Fusing Engineered CYP109E1 and a New Reductase Domain for 25(OH)VD₃ Biosynthesis

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 CYP109E1^M2, 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 CYP109E1^M2. 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 <i>Escherichia coli</i> 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.