Unveiling Aziridine‐Containing Natural Products by Genomic and Spectroscopic Approaches

Aziridine‐containing natural products are prized for their potent bioactivities, yet their scarcity and poorly understood biosynthesis have limited systematic exploration. Here, we address this by integrating genome mining with a 1 H– 13 C coupled HSQC metabolomic approach that exploits the distinctive NMR signatures of aziridines, enabling their direct detection from complex extracts. This strategy unveiled the desertolides, the first macrolides incorporating a rare terminal 2‐methyl‐aziridine‐2‐carboxylate moiety. Genetic and isotopic studies identified a dedicated biosynthetic subcluster ( desA – desN ) that assembles and installs this unit from glutamate, and heterologous expression confirmed the self‐sufficiency of this subcluster. Direct MS evidence reveals the aziridine moiety covalently bound to the active‐site Cys113 of DesN, establishing this KAS III homolog as the first dedicated aziridine‐transferase and a promising tool for polyketide engineering. Bioinformatic analysis uncovered over 50 biosynthetic gene clusters, suggesting that this aziridine‐associated biosynthetic logic may be more widespread than currently appreciated. This work establishes a tractable platform for the targeted discovery and engineered biosynthesis of aziridine‐containing natural products, opening this underexplored pharmacophore to systematic interrogation.