Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in <i>Saccharomyces cerevisiae</i>

The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL₁₂) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from <i>Lasiodiplodia theobromae</i> and resorcylide from <i>Acremonium zeae</i> revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in <i>Saccharomyces cerevisiae</i> provided a convenient access to the RAL₁₂ scaffolds desmethyl-lasiodiplodin and <i>trans</i>-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an <i>O</i>-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-<i>S</i>-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during <i>in vivo</i> heterologous biosynthesis of RAL₁₂ metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions