Study On The Biosynthesis Of 9?-hydroxy-4-and Rostene-3,17-dione

Steroidal drugs which have been extensively used in clinical application are second only to antibiotic drugs.At present,the sreroids were synthesized by chemical semi-synthsis and microbial methods.Microorganisms are often used in the preparation of precursors such as 4-androstene-3,17-dione(AD),1,4-androstene-3,17-dione(ADD)and 9a-hydroxy-4-androstene-3,17-dione(9a-OH-AD).9a-OH-AD can be used clinically as corticosteroid,anti-androgen,anti-estrogen or contraceptive drug with significant commercial value.Currently,the strains from Mycobacteria and Rhodococcus are extensively studied in the bio-synthesis of 9a-OH-AD.However,a variety of problems such as serious environmental pollution and low yield are still need to be resolved in these methods.Escherichia coli has a great advantage in terms of strain safety,cell growth rate,experimental operation and industrial production.The preparation of 9a-OH-AD by using an engineered E.coli strains has become the hotspot of current research on sreroidal synthesis.In this study,3-ketosteroid-9a-hydroxylase(KshA)and 3-ketosteroid-9a-hydroxylase reductase(KshB)were highly expressed in E.coli.Moreover,the coexpression of KshA and KshB and the fusion expression of KshA and P450 BM-3 were performed,followed by the co-expression of coenzyme regeneration system(Formate dehydrogenase:FDH).The enzyme activities of the above strains were measured by the yield rate of 9a-OH-AD which was transformed from substrate AD.1)The KshA enzyme from Mycobacteria and Rhodococcus was efficiently expressedin E.coli.The KshA enzyme from Rhodococcus has relatively higher efficience for transformation of AD to 9a-OH-AD.The reaction conditions were then optimized,and the yield of 9a-OH-AD reached 97.09%at the substrate concentration of 500 ?M.2)The KshB enzyme from Rhodococcus was efficiently expressed and purified using Ni-column affinity chromatography.Furthermore,we demonstrate that KshB is a coenzyme NADH-dependent reductase.3)An engineered E.coli co-expressing KshA,KshB and an engineered E.coli expressing KshA-P450 fusion protein were constructed.Moreover,the enzyme activity of the strain expressing the fusion protein KshA-P450 was 2.18 times higher than the strain expressing KshA alone.4)The gene fdh3m was generated from formate dehydrogenase gene(fdh)by site-directed mutagenesis.And the plasmid pETDuet-fdh3m was obtained,which was used for the construction of NADH and NADPH coenzyme regeneration system.The plasmids co-expressing kshA,kshB,fdh3m and plasmids co-expressing kshA-p450,fdh3m were subsequently constructed,and the protein expression was successfully carried out.The enzyme activity of 9a-hydroxylation of AD was also detected.