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

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 (BjFMO) 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, BjFMOM9. Under optimal conditions, BjFMOM9 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, BjFMOM9 consistently outperformed the wild type, achieving >80% conversion within 10 h and >90% ee for the (S)-product.