代谢工程大肠杆菌高效利用甘油合成丁二酸  

High-efficiency production of succinic acid from glycerol by metabolically engineered Escherichia coli

作　　者：李卓亚 王杰 田康明 董自星[1,2] 金鹏 刘晓光 王正祥 

机构地区：[1]天津科技大学生物工程学院与工业发酵微生物教育部重点实验室,天津300457 [2]天津科技大学化工与材料学院生物化工系,天津300457

出　　处：《食品工业科技》2018年第5期94-100,共7页Science and Technology of Food Industry

摘　　要：为了构建高产丁二酸的重组大肠杆菌,以删除了乙酸激酶和磷酸乙酰转移酶基因(ackA-pta)、乳酸脱氢酶基因(ldhA)和丙酮酸甲酸裂解酶基因(pflB)的大肠杆菌CICIM B0013-025为出发菌株,将其磷酸烯醇式丙酮酸羧化酶(ppc)基因的启动子替换为温度诱导的λ噬菌体启动子P_L-P_R,获得温度调控型的丁二酸合成菌株B0013-026。继而通过发酵条件优化,建立了两阶段发酵法:菌株的生长温度和诱导温度分别为37和42℃,以甘油为碳源并添加入5 g/L蛋白胨,发酵产酸阶段在微供氧(100 r/min)条件下进行。在5 L发酵罐中采用最优条件进行发酵,丁二酸的产量、生产强度和甘油转化率分别为62.5 g/L、1.04 g/(L·h)和64.2%,而且发酵液中仅有少量的α-酮戊二酸(3.0 g/L)和乙酸(1.8 g/L)等副产物积累,实现了以甘油为唯一碳源高效合成丁二酸,为其工业化生产提供了重要参考。To construct a high-yield succinate producing strain, the recombinant strain Escherichia coli CICIM B0013-025 lacking genes ackA-pta, ldhA and pflB served as the starting strain.The promoter of phosphoenolpyruvate carboxylase in this bacterium was then replaced by the temperature- inducible promoter PL and PR of lambda phage, generating the thermoregulatmy succinic acid-producing strain B0013-026.After optimization, a dual-phase fermentation process was established as follows：the temperature used for cell growth and induction was 37 ℃ and 42 ℃ ,respectively;suecinic acid was produced under micro-aerobic condition,with glycerol and 5 g/L of tryptone as the sole carbon source and organic nitrogen, respectively.After cultured in a 5 L fermentor under optimal conditions,the succinate titer,overall productivity and conversion rate of glycerol reached 62.5 g/L, 1.04 g/（L·h） and 64.2% , with only small amounts of α-ketoglutaric acid （3.0 g/L） and acetate（ 1.8 g/L）accumulated.Therefore, using glycerol as the sole carbon source, we achieved the efficient production of succinic acid, providing an important reference for its industrial production.

关 键 词：丁二酸 大肠杆菌 温敏型启动子 磷酸烯醇式丙酮酸羧化酶 两阶段发酵法 

分 类 号：TS201.1[轻工技术与工程—食品科学]