Redesigning CYP109E1 for Improving Catalytic Performance in 25-Hydroxyvitamin D₃ Synthesis Through Synergistic Enhancement of Electron Transfer and NADPH Regeneration

P450 enzymes are promising biocatalysts and play an important role in the field of drug synthesis due to their high catalytic activity and stereoselectivity. CYP109E1 from <i>Bacillus megaterium</i> was used to convert VD₃ for the production of 25(OH)VD₃. However, the industrial production was still limited due to the low catalytic performance of CYP109E1. To overcome this, we constructed an engineered strain containing a modified CYP109E1 coupled with an efficient electron transfer chain and NADPH regeneration system. First, Adx_4-108T69E-Fpr was identified as the most compatible redox partner for the enzyme based on in-silico analysis. Then, targeted mutations were introduced at the substrate channel of CYP109E1, resulting in higher production efficiency. Next, the production of 25(OH)VD₃ was increased by 13.1% after introducing a double Adx_4-108T69E expression cassette. Finally, an NADPH regeneration system was introduced by overexpressing <i>zwf</i>, which increased the yield of 25(OH)VD₃ 48.7%. These results demonstrate that recombinant <i>Escherichia coli</i> BL21 (DE3) coexpressing CYP109E1_R70A-ZWF and 2Adx_4-108T69Es-Fpr is an efficient whole-cell biocatalyst for the synthesis of 25(OH)VD₃, illustrating an attractive strategy for improving the catalytic efficiency of P450 enzymes.