Cooperative Redox Reactions Encoded by Two Gene Clusters Enable Intermolecular Cycloaddition Cascade for the Formation of Meroaspochalasins

Abstract Meroaspochalasins (mAPOs) are a group of intricate heteromers comprising two distinct subunits, dienophile aspochalasin, and diene isobenzofuran, of which the biosynthetic mechanism is of great interest yet unrevealed. In this study, two independent biosynthetic gene clusters (BGCs), flas and epi , being responsible for the biosynthesis of aspochalasin B ( 7 ) and pre‐diene hemiacetal 21 (or 26 ), respectively, were identified in the filamentous fungus Aspergillus flavipes . In vivo and in vitro studies proved that a flavin adenine dinucleotide (FAD)‐dependent oxidase FlasF in the flas cluster catalyzes the crucial oxidation to generate diverse aspochalasin monomers, particularly the dienophile 7 . Interactive reduction catalyzed by the short‐chain alcohol dehydrogenase/reductase (SDR) FlasG and endogenous NADPH further increases the complexity of this anabolic network. The cytochrome P450 enzyme EpiC and SDR enzyme EpiD in the epi cluster collaboratively catalyze the formation of pre‐diene 21 (or 26 ), which can spontaneously dehydrate to yield a diene, leading to the nonenzymatic cascade of [4 π + 2 π ] Diels–Alder and formal [5 π + 2π] cycloaddition reaction to generate mAPO dimers and trimer progressively. Moreover, the FAD‐dependent oxidase EpiG catalyzes the hydroxylation at the C3 position of the diene as a critical step in the formation of mAPO trimers.