25-hydroxyvitamin D3 (25(OH)VD3) is an important daily nutritional supplement, and direct enzymatic C25 hydroxylation of vitamin D3 (VD3) to 25(OH)VD3 is a green and sustainable method. However, the low catalytic activity and electron transfer efficiency of redox partners of cytochrome P450 limited its production. Here, structure-guided semirational design of CYP109E1 led to the mutant CYP109E1M2, which showed a 2-fold increase in 25(OH)VD3 production compared to the wild-type. Furthermore, the novel reductase domain BmRD was first employed to construct a fusion protein with CYP109E1M2. Notably, truncating only two amino acids at the N-terminus of BmRD generated a fusion protein Chimera-2, resulting in a 38.5% increase in 25(OH)VD3 production. Under optimized conditions, the engineered strain Escherichia coli 03-2 produced 491.3 mg/L 25(OH)VD3 with a conversion of 49.0% and a space-time yield of 61.4 mg/(L·h). This work demonstrates the modification and optimization potential of P450 and lays a theoretical foundation for the industrialization of the 25(OH)VD3 biosynthesis.
Jiaying Ai,Ziyang Yin,Jikai Gao,Wenjing Wang,Fuping Lu,Hui-Min Qin,Shuhong Mao
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 Bacillus megaterium 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, Adx4–108T69E-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 Adx4–108T69E expression cassette. Finally, an NADPH regeneration system was introduced by overexpressing zwf, which increased the yield of 25(OH)VD3 48.7%. These results demonstrate that recombinant Escherichia coli BL21 (DE3) coexpressing CYP109E1_R70A-ZWF and 2Adx4–108T69Es-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.
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