In vivo characterization of the dia biosynthetic gene cluster reveals diaporthinic acid as its main product

The biosynthetic gene cluster (BGC) responsible for producing diaporthinic acid has remained genetically unassigned despite repeated isolation of this metabolite from several fungal species. In this study, we activated the dia BGC in Trichoderma reesei by overexpressing the cluster-associated zinc-cluster protein DiaR1 to identify the BGC’s in vivo metabolic output and reconstruct the corresponding biosynthetic pathway. Metabolite production was analyzed by HPLC-MS/MS, and the major product was isolated and structurally confirmed by NMR spectroscopy. Individual genes of the dia cluster were deleted in the activated background to assess their functional roles, and transcript levels were quantified by RT-qPCR. Activation of the cluster resulted in the predominant accumulation of diaporthinic acid, accompanied by several related isocoumarin derivatives, while antibacterial and antifungal assays showed no detectable activity of diaporthinic acid under the tested conditions. Deletion analyses demonstrated that the polyketide synthase Dia1, the bifunctional halogenase/methyltransferase Dia5, and the FAD-dependent oxidoreductase Dia4 are essential for diaporthinic acid formation, whereas Dia2 and Dia3 are dispensable in vivo despite the previously proposed roles of their Aspergillus oryzae homologs based on in vitro studies. On the basis of intermediate accumulation patterns, we propose that Dia4 catalyzes the oxidation of dichlorodiaporthin to diaporthinic acid. Together, these results genetically link diaporthinic acid to the dia BGC and refine the previously proposed biosynthetic model derived from A. oryzae.