Molecular modification and application of the key enzyme CYP109E1-H in 25-hydroxyvitamin D3 biosynthesis

Abstract

25-Hydroxyvitamin D₃ (25-OH-VD₃) is one of the primary active forms of vitamin D₃ (VD₃) in the human body and has significant medicinal value. The traditional organic synthesis method of 25-OH-VD₃ is very complex and the yield is very low. Biocatalysis can simplify the synthesis steps, with relatively mild reaction conditions and easy access to raw materials. Therefore, the production of 25-OH-VD₃ through microorganisms has been considered a promising alternative to chemical synthesis. However, the recombinant expression system demonstrated poor catalytic activity and low substrate conversion efficiency, hindering the further application of 25-OH-VD₃ biological synthesis. The purpose of this study is to improve the efficiency of biotransformation and increase the production of 25-OH-VD₃ by modifying VD₃ hydroxylase. Initially, the positive mutant CYP109E1^I85F-H was obtained through saturation mutation. The concentration of 25-OH-VD₃ was increased to 1.57 mg/L, which was 1.9 times that of the wild-type (WT) strain. Then, the electron transfer engineered strain WB600-pMA5-CYP109E1^I85F-H-Fdx-FdR was constructed to further improve the efficiency of electron transfer, with a 14.5 % increase in product concentration. Finally, 14.53 mg/L of 25-OH-VD₃ was achieved in optimized SR medium supplemented with dimethyl sulfoxide (DMSO) as co-solvent. The combination of semi-rational molecular modification strategy and optimization of the catalytic system increased the production of 25-OH-VD₃ by 16.9 times compared with the WT strain, providing guidance for CYP109E1-H microbial synthesis of 25-OH-VD₃.