Torularhodin, astaxanthin, and canthaxanthin are xanthophylls that, in addition to carbon and hydrogen, also contain oxygen in their chemical structure (Mussagy et al. 2019). The biosynthesis of microbial carotenoids (Fig. 13.11) is derived from acetyl CoA, obtained from fatty acids via the β-oxidation pathway in the microorganism mitochondria (Mussagy et al. 2019). Phytoene is the first carotenoid formed from two geranylgeranyl pyrophosphate (GGPP) molecules a reaction catalyzed by phytoene synthase. Depending on the biocatalytic reactions (cyclization, substitution, elimination, addition, and rearrangement), the phytoene molecule can originate different molecular structures of carotenoids. The desaturation of phytoene by the phytoene desaturase results in the lycopene molecule. β-carotene is formed through lycopene cyclization, where the lycopene β-cyclase introduces two β-ionone end-groups into the chemical structure (Cacciola and Sandmann 2022; Mussagy et al. 2019). Xanthophylls results from hydroxylation reactions in the carotene ring. β-carotene is converted into zeaxanthin through two enzymatic reactions by β-carotene hydroxylase (Mussagy et al. 2019).

Fig. 13.11

Biosynthesis of carotenoids, derived from acetyl CoA (Source: Mussagy et al. 2019)

The beneficial properties of carotenoids allow their use in various industries, from the food industry to the most recent applications in the pharmaceutical and nutraceutical industries. Carotenoids can be used as colouring foods, food additives and supplements with beneficial properties for human health such as antioxidant, antitumor, among others (Amengual. 2019; Mussagy et al. 2019). In recent years, Biotechnology has made progress regarding the use of fungi to produce carotenoids. β-carotene is produced on a large scale by the mould Blakeslea trispora, and significant advances (at the laboratory scale) have been made in astaxanthin production, using the yeast Xanthophyllomyces dendrorhous (Gassel et al. 2014). This development in the use of fungi, particularly natural or transgenic yeasts, in the production of carotenoids, has been essential in applying concepts that are highly valued today, namely sustainability and circular economy, producing these natural compounds through the cultivation of agro-industrial residues (Cacciola and Sandmann 2022).

2.3.2 Sesterterpenoids

Sesterterpenoids that have been isolated from fungi are pentaprenyl terpenoids whose often complex polycyclic structures are derived from the linear precursor geranylfarnesyl diphosphate (GFPP). These compounds are relatively rare among terpenoid natural products (K. Li and Gustafson 2021; Okada et al. 2016).

These molecules are generated from terpenes, and based on the number of C5 isoprene units, they are classified as hemi- (C5), mono- (C10), sesqui- (C15), di- (C20), sester- (C25), tri (C30), and tetraterpenes (C40). Among these, sesterterpenes and their derivatives, known as sesterterpenoids, are ubiquitous secondary metabolites in fungi (Evidente et al. 2015). Their structural diversity encompasses carbotricyclic ophiobolanes, polycyclic anthracenones, polycyclic furan-2-ones, or polycyclic hydroquinones (Evidente et al. 2015).

Forty-seven sesterterpenoids have been found in Aspergillus fungi, including ophyobolins, asperanes, and other type sesterterpenoids (Zhao et al. 2022). This section will focus on the genus Aspergillus, where the production of these compounds has been studied, revealing promising biological activities in several areas (Zhao et al. 2022).

2.3.2.1 Tricarbocyclic Sesterterpenoids (5/8/5-Membered Ring System)

Ophiobolin A (Fig. 13.12) is a fungal secondary metabolite with cytotoxic properties. It is produced through diverse cyclizations of linear C₂₅ precursors, that share the same 5-8-5 carbotricyclic skeleton with fusicoccins and cotylenins, two groups of diterpenoids produced by Fusicoccum amygdali and by Cladiosporum sp. 501-7 W (Masi et al. 2019; Okada et al. 2016).

Fig. 13.12

Structure of ophiobolin A (Source: Kinghorn 2020)

Several additional analogues of ophiobolin A were isolated from different fungi strains, and the types of ophiobolins produced vary with the culture conditions (Kinghorn 2020; Okada et al. 2016). Until now, more than 50 naturally occurring ophiobolins have been reported, with the majority coming from Bipolaris and Aspergillus species (Cai et al. 2019). Several ophiobolin-type sesterterpenoids were isolated from Aspergillus ustus and Aspergillus spp. These include (5α,6α)-ophiobolin H, (5α,6α)-5-O-methylophiobolin H, 5-O-methylophiobolin H, (6α)-21,21-O-dihydroophiobolin G and (6α)-18,19,21,21-O-tetrahydro-18,19-dihydroxyophiobolin, (6α)-21-deoxyophiobolin G, (6α)-16,17-dihydro-21-deoxyophiobolin G, ophiobolins U–W, ophiobolin O, 6-epi-ophiobolin O, ophiobolins X-Z, 21-dehydroophiobolins U and K, 21-epi-ophiobolins Z and O (184) (Zhao et al. 2022).

Ophiobolin B is produced from Bipolaris oryzae, ophiobolin C from B. zizanie, ophiobolin D from Cephalosporium caerulens and ophiobolin F from B. maydis (Okada et al. 2016). The formation of the 5/8/5-membered ring system (Fig. 13.13) starts with the cyclization mechanism of geranylfarnesyl diphosphate, where an 11/5-membered ring system is first generated. Subsequently, a 1,5-hydride shift and the formation of another 5-membered ring occur (Kinghorn 2020).

Fig. 13.13

Cyclization mechanism for the formation of the 5/8/5-membered ring system (Source: Kinghorn 2020)

2.3.2.2 Tetracarbocyclic Sesterterpenoid (7/6/6/5-Membered Ring System and 5/8/6/6-Membered Ring System)

Aspergilloxide is a tetracarbocyclic sesterterpenoid, isolated from the Aspergillus sp. with a 7/6/6/5-membered ring system, and the proposal cyclization mechanism starting from geranylfarnesyl diphosphate is shown in Fig. 13.14 (Kinghorn 2020).

Fig. 13.14

Cyclization mechanism for the formation of the 7/6/6/5-membered ring system of aspergilloxide (Source: Kinghorn 2020)

Asperterpenol A is an acetylcholinesterase inhibitor, reported from endophytic fungus Aspergillus sp. 085242, and the tetracarbocyclic skeletons are formed as shown in Fig. 13.15 (Kinghorn 2020).

Fig. 13.15

Cyclization mechanism for the formation of the 5/8/6/6-membered ring system of asperterpenol A (Source: Kinghorn 2020)

2.3.2.3 Pentacarbocyclic Sesterterpenoids (5/7/3/6/5-Membered Ring System; 5/3/7/6/5 and 5/4/7/6/5-Membered Ring)

Asperterpenoid A is a potent inhibitor of the Mycobacterium tuberculosis protein-tyrosine phosphatase B (PtpB), and it is isolated from the endophytic Aspergillus sp. 16-5c. The possible cyclization reaction for the formation of the 5/7/3/6/5-membered ring system is shown in Fig. 13.16 (Kinghorn 2020).

Fig. 13.16

Possible cyclization reaction for the formation of the 5/7/3/6/5-membered ring system of asperterpenoid A (Source: Kinghorn 2020)

Aspterpenacid A, isolated from the fungi A. terreus H010, has a 5/3/7/6/5-membered ring system, while astellatol, isolated from A. variecolor, possesses a 5/4/7/6/5-membered ring system. The proposed pathway for synthesizing these compounds starting from geranylfarnesyl diphosphate is represented in Fig. 13.17 (Kinghorn 2020).

Fig. 13.17

Possible cyclization reactions for the formation of the basic carbon skeletons of aspterpenacid A and astellatol (Source: Kinghorn 2020)

2.3.2.4 Hexacarbocyclic Sesterterpenoids (5/5/5/5/3/5-Membered Ring Systems)

Niduterpenoid A and niduterpenoid B were first isolated from Aspergillus nidulans and possess hexacarbocyclic sesterpenoids. The cyclization reaction is quite complicated and starts from geranylfarnesil diphosphate. The possible cyclization reactions for forming the hexacarbocyclic skeleton of niduterpenoid A and niduterpenoid B is shown in Fig. 13.18 (Kinghorn 2020).

Fig. 13.18

Possible cyclization reactions for the formation of the hexacarbocyclic skeleton of niduterpenoid A and niduterpenoid B. The intermediate A possesses a 5/5/5/6/5-membered ring system (Source: Kinghorn 2020)

2.3.3 Meroterpenoids and Isoprenoids

Meroterpenoids and isoprenoids, like other natural compounds, may be confused with sestertepenoids; but not all compounds with 25 carbon atoms are sesterterpenoids.

Meroterpenoids are natural products with a C10 polyketide moiety (e.g., preterretonin A, protoaustinoid A, and andrastin E), but they are not biosynthesized via geranylfarnesyl diphosphate (GFPP). Instead, they are generated from a C15 terpenoid moiety and a C10 polyketide moiety. These C15 and C10 moieties are combined in their biosynthesis to form the C25 basic carbon skeleton (Kinghorn 2020) as showed in Fig. 13.19.

Fig. 13.19

Biosynthesis of preterretonin A, protoaustinoid A, and andrastin E (Source: Kinghorn 2020)

Highly branched isoprenoids are a member of the terpenoids family, with a 25 carbon atoms skeleton. Isoprenoids are not considered sesterterpenoids, because they do not derive from the C25 polyprenyl diphosphate, but from (C10) geranyl diphosphate (GPP) and (C15) farnesyl diphosphate (FPP) (Kinghorn 2020) as showed in Fig. 13.20.

Fig. 13.20

Biosynthesis pathway of Highly branched isoprenoid (Source: Kinghorn 2020)

2.4 Indole Alkaloids

Indole alkaloids are one of the largest classes of nitrogen-containing secondary metabolites that are widely found in plants, bacteria, fungi, and animals (Fig. 13.21). About 12,000 alkaloids have been discovered, many of which are pharmacologically active and traditionally used as antitussives, purgatives, sedatives, and anticancer drugs (Oudin et al. 2007). Previous phytochemical investigations have led to the characterization of indole alkaloids with cytotoxic, anti-diabetic, and anti-inflammatory activities (Khyade et al. 2014). Therefore, this important class of secondary metabolites has aroused great interest in natural products research due to its structural complexity and significant pharmacological activities (Z. W. Wang et al. 2021; Yu et al. 2021). Fungi, especially Ascomycota, have been reported as prolific producers of indole alkaloids (Hanson 2008). The availability of fungal genome sequences has, in recent years, significantly accelerated the identification of the biosynthetic genes involved in the biosynthesis of secondary metabolites from fungi (Wiemann and Keller 2014; Yaegashi et al. 2014).

Fig. 13.21

Basic structure of the indole nucleus

Many fungal metabolites, collectively designated as indole alkaloids, contain in their structures a prenylated indole nucleus (Fig. 13.21) that derives from l-tryptophan and mevalonate. These metabolites include two large groups: (a) the ergot alkaloids produced by the plant parasitic Claviceps species (Tudzynski et al. 2001), and (b) the indole alkaloids produced by species of Aspergillus, Penicillium, and Neosartorya, among others (S. M. Li 2009). These alkaloids differ: (i) in the carbon atom of the indole molecule bearing the isopentenyl group, (ii) in modifications of the diketopiperazine ring, and (iii) in modifications of the N1 atom of indole, that are introduced by “late” modification enzymes encoded by additional genes in the clusters. One of the best-known indole alkaloid groups is that of the ergot alkaloids (Tudzynski et al. 2001) and another important group is that of roquefortine C (mycotoxin) and related indole alkaloids (glandicoline, meleagrin, neoxaline) (García-Estrada et al. 2011; Sumarah et al. 2005). Several of these compounds are produced by Penicillium species of the Corymbifera family (Martín et al. 2014).

Indole alkaloids are usually derived from tryptophan and dimethylallyl pyrophosphate, although sometimes amino acids other than tryptophan are used as precursors (Keller et al. 2005). Different strategies to incorporate indole moieties into the final alkaloid structures are found in fungal secondary metabolism. Not surprisingly, most of the indole precursors are related to l-tryptophan (1), the most abundant indole-containing species in the cell. The biosynthesis of (1) itself starts from chorismate in the shikimic acid pathway and involves the intermediates anthranilate and indole-3-glycerol-phosphate. Phosphate intermediate is transformed into indole, which can be coupled with serine to form (1) (Dunn et al. 2008). Tryptophan (1) can be decarboxylated and converted into tryptamine, (Lovenberg et al. 1962) or be prenylated at C4 to yield 4-dimethylallyl tryptophan (4-DMAT) (2), as summarized below in Fig. 13.22 (Lee et al. 1976; Unsöld and Li 2005). Feeding experiments with isotope-labeled precursors have shown that l-tryptophan and indole-3-glycerol-phosphate, tryptamine and 4-DMAT, can each serve as the biosynthetic precursor for the indole/indoline moieties in fungal indole alkaloids (Flieger et al. 1997; Xu et al. 2014).

Fig. 13.22

Formation of the ergoline scaffold-biosynthetic pathway (Gerhards et al. 1950)

The best-understood pathway is ergotamine synthesis in Claviceps purpurea and related species (Králová et al. 2021; Tudzynski et al. 1999). The biosynthetic pathway is shown in Fig. 13.22, which starts with the C4-prenylation of l-tryptophan (1) with dimethylallyl diphosphate (DMAPP) as prenyl donor. This reaction is catalyzed by the prenyltransferase 4-dimethylallyltryptophan synthase (DMATS), also named FgaPT2 in A. fumigatus (Coyle and Panaccione 2005; Lee et al. 1976; Unsöld and Li 2005). Biochemical and structural elucidations clearly show the formation of 4-γ,γ-dimethylallyltryptophan (DMAT (2)) as a product (Metzger et al. 2009; Steffan et al. 2007; Steffan and Li 2009). Metzger et al. reported the X-ray structure of FgaPT2 in complex with L-tryptophan, proposing a three-step mechanism: the formation of a dimethylallyl cation, a nucleophilic attack of the indole nucleus to that cation and a deprotonation step, which led to a better understanding of the reaction mechanism (Luk and Tanner 2009; Metzger et al. 2009). Evolutionary investigations have indicated that the gene fgaPT2 from A. fumigatus has the same origin as prenyltransferase genes from another Ascomycota, including the ergot-alkaloid-producing Clavicipitaceae (Gerhards et al. 1950; Liu et al. 2009).

After the reaction, the pathway reaches a branch point. Several products arise from (5), depending on the fungus. For example, the next intermediate in A. fumigatus is festuclavine (6), in P. commune pyroclavine (7) and in C. purpurea agroclavine (8) (Matuschek et al. 2012). The branch point is mainly controlled by the old yellow enzyme EasA (also termed FgaOx3), and the functional differences in this enzyme result in divergent ergot alkaloid pathways (Coyle et al. 2010). For the formation of festuclavine in A. fumigatus, a second enzyme (the festuclavine synthase FgaFS) is required, as shown by Wallwey et al. (Wallwey et al. 2010; Xie et al. 2011). Cheng et al., reported the formation of agroclavine catalyzed by an enzyme from E. festucae var. lolii (Cheng et al. 2010). However, in C. purpurea in vitro investigations on the respective reaction showed that EasG (a homologue of FgaFS from A. fumigatus) can catalyze the formation of (8) via a non-enzymatic adduct with reduced glutathione (Gerhards et al. 1950; Matuschek et al. 2011). As shown by Matuschek et al., the formation of pyroclavine in P. commune requires both homologues: FgaOx3_PC and FgaFS_PC (Matuschek et al. 2012).

Other tryptophan-derived alkaloids such as the fumigaclavines and fumitremorgens of A. fumigatus undergo one or more prenylation steps. The details of these pathways are yet to be elucidated, but it is likely that the fumigaclavine biosynthetic pathway proceeds through agroclavine and might therefore have some early steps in common with the ergotamine pathway (Keller et al. 2005; von Nussbaum 2003).

The biosynthetic pathway for indole alkaloids has been investigated extensively in Claviceps species and A. fumigatus and the elucidation of the pathway is of interest especially because of broad range of pharmaceutical uses, being able to increase knowledge concerning the genes and enzymes. Therefore, molecular genetic manipulations may be used to improve industrial production of medically important indole alkaloids, and novel forms that could act as drugs with new or improved pharmacological activities and minimal side effects might be created by synthetic microbiology or other related techniques.

3 Conclusions

In this chapter, the most abundant secondary metabolites from fungi, namely their biosynthesis, were discussed.

Fungal secondary metabolites exhibit impressive chemical structures and biological activities, but their biosynthetic pathways share some key points with primary metabolites or even with each other. Four main classes of fungal secondary metabolites can be considered, originating through acetyl-CoA and via the shikimate pathway, i.e. polyketides, non-ribosomal peptides, terpenoids, and indole alkaloids.

Although some of these compounds are associated with adverse effects, such as mycotoxins, the truth is that others have brought benefits that have revolutionized the world of pharmacy/medicine and agriculture, namely antibiotics or pesticides. This dichotomy regarding fungal secondary metabolites is thus indicative of the enormous diversity of natural products that fungi can produce.

Conflicts of Interest

The authors declare no conflict of interest.