Biosynthesis Of Hydroxylated Steroids

Many natural and semi-synthetic steroids play an important role in the pharmaceutical and food industries.It is difficult to conduct further research because of the rare natural content,extraction cumbersome,and the difficulty to conduct chemical synthesis of hydroxylated steroids which worked as essential drug intermediates.Finding a new way to solve sources of natural product is imminent,and we hope to solve these problems through a biocatalytic approach.Cytochrome P450 oxidase is a typical representative of steroid hydroxylases,especially P450 BM3 derived from Bacillus megaterium.The study of P450s is beneficial to obtain hydroxylated steroid intermediates by using synthetic biology means in economical and environmentally friendly way.In this paper,the functional identification and enzymatic properties of P450 BM3 mutants were discussed,and the cloning and expression of P450 genes derived from Ornithogalum saundersiae were studied.The main results are as follows:1.Probing steroidal substrate specificity of cytochrome P450 BM3 variantsIn the present investigation,a total of 13 steroids were used as substrates to probe the hydroxylation capacity of three representative BM3 mutants M01A82W、M01A82WS72I and M11A82W.Enzymatic catalytic results showed that the three BM3 proteins were indeed able to metabolize 3-keto-△4-steroids to monohydroxylated metabolites.On the contrary,the three BM3 mutants had no oxidative activity on 3-hydro-△5-steroids.These results suggest a substrate preference of BM3 mutants towards 3-keto-△4-steroids.2.Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3Here,BM3 mutant 139-3 and its mutants were used as the biocatalyst to screen 13 steroids.Results revealed 139-3 was able to specifically hydroxylate androstenedione at 1α-position,namely 1α-OH-androstenedione.To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used,an optimization of catalyzing conditions was performed.Accordingly,the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 ℃,under pH 7.0 for 4 h,with 37%transformation rate.Moreover,four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope.Surprisingly,these mutants,sharing a common mutated site R379S,lost their activities towards androstenedione.These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3.3.Cloning and expression of P450 genes from O.saundersiaeTotal RNA was extracted from sterile bulb tissue of O.saundersiae and had been used as a template for reverse transcription to obtain the cDNAs.Three Cytochrome P450 oxidase genes(Os5155、Os813 and Os9107)were cloned using nest PCR.And then,according to the method of In-Fusion,primers were designed for expression vector construction of target genes.Firstly,the eukaryotic expression vector was constructed.Then,the prokaryotic expression vector of truncated P450 gene was constructed according to the experimental requirement.Soluble prokaryotic expression of t27pET28a/5155 and t29pET28a/9107 were obtained by co-expression with molecular chaperone dnaK-dnaJ-grpE,which lays the foundation for the functional identification of P450 genes.