1. Introduction
1.4. Specialized metabolism in fungal development
1.4.1. The monodictyphenone (mdp) / xanthone (xpt) secondary metabolite gene
The monodictyphenone (mdp) secondary metabolite gene cluster is found to contain twelve genes that are located near the rather silent telomeric region of chromosome VIII (Figure 4). It is known that chromatin remodeling factors are needed to influence the expression of genes that are responsible for the production of secondary metabolites (Gacek and Strauss 2012).
Bok and co-workers showed that CclA, a member of the histone 3 lysine 4 methylating COMPASS (complex associated with Set1) complex regulates the expression of secondary metabolite gene clusters such as the monodictyphenone (mdp) gene cluster (Bok et al., 2009). The deletion of cclA alters the expression of genes necessary for the production of monodictyphenone. Monodictyphenone represents a precursor for the synthesis of prenyl-xanthones. The genes xptA, xptB and xptC required for the conversion of monodicytphenone into xanthones are not embedded in the monodictyphenone gene cluster (Pockrandt et al., 2012, Sanchez et al., 2011).
Instead, they are localized on two different chromosomes. The genes that encode the two prenyltransferases XptA and XptB are localized on chromosome I and II. The gene xptC that encodes an oxidoreductase that is localized on chromosome II and is separated from the gene that encodes the prenyltransferase XptB by the gene AN7998. Sanchez and co-workers showed that mdpE (encodes a putative C6 zinc finger transcription factor), mdpI (encodes a putative AMP-binding CoA ligase) and AN7998 (encodes a putative oxidoreductase) are not involved in the synthesis of prenyl-xanthones (Sanchez et al., 2011).
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1.4.1.1. Monodictyphenone is a precursor for the synthesis of xanthones
Xanthones are organic compounds found in different species like the mangosteen tree Garcinia mangostana and are responsible for a yellow pigmentation. The chemical building block of xanthones is composed of a core structure named xanthone nucleus (9H-Xanthen-9-on) that is an aromatic oxo compound. Different modifications of the xanthone nucleus lead to a variety of different xanthones. A possible modification of the xanthone nucleus can be due to a prenylation event. Xanthones found in Garcinia mangostana are known for anticancer activities (Alam and Khan 2018).
The polyketide synthase MdpG encoded by the monodictyphenone (mdp) gene cluster is required for the synthesis of anthraquinone emodin, monodictyphenone and related compounds (Klejnstrup et al., 2012). The sequence of the polyketide synthase MdpG contains as many as 1806 amino acids with a predicted molecular mass of 196,8 kDa.
Figure 4. Secondary metabolite gene clusters which are involved in the production of monodictyphenone and xanthones.
A) The monodictyphenone (mdp) gene cluster is located at the telomeric region of chromosome VIII and contains twelve genes. The gene mdpG (blue) encodes a polyketide synthase that is involved in the production of the polyketide backbone core structure of monodictyphenone. The enzymes encoded by the residual genes modify the core structure resulting in the production of emodin and finally in monodictyphenone and prenyl-xanthones. The genes mdpE (encodes a C6 zinc finger transcription factor) and mdpI (encodes an AMP-binding CoA ligase) in grey are not essential for the production of
prenyl-xanthones.
B) Monodictyphenone represents a precursor for the production of prenyl-xanthones. Two prenyltransferases XptA, XptB and an oxidoreductase XptC are involved in the conversion of monodictyphenone into prenyl-xanthones. The genes that encode the prenyltransferases XptA and XptB are localized on chromosome I and II. The oxidoreductase XptC is localized on chromosome II and is situated next to the gene AN7999 (grey) which encodes an oxidoreductase and is not essential for the production of prenyl-xanthones (Sanchez et al., 2011, Chiang et al., 2010).
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The polyketide synthase MdpG synthesizes the main polyketide backbone core structure. MdpG polyketide synthase consists of several domains with defined functions mentioned in Figure 5. These domains of MdpG are involved in the synthesis
of the polyketide backbone.
Emodin and monodictyphenone are precursors for prenyl-xanthone. Sanchez and co-workers deleted mdpG and revealed that the product monodictyphenone and other compounds such as prenyl-xanthones are no longer produced (Sanchez et al., 2011).
The first step in the production of emodin and monodictyphenone requires MdpG.
Malonyl-CoA represents a substrate for MdpG which synthesizes the polyketide backbone. The polyketide synthase MdpG lacks a thioesterase (TE) domain which hydrolyzes the newly formed polyketide backbone off the synthase. The gene mdpF that encodes a putative zinc dependent hydrolase, catalyzes most probably the release of the polyketide backbone from MdpG (Chiang et al., 2010). The gene mdpH that encodes a decarboxylase, catalyzes the conversion of atrochrysone carboxylic acid into atrochrysone (Klejnstrup et al., 2012). The deletion of mdpH results in an inability of the above-mentioned conversion to atrochrysone (Chiang et al., 2010). In order to convert atrochrysone to emodin two unknown dehydrating and modifying enzymes are necessary. The following enzymes finally convert emodin into monodictyphenone, a dehydratase (MdpB), a ketoreductase (MdpC), a glutathione S transferase (MdpJ), an oxidoreductase (MdpK) and a Baeyer-Villiger oxidase (MdpL) (Simpson 2012, Klejnstrup et al., 2012). The monooxygenase MdpD is required for the hydroxylation of monodictyphenone (Bok et al., 2009).
As a next step the hydroxylated monodictyphenone is converted into prenyl-xanthones. Klejnstrup and co-workers demonstrated that the two prenyltransferases XptA and XptB are involved in the prenylation of hydroxylated monodictyphenone (Klejnstrup et al., 2012). The biosynthesis of the stereoisomers shamixanthone and epishamixanthone is finally catalyzed by the oxidoreductase XptC (Sanchez et al.,
2011).
It is known that monodictyphenone and xanthones are antimicrobial agents that serve against fungivory and other environmental threats (Bok et al., 2009, Regulin and Kempken 2018). In a transcriptomic and metabolomic profiling study it was observed that after the addition of choline Hülle cell formation occurred in a vegetative mycelium and the secondary metabolite monodictyphenone was present in this liquid culture (Alves et al., 2016).
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Figure 5. Biosynthesis of monodictyphenone and xanthones.
The polyketide synthase MdpG synthesizes the main polyketide backbone core structure and uses malonyl-CoA as a substrate. Several domains of MdpG are involved in the synthesis of the poyketide backbone (SAT domain an ACP-transacylase as a starter unit, KS a ß-ketoacyl synthase domain and AT an acetyltransferase domain. These three domains are involved in the synthesis of a polyketide backbone intermediate. The internal product template (PT) domain is responsible for folding and cyclization of the polyketide backbone intermediate. The ACP is an acryl carrier protein domain and represents a transiently holding domain for the polyketide backbone).
The putative zinc dependent hydrolase MdpF catalyzes most probably the release of the polyketide backbone from MdpG. The decarboxylase MdpH catalyzes the conversion of atrochrysone carboxylic acid to atrochrysone. In the conversion of atrochrysone to emodin unknown enzymes are involved.
Several enzymes (MdpB (dehydratase), MdpC (ketoreductase), MdpJ (glutathione S transferase), MdpK (oxidoreductase), MdpL (Baeyer-Villiger oxidase) are required for the further synthesis of emodin to monodictyphenone. The monooxygenase MdpD hydroxylates monodictyphenone. The two prenyltransferases XptB and XptA are required for the prenylation of hydroxylated monodictyphenone.
Giving rise to the compounds variecoxanthone A and emericellin. The oxidoreductase XptC converts emericellin into the stereoisomers shamixanthone and epishamixanthone (Simpson 2012; Klejnstrup et al., 2012).
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