Structural and Mechanistic Basis of Dual Substrate Promiscuity in the Acyltransferase AntB Enables Late‐Stage Diversification of Natural Products

ABSTRACT Biocatalytic regioselective acylation is a powerful strategy for diversifying natural products and optimizing their pharmacological properties. Compared with plant acyltransferases (ATs), bacterial tailoring ATs remain poorly understood. AntB from the antimycin pathway was previously reported to possess acyl‐donor promiscuity toward limited acyl‐CoA thioesters, yet its acyl‐acceptor promiscuity, structural basis, and catalytic mechanism had not been elucidated. Here, we systematically characterize AntB and uncover its remarkable dual substrate promiscuity. The enzyme accepts eight structurally diverse natural product acceptors—including terpenoids, alkaloids, polyketides, and aromatics—and supports iterative acylation, while also processing 43 chemically distinct acyl donors, including 22 previously unreported. Structural analysis revealed the molecular basis of its dual substrate promiscuity and catalysis. Engineering afforded the S17A/M265L variant with a 4.45‐fold increase in activity toward SPN‐J acylation. To harness this catalytic flexibility, we developed a one‐pot biocatalytic platform by combining AntB with engineered acyl‐CoA synthetases UkaQ FAV and AliA 100 for in situ acyl‐CoA regeneration. Application of this system to the late‐stage modification of the agrochemical precursor deacyl‐ UK‐2A generated a panel of acylated analogues, including three novel derivatives with enhanced antifungal activity. This study advances understanding of bacterial tailoring ATs and establishes AntB as a versatile biocatalyst for late‐stage natural product diversification.