Activity-driven engineering of Baeyer-Villiger monooxygenase from Thermobifida fusca for the synthesis of methyl (2S)-5-chloro-2-hydroxy-1-oxo-2,3-dihydro-1H-indene-2-carboylate: a key precursor of (S)-indoxacarb.

The synthesis of key chiral intermediates via biocatalysis is of great importance in pharmaceutical manufacturing. In this study, we engineered a phenylacetone monooxygenase from Thermobifida fusca to enhance its catalytic activity for the synthesis of methyl (2S)-5-chloro-2-hydroxy-1-oxo-2,3-dihydro-1H-indene-2-carboxylate, a crucial precursor of the insecticide (S)-Indoxacarb. By employing a computational strategy integrating molecular dynamics (MD) simulations and machine learning (ML), we rationally designed and identified a quintuple variant, BVMO-4-H116N-M497E-L230N-V17I-Q526V (BVMO-4-M5). This variant exhibited a 4.1-fold increase in catalytic activity, which was attributed to optimized substrate binding and enhanced nucleophilic attack efficiency as revealed by structural analysis. In a preparative-scale 50L bioreactor, using 10.0g/L (dry cell weight) whole cells of BVMO-4-M5 as biocatalyst, 101.7g/L of the target chiral ester was produced from 100g/L prochiral substrate, with an enantiomeric excess (ee) of >98.2% and a space-time yield (STY) of 203.4g/(L·d). This work demonstrates the power of computational enzyme design in developing efficient biocatalysts for the industrial synthesis of high-value chiral chemicals.