Gene Mining Of A Novel Progesterone Hydroxylase And Its High-Efficiency Biotransformation System

Steroidal drugs are the second largest group of drugs after antibiotics,known as the"key to life"and"hormones of life".Progesterone and its derivatives are important steroid drugs.17α-hydroxyprogesterone（17-HP）is a class of derivatives that are hydroxylated at the C17position of progesterone and are widely used as anti-androgen drugs.The traditional synthesis of 17-HP has the problems of high pollution and high separation cost.Using progesterone as a substrate,the synthesis of 17-HP by microbial catalysis has the advantages of low pollution and mild.However,most of the strains used for the hydroxylation of progesterone are molds,which have problems such as long growth cycles,difficult molecular modification,and pathogenicity.Therefore,it is of great significance to screen a novel progesterone 17α-hydroxylase（CYP17A）and construct a safe and efficient heterologous expression system with short growth cycle,easy molecular modification.In this study,a novel enzyme was obtained through gene mining,and an efficient heterocatalytic system was constructed.The specific results are as follows:（1）By means of genome mining,the HS＿CYP17A1 from Homo sapiens was used as the probe.After searching in the NCBI database and removing the redundant sequences,the evolutionary tree was constructed using MEGA software,and 3 sequences were selected for codon optimization and gene synthesis.The enzyme BT＿CYP17A1 derived from Bovine taurus has the hydroxylation ability of progesterone C17 after allogeneic expression in Pichia pastoris.The 17-HP yield of P.pastoris GS115-BT＿CYP17A1 was 25.21±1.56 mg·L^-1,the androstenedione yield was 85.19±6.81 mg·L^-1,and the molar ratio of the 17-HP product was22.83%.The molar conversion efficiency was 36.8%(for 1 mmol·L^-1 progesterone).（2）By multiple sequence alignment between enzyme BT＿CYP17A1 and CYP17A1,including probe sequence HS＿CYP17A1,it was found that the amino acid residues leading to the loss of HS＿CYP17A1 lyase activity were conserved sites,and the corresponding sites in BT＿CYP17A1 were selected for site-specific mutation.The molar ratio of 17-HP in the product of BT＿CYP17A1^R347A increased to 96.39%,and the 17-HP yield reached 113.06±6.72mg·L^-1.Through homology modeling of BT＿CYP17A1 and molecular docking of enzyme and substrate progesterone,it was found that when the mutant BT＿CYP17A1^R347A interacts with substrate,the distance between arginine 239 and substrate progesterone C20 before and after mutation changes from 3.1（?）to 7.0（?）,and the original hydrogen bond force disappears after mutation.This may be the cause of the loss of lyase activity.（3）The electron transport chain of progesterone catalyzed system of recombinant P.pastoris GS115-BT＿CYP17A1^R347A was optimized and co-expressed by cytochrome P450oxidoreductase(CPR_RAT)from Rattus norvegicus.Glucose-6-phosphate dehydrogenase（ZWF_K）from Kluyveromyces lactis was introduced into the NADPH regeneration system,and the recombinant strain BT＿CYP17A1^R347A-CPR_RAT-ZWF_K was constructed.Results showed that the 17-HP yield of recombinant strains BT＿CYP17A1^R347A-CPR_RAT-ZWF_K reached195.86±9.68 mg·L^-1,increased by 72.29%than that of GS115-BT＿CYP17A1^R347A.（4）In order to further increase the yield of 17-HP in recombinant strain,a new steroid transporter was screened to improve the catalytic efficiency the of heterologous expression system.The results showed that the expression of steroid transporter TP_CH from Cochliobolus heterostrophus significantly improved the transport efficiency of progesterone and increased17-HP production by 22%.The TP_CH was introduced to GS115-BT＿CYP17A1^R347A-CPR_RAT-ZWF_K,the 17-HP titer of GS115-BT＿CYP17A1^R347A-CPR_RAT-ZWF_K-TP_CH reached234.82±10.13 mg·L^-1,The substrate conversion efficiency was 78.81%,which was the heterologous expression system with the highest yield of 17-HP reported in the literature.