新金色分枝杆菌ksdD基因的敲除及其对菌株AD(D)合成的影响

新金色分枝杆菌(Mycobacterium neoaurum)能将植物甾醇转化为药物中间体4-烯-雄甾-3,17-二酮(AD)和1,4-二烯-雄甾-3,17-二酮(ADD),其中3-甾酮-△1-脱氢酶(KSDD)是AD转化为ADD的关键酶.本实验室在筛菌过程中筛选到一株能将甾醇转化为AD(D)的菌株,经鉴定为M.neo...

Knockout of ksdD gene in Mycobacterium neoaurum and its effects on synthesis of AD(D)

Mycobacterium neoaurum can convert phytosterol into pharmaceutical intermediates such as androst-4-end-3, 17-dione（AD） and androst-1,4-end -3, 17-dione （ADD）. 3-ketosteroid-△1-dehydrogenase（KSDD） is the key enzyme in the process of AD converted into ADD. M. neoaurum used as the initial strain, 1.7kb complete sequenceof ksdD was obtained by PCR for encoding KSDD enzyme. After the kanamycin-resistant gene （Kin） cassette from plasmid pET28a was inserted into the center of ksdD gene, the ksdD. ：Kin cassette was then electroporated into the competent cell of M. neoaurum. PCR was performed to confirm whether the Km gene was integrated into the ksdD gene of these clones and the recombinant strains of ksdD- disrupted were screened out. Compared to initial strain, the KSDD enzyme activity of the ksdD-disrupted strain decreased 85 percent, the yield of AD increased one fold and the yield of ADD decreased 35.2 %. The results confirm that the KSDD enzyme encoded by ksdD gene is the key enzyme in the process of AD converted into ADD, laid a foundation of further study on characterization of KSDD enzyme and the metabolic mechanism of AD converted into ADD.