Grayanic acid

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.

Lichen secondary metabolites have tremendous pharmaceutical and industrial potential. Although more than 1000 metabolites have been reported from lichens, less than 10 have been linked to the genes coding them. The current biosynthetic research focuses strongly on linking molecules to genes as this is fundamental to adapting the molecule for industrial application. Metagenomic-based gene discovery, which bypasses the challenges associated with culturing an organism, is a promising way forward to link secondary metabolites to genes in non-model, difficult-to-culture organisms. This approach is based on the amalgamation of the knowledge of the evolutionary relationships of the biosynthetic genes, the structure of the target molecule, and the biosynthetic machinery required for its synthesis. So far, metagenomic-based gene discovery is the predominant approach by which lichen metabolites have been linked to their genes. Although the structures of most of the lichen secondary metabolites are well-documented, a comprehensive review of the metabolites linked to their genes, strategies implemented to establish this link, and crucial takeaways from these studies is not available. In this review, I address the following knowledge gaps and, additionally, provide critical insights into the results of these studies, elaborating on the direct and serendipitous lessons that we have learned from them.