Deciphering the biosynthetic pathways of lichen acids.

Depsides and depsidones are polyketide-derived lichen acids widely distributed in lichen thalli, yet the biosynthetic gene clusters (BGCs) responsible for their production remain poorly understood. To address this gap, we investigated the diversity and evolutionary relationships of polyketide BGCs in lichens. We conducted genome mining and phylogenetic analyses of polyketide synthases (PKSs) across 100 lichen genomes, followed by BGC network analysis to assess BGC diversity. Functional validation was performed through heterologous expression of PKS and tailoring enzyme genes in heterologous hosts. Lecanorales species showed the highest diversity of PKSs. Phylogenetic analysis revealed that pks1 is closely related to pks23 responsible for atranoric acid biosynthesis. The pks1 BGCs exhibited gene content variation between species producing different depsides/depsidones. Heterologous expression of pks1 produced 4-O-demethylbarbatic acid, the same compound from pks23. Co-expression of pks1 with tailoring enzymes yielded virensic acid, a depsidone precursor. High pks1 expression in barbatic acid-containing thalli supported its role in medullary depside/depsidone biosynthesis. These findings establish pks1 and pks23 as paralogous PKSs and reveal how tailoring enzyme diversity contributes to chemical diversity of lichen acids. This study provides new insight into the biosynthetic and evolutionary mechanisms driving structural diversification of lichen acids.