Molecular Modification And Mechanism Analysis Of Steroid P450 Dihydroxylase CYP-cl3 For Catalytic Efficiency Enhancement

3β,7α,15α-trihydroxy-5-androstene-17-one（7α,15α-di OH-DHEA）is a dihydroxylated derivative of dehydroepiandrosterone（DHEA）,which can be used as an intermediate in the synthesis of oral contraceptive drospirenone.The microbial biotransformation of DHEA to7α,15α-di OH-DHEA has great research and application value due to its high efficiency and green characteristics.In the early work of our laboratory,the P450 CYP-cl3 with C7αand C15αdihydroxylation activities on DHEA was obtained from the filamentous fungus Colletotrichum lini ST-1 and expressed in Pichia pastoris GS115.The research of catalytic process showed that the dihydroxylation of DHEA catalyzed by CYP-cl3 was a two-step sequential reaction of C7αfollowed by C15α.However,due to the low enzymatic activity of CYP-cl3 in heterologous hosts P.pastoris GS115,the substrate conversion efficiency was not ideal.Besides,the second-step hydroxylation rate of the dihydroxylation reaction was relatively low,resulting in the accumulation of the intermediate 7α-OH-DHEA,which restricted the further synthesis of the target product 7α,15α-di OH-DHEA.In order to solve the above problems,we firstly adopted the directed evolution strategy to improve the activity of CYP-cl3,and then improved the efficiency of the second-step C15αhydroxylation reaction through semi-rational design.On this basis,the high-efficiency expression and substrate transformation of the dominant combination mutant were carried out in a 5 L fermenter,which effectively improved the ability of the recombinant strain to synthesize 7α,15α-di OH-DHEA.Combined with mutation data,the contribution of key residues to enzyme activity was further explored,and the molecular mechanism of CYP-cl3 catalytic activity was preliminarily analyzed.The main conclusions are as follows:（1）Directed evolution of steroid P450 dihydroxylase CYP-cl3 for catalytic activity enhancement.A chromogenic method for rapid identification of 7α,15α-di OH-DHEA,and 7α-OH-DHEA in biotransformation samples was established and applied to the screening of mutants with high enzymatic activity in the CYP-cl3 directed evolution library.Compared with traditional liquid-phase methods,the screening speed of the chromogenic method was increased by 600 times.A directed evolution mutation library of the CYP-cl3 gene was constructed by random mutagenesis and expressed in P.pastoris GS115 for the biotransformation of DHEA.Biotransformation samples were rapidly screened by chromogenic method and revalidated by high performance liquid chromatography（HPCL）to identify positive mutations.After three rounds of directed evolution,the total product molar yield of mutant（CYP-cl3-A83P/E264I/V281A）increased to 77.87%,which was 38%higher than the natural CYP-cl3.（2）Semi-rational design modification of steroid P450 dihydroxylase CYP-cl3 for the second-step catalytic efficiency enhancement.Based on the CYP-cl3/7α-OH-DHEA docking model,sixteen single mutants of CYP-Cl3 with increased affinity for the intermediate 7α-OH-DHEA were designed by computer-simulated saturation mutation method,and then potential sites（L308 and T315）affecting the efficiency of the second-step hydroxylation were discovered by whole-cell transformation.The product distribution of L308M and T315P mutants showed an increase in the efficiency of the second-step hydroxylation reaction,and the proportion of7α,15α-di OH-DHEA in the total product increased by 13.2%and 17.8%,respectively.The beneficial mutations were introduced into the best mutant obtained by directed evolution,the proportion of 7α,15α-di OH-DHEA in the total product of CYP-cl3-A83P/E264I/V281A/T315P mutant increased to 43.5%.The expression optimization and substrate transformation of the above combination mutant were further carried out in a 5 L fermentor.By optimizing the methanol flow rate,when the substrate feeding amount was 4 g·L^-1,the proportion of 7α,15α-di OH-DHEA in the total product was up to 99.9%.At the same time,the concentration of7α,15α-di OH-DHEA was up to 0.782 g·L^-1,which was 218.7%higher than that of the shake flask.（3）Preliminary analysis of the relationship between the structure and catalytic ability of steroid P450 dihydroxylase CYP-cl3.Based on the mutation data obtained by directed evolution,semi-rational design and the homology model of CYP-cl3,the effects of substrate anchor sites,active pocket sites,and distal sites on CYP-cl3-catalyzed DHEA dihydroxylation were studied.The results showed that for the catalytic activity of CYP-cl3,the absence of hydrogen bonds between sites 122,494 and the substrate DHEA resulted in partial or complete loss of catalytic activity.The A83P,E264I,and V281A residues obtained by directed evolution were located at the distal end of the active pocket,which might improve the binding of CYP-cl3 to cytochrome P450 reductase and thus showed increased activity.Sites 126,311,and 371 were not completely conserved,and the substitution of similar residues can maintain the catalytic activity of the enzyme to a certain extent,which was of great significance to the stability of CYP-cl3 function.For the efficiency of the second-step hydroxylation reaction,the change of polarity and larger steric hindrance caused by mutations at sites 227 and 312 might be the reasons for the weakened affinity of the enzyme to the intermediate 7α-OH-DHEA,which led to the reduction of the efficiency of this step.The rigid loop structure of the T315P mutant locally offset the I helix towards the active center,shrinking the substrate-binding pocket and making the intermediate7α-OH-DHEA bind more tightly to the active center,thereby increasing the efficiency of the second-step hydroxylation.In addition,most of the other site mutations in the active pocket had no positive effect on improving the second-step hydroxylation efficiency.It is speculated that the conserved nature of the substrate binding pocket limits the continuous reaction of CYP-Cl3to DHEA.