Expression Of3-ketosteroid-Δ~1-dehydrogenase From Mycobacterium Neoaitrum In Bacillus Subtilis And Its Application

Steroid drugs are important in the pharmaceutical industry and have been widely used inclinical applications. The steroids,4-androstene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD), are the most important steroidal derivatives in the process of steroiddegradation by microbial strains. The enzyme3-ketosteroid-â–³~1-dehydrogenase (KSDD),involved in steroid metabolism, catalyzes the transformation of4-androstene-3,17-dione (AD)to androst-1,4-diene-3,17-dione (ADD) specifically. Previously, we isolated Mycobacteriumneoaurum JC-12that is capable of producing the sole product ADD from phytosterol in ourlaboratory. In this work, the study on KSDD from M. neoaurum JC-12and bioconversion ofAD to ADD were performed.(1) The ksdd gene was obtained from M. neoaurum JC-12by PCR and the result of genesequencing was analyzed. The forecast of conserved sequence and transmembrane domain ofKSDD were performed. The recombinant plasmid pET22b-ksdd was constructed. Then therecombinant plasmid was transformed into Escherichia coli BL21to construct therecombinant strain E. coli BL21/pET22b-ksdd. Induced by IPTG, the successfully expressedKSDD was analyzed by SDS-PAGE. The activities of the recombinant enzyme in E. coli were0.84U/mg. And the recombinant KSDD were purified by Ni-NAT. The recombinant E. colicatalyzed the biotransformation of AD to ADD in a transformtion rate of23.86%and showedabout2folds higher than M. neoaurum JC-12. The time required for transformation of AD toADD was about24h by the recombinant E. coli, much shorter than that of the wild-typestrain.(2) The recombinant plasmid pMA5-ksdd was constructed. Then the recombinantplasmid was transformed into Bacillus subtilis168to construct the recombinant strain B.subtilis168/pMA5-ksdd. The activities of the recombinant enzyme in B. subtilis168/pMA5-ksdd were1.75U/mg. When using the whole-cells as catalysts, the products wereanalyzed by tin-layer chromatography and high-performance liquid chromatography. Therecombinant B. subtilis168/pMA5-ksdd catalyzed the biotransformation of AD to ADD in atransformation rate of65.7%(ADD yield is0.65g/L) and showed about19folds higher thanM. neoaurum JC-12. The time required for transformation of AD to ADD was about10h bythe recombinant B. subtilis, much shorter than that of the wild-type strain and other reportedstrains. In shake flask scale, the optimization of medium composition, culture condition andsubstrate cosolvent of B. subtilis168/pMA5-ksdd were performed. Finally, ADD yield of B.subtilis168/pMA5-ksdd is1.80g/L.