Biocatalytic Studies On 17α Hydroxylation Of Progesterone

17α-hydroxyprogesterone(17OHP4)is an important intermediate of steroid hormone drugs.Its traditional chemical synthesis method is expensive and complicated,and biosynthesis is more green and environmentally friendly.biosynthesis has gradually become a research hotspot.The biosynthesis of 17OHP4 mainly depends on 17α-hydroxylase(CYP17),which belongs to cytochrome P450 monooxygenase(cytochrome P450 enzymes,CYP450).In order to obtain high yield of 17OHP4,it is an urgent problem to improve the activity and specificity of CYP17 on the hydroxylation of progesterone at position 17.In this experiment,17α-hydroxylase was screened,a recombinant strain with high hydroxylation activity was constructed,and the effect of cytochrome oxidoreductase(CPR)adaptation on17α-hydroxylase was studied to achieve the purpose of improving the yield of 17OHP4.In this experiment,17α-hydroxylase genes from different sources were first expressed in Saccharomyces cerevisiae strains,and CYP17 recombinant strains with high hydroxylase specificity were obtained according to the analysis of their hydroxylation products.The results showed that among the six 17α hydroxylases screened,the human 17α hydroxylase exhibited significant 17α hydroxylation activity,and the yield of its conversion to 17OHP4 was 28.5%.Secondly,the h CYP17 gene and its mutant A105 L were cloned into different host cells.By analyzing the transformation products,it was found that h CYP17 can catalyze the conversion of progesterone to 17α-hydroxyprogesterone in S.cerevisiae and Pichia pastoris,but not expression in E.coli.Compared with the expression system of S.cerevisiae,the CYP17 and A105 L mutants have higher substrate conversion rates and fewer by-products in P.pastoris.In S.cerevisiae,the 17OHP4 yield of the A105 L mutant was increased by 6%,but did not show a significant increase in the yield in P.pastoris.In order to further improve the activity of hydroxylase,five different sources of CPR were selected to co-express with h CYP17 and A105 L,and the effects of different CPRs on the biocatalytic activity of human17α-hydroxylase were analyzed.The results showed that in recombinant S.cerevisiae,five CPR strains co-expressed with h CYP17 and mutant A105 L could increase the substrate conversion rate,but the production rate of the main product was less,and more by-products were generated;in P.pastoris,different All the sources of CPR can increase the catalytic activity of h CYP17,and at the same time increase the production rate of the main product17OHP4.Among them,h CPR has better compatibility with h CYP17,and the yield of17OHP4 is increased by 42%,respectively,the other four sources of CPR(Sc CPR,rat CPR,An CPR,Sg CPR)co-expression yields increased by 40%,21%,17.7%,and 16%.This study can improve the activity of 17α-hydroxylase by changing the oxidoreductase of key electron transfer,which has application significance for the heterologous expression and industrial production of steroid drugs.