Naphthopyranones represent a structurally diverse family of fungal polyketides exhibiting a broad range of biological activities, including antibacterial, antifungal, and cytotoxic properties. Despite extensive investigations of terrestrial-derived naphthopyranones, the biosynthetic machinery responsible for their production in marine fungi has remained unexplored. Here, we report the first characterization of naphthopyranone biosynthetic gene clusters (BGCs) from a deep-sea-derived fungus. Genome mining of the cold seep-associated Penicillium javanicum OUCF108 revealed two highly homologous polyketide synthase gene clusters, pig1 and pig2. Comparative transcriptomics combined with targeted disruption of the core PKS gene pigA2 demonstrated that pig2 is the essential BGC responsible for (R)-semivioxanthin (1) production. Stepwise reconstruction of the pig2 pathway in Aspergillus oryzae NSAR1 unraveled the complete biosynthetic route from the heptaketide precursor nor-toralactone (2) to (R)-semivioxanthin (1) and its dimeric derivatives. In vitro biochemical characterization revealed that the O-methyltransferase PigN2 catalyzes regioselective 6-O-methylation with relaxed substrate specificity, that the laccase PigF2 mediates oxidative dimerization of 1 to afford dimeric derivatives, and that the fasciclin-like protein PigG2 alters this default regiochemistry, affording abundant alternative regioisomeric dimers alongside the 5,5'-linked product. Notably, a new naphthopyranone derivative, nor-4-hydroxy-toralactone (4), was isolated and structurally elucidated. Antimicrobial evaluation of all isolated compounds revealed that 4 exhibits moderate antifungal activity against the multidrug-resistant pathogen Candida auris (MIC = 12.5 μg mL-1). Structure-activity relationship analysis identified the C-4 hydroxyl moiety is critical for activity. This study highlights the potential of deep-sea fungi as an untapped reservoir of bioactive naphthopyranones and provides enzymatic insights for the construction of regioselectively coupled biaryl scaffolds.
🌊
