Total Biosynthesis of Melleolides from Basidiomycota Fungi: Mechanistic Analysis of the Multi‐Functional GMC Oxidase Mld7

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock‐in method to elucidate the biosynthetic pathway leading to 1a‐hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation on the D6‐protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short‐chain dehydrogenase/reductase family proteins. The highlight on the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation triggered double bond shift accompanying dehydration and acyl group assisted substitution with two different nucleophiles at the C6 position to afford the D7‐protoilludene derivatives such as melleolide and armillarivin. The complex reaction mechanism was proposed by density functional theory (DFT) calculations. Of particularly importance is that product distribution is regulated by the interaction with cell membrane. This proposed biosynthetic pathway provides an opportunity to understand the structural diversification mechanisms of melleolides and protoilludane sesquiterpenes.