Heterologous Expression of 20α-Hydroxysteroid Dehydrogenase in Escherichia coli for the Production of 20α-Hydroxyprogesterone

20α-Hydroxyprogesterone (20α-DHP) is an endogenous progestogenic metabolite of progesterone, biosynthesized primarily through the catalytic action of 20α-hydroxysteroid dehydrogenase (20α-HSD). To screen for highly active 20α-HSDs, five enzymes derived from phylogenetically diverse species—human, orangutan, mouse, bovine, and porcine—were systematically evaluated using multiple sequence alignment, molecular docking, and molecular dynamics simulations. The corresponding genes were subsequently expressed heterologously in E. coli BL21(DE3), and activity screening identified the human-derived 20α-HSD as the superior candidate. Enzymatic characterization revealed maximal activity at pH 8.0 and 35°C, negligible sensitivity to metal ions, and high substrate affinity and catalytic efficiency. Expression conditions were further optimized, with 0.2   mM IPTG at 20°C for 20   h established as the optimal induction parameters. Systematic optimization of reaction parameters—including organic solvent types and optimal concentration of the organic solvent, catalytic time, NADPH concentration, and recombinase concentration —achieved a progesterone conversion rate of 66.75%. To minimize cofactor consumption and enhance process economics, a glucose dehydrogenase-mediated NADPH in situ regeneration system was constructed, sustaining a conversion rate of 64.62%. Scale-up to a 100   mL preparative reaction yielded a conversion rate of 60.83%. Following column chromatography purification, 20α-DHP was isolated in 45.36% yield with 95% purity, as confirmed by HPLC and ¹H NMR analyses. In summary, this study achieved the successful heterologous expression of 20α-HSD in E. coli and established an efficient, green, and highly selective biosynthetic route for 20α-DHP, providing a viable technical foundation for its potential industrial-scale production.