Abstract
In this study, the conversion of vitamin D3 (VD3) to its two active forms 25(OH)VD3 and 1α, 25(OH)2VD3 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 VD3 and convert VD3 to its active forms. Finally, a one-bacterium-multi-enzyme system was constructed and used in whole-cell catalytic experiments. The results indicated that 2.491mg/L of 25(OH)VD3 and 0.698mg/L of 1α, 25(OH)2VD3 were successfully produced under the condition of 1.0% co-solvent DMSO, 1mM coenzyme NADH and 35g/L biocatalyst loading. This study contributes to a basis for the industrial production of active VD3 in future.
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