Discovery of four novel furanosteroids and a new 4-methylprogesteroid from demethoxyviridin pathway-disrupted mutants of Nodulisporium sp. through cultivation time extension

Abstract

Furanosteroids are a structurally distinctive class of fungal metabolites with significant biological activities, particularly well-known as potent phosphatidylinositol 3-kinase inhibitors. Previously, we have elucidated the biosynthetic pathway of demethoxyviridin, one of the most representative furanosteroids. Inspired by our previous finding that some non-specific tailoring enzymes in Nodulisporium sp. 65-12-7-1, the producer of demethoxyviridin, are able to redirect the metabolic flux of demethoxyviridin towards off-pathway products, we inferred that the metabolic potential of the demethoxyviridin biosynthetic gene-disrupted mutants could also be harnessed to give novel derivatives. In this study, in order to enhance the catalytic function of non-specific enzymes, we extended the cultivation period from two to five days, and then isolated and identified seven compounds from two mutant strains with knockout of the cytochrome P450 enzyme gene vidR and the short-chain dehydrogenase/reductase gene vidO. Among them, five compounds are novel, including four furanosteroids (1–4) and a 4-methylprogesteroid (5), which may serve as a biosynthetic precursor of furanosteroids. Intriguingly, structural analysis of these new compounds enabled us to propose functional hypotheses for the previously uncharacterized enzymes: VidR is potentially involved in C-7 oxidation, and VidO likely acts downstream of VidR. The cytotoxicity assay revealed that compound 2 displayed remarkable cytotoxic effects on human PANC-1 cells. This work demonstrated that combining biosynthetic gene disruption with extended fermentation is an effective approach to generate novel analogues by redirecting biosynthetic flux.