Heterologous expression of an in planta-upregulated gene cluster in the wheat pathogen Parastagonospora nodorum establishes the biosynthesis of methylene-bridged depsides.

Depsides are polyphenolic natural products commonly found in lichens, but also produced by some free-living fungi. Here, we report the discovery and functional characterisation of an in planta-upregulated biosynthetic gene cluster (nds) in the wheat fungal pathogen Parastagonospora nodorum SN15, comprising a non-reducing polyketide synthase (NR-PKS; NdsA), a non-ribosomal peptide synthetase (NRPS; NdsB) and a cytochrome P450 oxygenase (NdsC). Heterologous expression of the nds cluster in Aspergillus nidulans LO8030 yielded previously reported depsides, CJ-20,557 (1) and duricamidepside (2), alongside two novel dimeric methylene-bridged depsides, nodoraside A (3) and B (4). Combinatorial gene expression and precursor feeding experiments revealed that hydroxylation of CJ-20,557 by the cytochrome P450 NdsC, followed by non-enzymatic dehydration, generates an ortho-quinone methide intermediate, which reacts with a second depside unit to form methylene-bridged depsides. Notably, this crosslinking occurs only in the presence of NdsA and NdsB in vivo, indicating a possible requirement for protein-protein interactions or enzyme co-compartmentalisation. Methylene-bridged depside natural products are rare, and this study provides the first insight into their biosynthetic origin.