Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D₃ C-25 hydroxylation capabilities

Calcidiol (25(OH)VD₃) and calcitriol (1α,25(OH)₂VD₃) are active vitamin D₃ with high medicinal value, which can maintain calcium and phosphorus balance and treat vitamin D deficiency. Microbial synthesis is an important method to produce high-value-added compounds. It can produce active vitamin D₃ through the hydroxylation reaction of P450, which can reduce the traditional chemical synthesis steps, and greatly improve the production efficiency and economic benefits. In this work, Bacillus megaterium H-1 was screened for its ability to produce 25(OH)VD₃ and 1α,25(OH)₂VD₃ from vitamin D₃. A new highly inducible vitamin D₃ hydroxylase CYP109E1-H was identified from B. megaterium H-1 through searching for transcripts with cytochrome P450 structural domains, combining the transcriptome sequencing with functional expression in Bacillus subtilis WB600. Biotransformation in recombinant B. subtilis confirmed that CYP109E1-H has C-25 hydroxylase activity towards vitamin D₃. CYP109E1-H is a natural mutant of CYP109E1 with greater stereoselectivity and it is a new vitamin D₃ mono-hydroxylase. The cloning and characterization of the CYP109E1-H gene provide useful information on the structural basis for improving the regional and stereoselectivity of the CYP109E gene.