Enhancing the production of physiologically active vitamin D3 by engineering the hydroxylase CYP105A1 and the electron transport chain

In this study, the conversion of vitamin D 3 (VD 3 ) to its two active forms 25(OH)VD 3 and 1α, 25(OH) 2 VD 3 was carried out by engineering the hydroxylase CYP105A1 and its redox partners Fdx and Fdr. CYP105A1 and Fdx–Fdr were respectively expressed in E. coli BL21(DE3) and purified. The electron transport chain Fdx–Fdr had higher selectivity for the coenzyme NADH than NADPH. HPLC analysis showed that CYP105A1 could hydroxylate the C25 and C1α sites of VD 3 and convert VD 3 to its active forms. Finally, a one-bacterium-multi-enzyme system was constructed and used in whole-cell catalytic experiments. The results indicated that 2.491 mg/L of 25(OH)VD 3 and 0.698 mg/L of 1α, 25(OH) 2 VD 3 were successfully produced under the condition of 1.0% co-solvent DMSO, 1 mM coenzyme NADH and 35 g/L biocatalyst loading. This study contributes to a basis for the industrial production of active VD 3 in future.