VD₃ hydroxylase (Vdh) is a key enzyme for the synthesis of active vitamin D₃.Two electron transfer chain proteins:ferredoxin (Fdx) and ferredoxin reductase (Fdr) are reqiured in the hydroxylation reaction of Vdh.Therefore,constructing an efficient catalytic system of VD₃ hydroxylase is the key for the industrial biosynthesis of active vitamin D₃.In this study,Pseudonocardia autotrophica CGMCC-5098 genomic DNA and Spinach genomic DNA were used as templates to amplify the fragments of the Vdh gene and Fdx-Fdr fusion gene by PCR.The recombinant plasmid pET28a-Vdh and pET28a-Fdx-Fdr was constructed,and then transformed into E. coli BL21 (DE3) to obtain recombinant bacteria containing Vdh gene and Fdx-Fdr fusion gene,respectively. Subsequently, the recombinant bacteria were induced by IPTG and freeze-dried,and then coupled with glucose dehydrogenase (GDH)-glucose coenzyme regeneration system to form a complete Vdh catalytic system.The optimal reaction conditions for this catalytic system are:Vdh (0.05 g),Fdx-Fdr (0.05 g),GDH (0.05 g),glucose (0.1 g),DNAP (50 μL,50 mM),VD₃ (100 μmol/L).Under the optimal reaction conditions,the system can efficiently catalyze the biosysthesis of VD₃ and its paricalcitol intermediates.After 12 h of reaction,the conversion rate of VD₃ is 40.10%,which is much higher than the 16.7% efficiency of P. autotrophica.The results of this study lay a foundation for the further efficient preparation of calcitriol drugs in the future.