Identifying sesterterpenoids via feature-based molecular networking and small-scale fermentation

Terpenoids are known for their diverse structures and broad bioactivities with significant potential in pharmaceutical applications. However, natural products with low yields are usually ignored in traditional chemical analysis. Feature-based molecular networking (FBMN) was developed recently to cluster compounds with similar skeletons, which can highlight trace amounts of unknown compounds. Fusoxypene A is a sesterterpene synthesized by Fusarium oxysporum fusoxypene synthase (FoFS) with a unique 5/6/7/3/5 ring system. In this study, the FoFS-containing biosynthetic gene cluster was identified from F. oxysporum FO14005, and an efficient FBMN-based strategy was established to characterize four new sesterterpenoids, fusoxyordienoid A-D (1-4), based on a small-scale fermentation strategy. A cytochrome P450 monooxygenase, FusB, was found to be involved in the functionalization of fusoxypene A at C-17 and C-24 and responsible for the hydroxylation of fusoxyordienoid A at C-1 and C-8. This study highlights the potential of FBMN as a powerful tool for the discovery and characterization of natural compounds with low abundance. KEY POINTS: Combined small-scale fermentation and FBMN for rapid discovery of fusoxyordienoids Characterization of four new fusoxyordienoids with 5/6/7/3/5 ring system Biosynthetic pathway elucidation via tandem expression and substrate feeding.