Molecular Modification Of Steroids 17 α Hydroxylase

17α-Hydroxyprogesterone is an important intermediate compound that can synthesize progesterone and corticosteroid,and has broad application prospects in pharmaceutical synthesis.Traditional chemical synthesis methods have prominent problems such as cumbersome synthetic routes,great potential environmental security implications and low economic benefits.Nowadays,steroid biotransformation has become a hotspot because of its unique advantages.However,how to improve the activity and specificity of the key enzyme CYP17 hydroxylase during biotransformation is a key problem that needs to be solved urgently.In this study,the C17α-hydroxylase gene from human(hCYP17A1)was cloned and expressed on the Saccharomyces cerevisiae expression platform,and the17α-hydroxyprogesterone product catalyzed by constructed hCYP17A1 was identified.72 recombinant mutants of S.cerevisiae were obtained by saturated mutation and site-directed mutation.The effects of amino acid mutation on the activity of C17α-hydroxylase were analyzed.A mutant hCYP17A1 A105 L with high specificity and activity was obtained by saturated mutation of A105.Compared with the wild type,the yield of17α-hydroxyprogesterone was increased by 1.84 folds and the yield of16α-hydroxyprogesterone was decreased by 67% of WT.The substrate conversion of mutant A105L-W121 S and A105L-D216 H were increased,and the yield of17α-hydroxyprogesterone was close to wild type,but the yield of 16α-hydroxyprogesterone in mutant A105L-D216 H was decreased.L232 W mutant did not produce17α-hydroxyprogesterone,but when combined with A105 L mutant,17α-hydroxyprogesterone was produced.Compared with S.cerevisiae recombinant mutants,the 17α-hydroxyprogesterone production of the recombinant Pichia pastoris pPIC3.5K A105L-D216 H was 2.46 folds higher than that of S.cerevisiae recombinant mutant.Furthermore,less by-product was obtained in this system.According to the crystal structure and substrate transformation results of hydroxylase hCYP17A1,A105 is located in the active center and has certain flexibility to the substrate.A105 L can reduce the substrate movement in the active site by forming a certain steric hinderance.W121 can be used as an electron donor.After mutation to S,the efficiency of electron acquisition by heme is improved and enzyme catalysis is promoted.The17α-hydroxyl activity of recombinant S.cerevisiae D216 H mutant was higher than hCYP17A1 WT,because the change of amino acid charge affected the electrostatic interaction near the site,making it easier for the substrate to enter the active catalytic binding site.L232 is located in the substrate entry channel,and L232 W mutant is enlarged due to addition of benzene ring in the side chain,which increases the steric hindrance for the substrate to enter the catalytic center of the enzyme molecule,resulting in loss of activity.The combination of L232 W and A105 L mutant increases its 17α-hydroxylation activity,highlighting the importance of A105 L mutant in hCYP17A1.