Unprecedented [5.5.5.6]Dioxafenestrane Ring Construction in Fungal Insecticidal Sesquiterpene Biosynthesis

Abstract Fenestranes, a specific class of natural products, contain four fused rings that share a central quaternary carbon atom. The fungal natural product penifulvin A ( 1 ) is a potent insecticidal sesquiterpene that features the [5.5.5.6]dioxafenestrane ring. Although the chemical synthesis of 1 has been achieved recently, the enzymes catalysing the cyclization and oxidation of FPP to 1 remain unknown. In this work, we identified a concise pathway that uses only three enzymes to produce 1 . A new sesquiterpene cyclase (PeniA) generates the angular triquinane scaffold silphinene ( 6 ). A cytochrome P450 (PeniB) and a flavin‐dependent monooxygenase (PeniC) catalyse a series of oxidation reactions to transform 6 into 1 , including oxidation of the C15 methyl group to a carboxylate moiety, oxidative coupling of the C15 carboxylate and the C1‐C2 olefin to form a γ‐lactone, and Baeyer–Villiger oxidation to form a δ‐lactone. Our results demonstrate the highly concise and efficient ways in which fungal biosynthetic pathways can generate complex sesquiterpene scaffolds.