Screen Of C11Α Hydroxylase Converting 17α-Hydroxy Progesterone To 11α,17α-Dihydroxy Progesterone And Analysis Of Key Amino Acid Sites

11α,17α-dihydroxy progesterone is an important intermediate in the production of steroid drugs,and the market demand of it is high.Currently,11α,17α-dihydroxy progesterone is obtained by biotransforming 17α-hydroxyprogesterone with Aspergillus ochraceus.However,the pigment accumulation of Aspergillus ochraceus at the later stage of transformation increased the cost of isolation and extraction of 11α,17α-dihydroxy progesterone.In this study,different sources of C11α hydroxylases were expressed in yeast cells and the most suitable C11α hydroxylase was screened out by substrate transformation experiment with the idea of synthetic biology.Then the key amino acid sites of 11αhydroxylase were analyzed by means of structure prediction,molecular docking and sitedirected mutation.Firstly,the conversion ability of 17α-hydroxy progesterone to 10 moulds commonly used in hydroxylation reaction was determined.Among them,Aspergillus ochraceus,Cunningpamycetes elegans,Absidia coerulea,Beauveria bassiana,Metarhizium anisopliae and Aspergillus niger can transforme 17α-hydroxyprogesterone.Among them,Aspergillus ochraceus had the strongest ability to transform 17α-hydroxyprogesterone.The maximum yield of 11α,17α-dihydroxy progesterone reached 78.55% when the transformation time was 60 h.Penicillium citreoviridin,Aspergillus flavus,Penicillium citrinum,and Penicillium raistrickii had no ability to transform 17α-hydroxyprogesterone.In this study,15 hydroxylases screened out by literature research and bioinformatics analysis expressing heterologously in Saccharomyces cerevisiae to analyze the conversion ability of 17α-hydroxyprogesterone.The results show that 17α-hydroxyprogesterone could be transformed by the recombinant strains of S.cerevisiae that heteroexpressed the CYP68J5(from the Aspergillus ochraceus),CYP68L8(from the Aspergillus nidulans),KZL87288.1(from the Colletotrichum incanum),XP＿018158286.1(from the Colletotrichum higginsianum IMI349063),KXH39116.1(from the Colletotrichum nymphaeae SA-01),CYP5311B1(from the Absidia coerulea),CYP106A2(from the Bacillus megacetobacter)and CYP509C12(from the Rhizopus oryzae).However,the recombinant strains of S.cerevisiae that heteroexpressed the CYP68L1(from the Aspergillus ochraceus),KAF5001838.1(from the Fusarium decemcellulare),KAF4828340.1(from the Colletotrichum tropicale),WP＿100532009.1(from the Bacillus mr C49),WP＿053347454.1(from the Peribacillus butanolivorans),KAG1470614.1(from the Rhizopus delemar),RCH90480.1(from the Rhizopus stolonifera)could not convert 17α-hydroxyprogesterone.Among them,CYP68J5(from the Aspergillus ochraceus)had the highest hydroxylation activity,converting 17α hydroxy progestin to 11α at 72 h,and the maximum product yield of 17α-dihydroxy progestin was85.28%.It is noteworthy that the hydroxylation function of three enzymes KZL87288.1,XP＿018159286.1 and KXH39116.1 was reported for the first time.Five hydroxylases(CYP68J5,KAF5001838.1,KZL87288.1,CYP106A2 and CYP509C12)with different expression activities were selected as research objects to explore the optimal host of hydroxylase expression.The results showed that the yeast system was suitable for the expression of hydroxylase,and Saccharomyces cerevisiae was better than Pichia pastoris,while Rhodococcus rhodochrous could not be used as the heterologous host of hydroxylase expression.The key amino acid sites of CYP68J5,the most active hydroxylase,were analyzed by means of structure prediction,molecular docking and sitedirected mutation.The results showed that D118,F216 and M488 near the substrate binding pocket were key amino acid sites of CYP68J5.The results provide new ideas and application examples for the production of 11α,17α-dihydroxy progesterone.