Molecular Modification Of Steroid C14α-hydroxylase And Construction Of Recombinant Engineered Yeast

Due to their unique physiological and pharmacological activities,steroid pharmaceuticals are widely used in clinical practice and are the second largest class of drugs after antibiotics.The structures of steroids are very complex,and their physiological and pharmacological activities depend on the substituents at specific sites of steroids.14α-Hydroxysteroids have anticancer activity,but it is difficult to selectively introduce a hydroxyl group at the C14 position of the steroid by chemical synthesis.Our previous work found that the filamentous fungus Curvularia lunata CICC40301 can convert progesterone to 14α-hydroxyprogesterone and 7α,14α-dihydroxyprogesterone and identified the C14α-hydroxylase CYP5103B5 involved in the hydroxylation reactions.However,the use of CICC40301 for 14α-hydroxyprogesterone production suffers from the presence of significant amounts of 7α,14α-dihydroxyprogesterone.In order to construct efficient steroid C14α-hydroxylation recombinant strains,this paper uses semi-rational method to carry out molecular modification of steroid C14α-hydroxylase CYP5103B5.According to the divergent evolution theory of enzymes,by comparing the homologous sequences of CYP5103B5 in the NCBI database,the non-homologous regions were designed as mutation regions,and series of CYP5103B5 mutants were constructed by site-directed mutagenesis and combinatorial mutagenesis.The mutant strains with significantly improved C14α-hydroxylation specificity were screened and identified by expressing the mutants in a heterologous host of Saccharomyces cerevisiae and evaluated by progesterone transformation experiments.The main research contents and results of this thesis are as follows:(1)Based on the sequence alignment of C14α-hydroxylase CYP5103B5 and highly homologous C11β-hydroxylase CYP5103B6,the site-directed mutation regions were selected,and 33CYP5103B5 mutants were constructed by PCR-based mutagenesis;(2)Mutants Y10(S129A/T130A/D131G/T132L/V133I/R134K/S135K),Y22(A291L)with significantly improved C14α-hydroxylation specificity were obtained by screening the progesterone transformation of recombinant yeast strains;(3)Analysis of individual amino acid mutations in the two regions showed that amino acid 131 of C14α-hydroxylase CYP5103B5 is a key residue affecting the specificity of C14α-hydroxylation,and mutant D131 G exhibits 93% C14α-hydroxylation specificity;(4)Time course experiments show that C14α-hydroxylase CYP5103B5 first undergoes hydroxylation at the 14α-position of progesterone,followed by hydroxylating at the 7α-position.The above studies show that using the divergent evolution theory of enzymes to design site-directed mutations by aligning homologous sequences is an effective way to identify key residues in the active site of CYP5103B5 and engineer C14α-hydroxylase mutants with high hydroxylation specificity.