Biosynthetic Elucidation and Structural Revision of Brevione E: Characterization of the Key Dioxygenase for Pathway Branching from Setosusin Biosynthesis

Abstract Brevione E ( 1 ) is a fungal hexacyclic meroditerpenoid with unique oxepane and cycloheptenone moieties. In this study, we identified the biosynthetic gene cluster of 1 and elucidated its biosynthetic pathway via heterologous expression of the biosynthetic genes and in vitro enzymatic reactions. Surprisingly, reexamination of the structure of 1 revealed a substituted tetrahydrofuran ring instead of the previously proposed oxepane system. Moreover, we determined that cycloheptenone synthesis involves skeletal rearrangement catalyzed by the α‐ketoglutarate‐dependent dioxygenase BrvJ. BrvJ is highly homologous to SetK, which engages in the biosynthesis of another fungal metabolite, setosusin, and accepts the same substrate as BrvJ but performs only simple hydroxylation. Finally, we identified the key amino acid residues critical for product selectivity of BrvJ and SetK, providing insight into how the biosynthesis pathways of 1 and setosusin diverge and how fungi diversify natural products.