新金色分枝杆菌ksd D基因的敲除及其对菌株AD(D)合成的影响  

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

作　　者：韩冷 田灵芝 马正 徐美娟[1] 夏海锋[1] 饶志明[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室江南大学生物工程学院,江苏无锡214122

出　　处：《工业微生物》2011年第4期37-42,共6页Industrial Microbiology

基　　金：国家"863计划"(No.2006AA020103;2007AA02Z207);国家"973项目"(2007CB707804);国家自然科学基金(No.20676053;30970056);霍英东青年基金(No.121020)

摘　　要：新金色分枝杆菌（Mycobacteriumneoaurum）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-株能将甾醇转化为AD（D）的菌株，经鉴定为M．neoaurum，本研究以该菌株为出发菌株，首先扩增出长度约1．7kb编码KSDD的ksdD基因全序列，在基因内部插入-段来源于质粒pET28a的卡那霉素抗性基因（Km），将得到的基因元件ksdD：：Km电转化M．neoaurum，通过PCR验证获得稳定遗传的ksdD基因缺失重组转化子。研究结果表明，与原始菌株相比，缺失株KSDD酶活下降了85％，AD产量相比提高了1倍，ADD产量下降了35．2％。该研究证实了ksdD基因编码的3-甾酮-△1-脱氢酶（KSDD）是AD转化ADD的关键酶，为进-步研究KSDD酶学性质及阐明AD合成ADD的代谢机理奠定基础。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.

关 键 词：新金色分枝杆菌 3-甾酮-△1-脱氢酶 ksdD 

分 类 号：Q78[生物学—分子生物学] TQ920.1[轻工技术与工程—发酵工程]