Study On The Key Enzyme Ksdd In The Bioconversion Of Ad Into Add In Mycobacterium Neoaurum

Steroidal drugs is the second largest category drugs after antibiotic, it has extensive applications in our life. Microbial phytosterol degradation is accompanied by the formation of steroidal pharmaceutical intermediates such as androst-4-end-3,17-dione and androst-1,4-end-3,17-dione, which are potential precursors in the synthesis of Steroidal drugs. 3-ketosteroid-△1-dehydrogenase (KSDD) is the key enzyme in the process of microbial phytosterol degradation, and it has a major impact in the process of biosynthesis of AD(D). A strain of Mycobacterium neoaurum was preserved in our laboratory which can convert hytpsterol into AD(D), so we do many research on the character and function of KSDD on the basis of this microorganism.1.7 kb complete sequence of ksdD gene was obtained by PCR which encode KSDD enzyme, then ksdD gene was ligated into pET-28a expression vector and the pET-28a-kad/D plasmid was created in E. coli BL21. Heterologous expression of ksdD gene in BL21/pET-28a-ksdD showed that the KSDD enzyme was expressed in the form of inactive inclusion bodies. KSDD enzyme still in the form of inclusion bodies by reducing the expression level of KSDD.To validate the function of gene ksdD in M. neoaurum JC-12, the kanamycin-resistant gene (Km) cassette which was obtained from plasmid pET-28a by PCR was inserted into the middle of ksdD gene. Nonreplicative integration vector pMD18-T-ksdD::Km was constructed to knock out ksdD gene. Electroporated the competent cell of M. neoaurum JC-12, after phenotypic and genotypic verification, targeted disruption of the ksdD gene in M. neoaurum JC-12 was confirmed. Growth curve shows that deletion of ksdD gene has no effect on the growth of recombinant strain, KSDD activity of recombinant strain is 15.6 mU/mg, it shows a decrease of 85% with respect to initial strain, the yield of AD of recombinant strain is 0.336 g/L, it shows a nearly one fold increase with respect to initial strain.To over expression ksdD gene in M. neoaurum JC-12, ksdD gene placed in the the downstream of Pkan promoter which can start the expression of Km gene and 16S rDNA conservative sequence was used as integration region, so the plasmid pETPkan-ksdD-16SrDNA was constructed. Electroporated the competent cell of M. neoaurum JC-12, after phenotypic and genotypic verification, the successful integration of Pkan-ksdD cassette into the genome of M. neoaurum JC-12 was confirmed. Growth curve shows that overexpression of ksdD gene has no effect on the growth of recombinant strain, KSDD activity of recombinant strain is 157.3 mU/mg, it shows an increase of 42.4% with respect to initial strain, the yield of ADD of recombinant strain is 1.246 g/L, it shows a decrease of 24.8% with respect to initial strain. These results suggest that KSDD is a key enzyme in the bioconversion of AD into ADD, and it lay the foundation for further research.