Engineering of P450 _PL2 Monooxygenase for the Stereoselective Synthesis of ( <i>R</i> )-Styrene Oxides

Asymmetric epoxidation of alkenes catalyzed by cytochrome P450 monooxygenases represents an efficient route to enantiopure epoxides. This work describes a colorimetric high-throughput screening (HTS) assay developed for the directed evolution of P450_PL2 monooxygenase. Utilizing this HTS method coupled with iterative saturation mutagenesis, we identified the single-mutant P450_PL2-SM4 (T179A), which exhibits enhanced activity and enantioselectivity for the asymmetric epoxidation of styrene. Furthermore, we constructed the recombinant strain P450_PL2-SM4-GDH, harboring a dual-plasmid system (pETDuet-1) expressing both P450_PL2-SM4 and glucose dehydrogenase (GDH). This strain demonstrated significantly enhanced activity (2.73-fold increase in the yield) for the epoxidation of styrene (<b>1a</b>), which can be attributed to NADPH recycling. Under optimized conditions, the recombinant strain P450_PL2-SM4-GDH was employed for the asymmetric epoxidation of styrenes <b>1a</b>-<b>1k</b>, affording the corresponding (<i>R</i>)-epoxides in yields of up to 80% and enantiomeric excess (ee) values of up to 99%.