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

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pMF41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/pMF41-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。

Abstract:

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 pMF41 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/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M. neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-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.