表达3-甾酮-△^(1)-脱氢酶降解植物甾醇合成雄甾-1,4-二烯-3,17-二酮  

Overexpressing 3-ketosteroid-△^(1)-dehydrogenase for degrading phytosterols into androst-1,4-diene-3,17-dione

作　　者：张乐乐[1] 张显[1] 邵明龙[1] 陈榕榕 饶志明[1] 李会[2] 许正宏[2] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学药学院,江苏无锡214122

出　　处：《生物工程学报》2015年第11期1589-1600,共12页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2012CB725202);国家高技术研究发展计划(863计划)(No.2011AA02A211);国家自然科学基金(No.21276110);中央高校基本科研业务费专项资金(No.JUSRP51306A);江苏高校优势学科建设工程项目资助~~

摘　　要：构建分枝杆菌表达载体pMTac并在分枝杆菌Mycobacterium neoaurum JC-12中加强表达甾醇降解过程中的关键酶3-甾酮-△1-脱氢酶(KSDD)以提高雄甾-1,4-二烯-3,17-二铜(ADD)的产量。将p MF41的启动子pACE替换成tac启动子构建载体pMTac,在分枝杆菌中分别表达报告基因绿色荧光蛋白(GFP)和关键酶KSDD,通过GFP亮度和KSDD酶活验证tac启动子在M.neoaurum JC-12中的效果,并发酵验证加强表达KSDD对产物ADD的影响。荧光显微照片表明两个载体均能在M.neoaurum JC-12表达GFP,但tac启动子的效果比pACE强。酶活测定结果为重组菌M.neoaurum JC-12/pMTac-ksdd破碎细胞上清液中KSDD酶活比原始菌提高了6.53倍,比M.neoaurum JC-12/pMF41-ksdd提高了4.36倍。摇瓶发酵显示重组菌M.neoaurum JC-12/pMTac-ksdd ADD的产量比原始菌提高了22.2%,由4.86 g/L提高到5.94 g/L,而AD的产量由0.92 g/L减少到0.17 g/L,降低了81.5%;与M.neoaurum JC-12/p MF41-ksdd比,ADD产量提高了12.7%,AD降低了71.2%。以20 g/L植物甾醇为底物,5 L发酵罐中重组菌M.neoaurum JC-12/pMTac-ksdd的ADD产量达到10.28 g/L。结果表明,构建的新型表达载体pMTac适用于在M.neoaurum JC-12中加强表达关键酶KSDD,而且在M.neoaurum JC-12中过量表达KSDD有助于ADD产量的提高,为目前报道的发酵法利用新金色分枝杆菌降解植物甾醇合成ADD的最高水平。We constructed plasmid pMTac to overexpress 3-ketosteroid-△^1-dehydrogenase（KSDD） in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione（ADD） production. To construct pMTac, pACE promoter on p MF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein（GFP） was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2 % compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD（4-androstene-3,17-dione） production of JC-12/p MTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M. neoaurum JC-12/p MF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/p MTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.

关 键 词：MYCOBACTERIUM neoaurum JC-12 3-甾酮-△^(1)-脱氢酶 tac启动子 雄甾-4-烯-3 17-二酮 雄甾-1 4-二烯-3 

分 类 号：TQ929[轻工技术与工程—发酵工程]