Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone

Abstract

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.

• Cladonia grayi
• depsides
• depsidones
• lichen fungi
• metabolic gene clusters
• polyketide synthases
• secondary metabolites
• symbiosis

We thank Susan May for technical assistance, David Swofford and Mike Nowak for help with the phylogenetic analysis, Fred Guengerich for his input on cytochrome P450, Vivian Miao, John Mercer and Louise Roth for critical reading of the manuscript, and the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) for contributing to the assembly and annotation of the genome data.

Notes

2 In this context we report for the record some of our earlier data that remained unpublished because no reliable PKS-to-metabolite connection could be made (Armaleo, Kua and Zhang reported as pers comm by Miao et al. (2001). A full length NR-PKS gene (designated CgrPKS2) similar to that identified by Chooi et al. (2008) and also encoding a MeT domain was found to be expressed in, and was cloned from, a race of Cladonia grayi that did not produce usnic acid or β-orcinol depsides and depsidones. The CgrPKS2 full-length mRNA was expressed from its own promoter and correctly spliced in the heterologous host Aspergillus nidulans, but no new secondary product could be identified. CgrPKS2 is also one of the genes whose induction tests and phylogeny are described here. The CgrPKS2 GenBank accession number is GU930714.