Discovery and Engineering of a Flavin‐Containing Monooxygenase From Bradyrhizobium sp. 200 for the Asymmetric Synthesis of an Indoxacarb Intermediate

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Indoxacarb is an oxadiazine insecticide bearing a single stereogenic center, of which only the ( S )‐enantiomer exhibits insecticidal activity. Therefore, the synthesis of the key intermediate ( S )‐5‐chloro‐2‐methoxycarbonyl‐2‐hydroxy‐1‐indanone ( 2 ) is crucial for the enantioselective total synthesis of Indoxacarb. In this study, a flavin‐containing monooxygenase from Bradyrhizobium sp. 200 ( Bj FMO) was employed for the asymmetric synthesis of this chiral intermediate, offering a biocatalytic approach as a potential complement to conventional chemical routes. Based on molecular docking and sequence alignment, several amino acid residues located in the FAD and NADPH‐binding domains were selected for site‐saturation mutagenesis. In combination with rational design, additional positions potentially affecting catalytic activity and stability were subjected to site‐directed mutagenesis. Beneficial single mutations were iteratively combined to generate the final variant, Bj FMO M9 . Under optimal conditions, Bj FMO M9 displayed a twofold increase in specific activity compared with the wild type. Its half‐life at 40 °C was extended from 1.3 to 3.0 h, and its tolerance toward isopropanol, ethanol, and acetone was markedly enhanced. In a preparative synthesis, Bj FMO M9 consistently outperformed the wild type, achieving >80% conversion within 10 h and >90% ee for the ( S )‐product.