利用重组大肠杆菌发酵甘油合成L-丙氨酸  

L-Alanine Production through Glycerol Fermentation by Recombinant Escherichia coli

作　　者：周丽[1] 邓璨[1] 崔文璟[1] 刘中美[1] 周哲敏[1] 

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

出　　处：《现代食品科技》2016年第6期163-169,共7页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31300087);江苏省自然科学基金项目(BK20130131)

摘　　要：本文探究以甘油为唯一碳源发酵合成L-丙氨酸的可行性。以删除了乙酸、甲酸、乙醇、琥珀酸、乳酸代谢产物合成途径的Escherichia coli B0016-050为出发菌株,用λpL启动子及其调控下的嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)来源的丙氨酸脱氢酶基因(ala D)替换B0016-050菌株染色体上丙氨酸消旋酶基因(dad X),获得温度控制型L-丙氨酸合成菌株B0016-060BC。菌株B0016-060BC以甘油为唯一碳源进行两阶段发酵(包括菌体生长阶段和L-丙氨酸合成阶段),表明在菌体生长至对数后期起始L-丙氨酸合成或者提高L-丙氨酸发酵阶段的通气量可提高L-丙氨酸合成水平。进一步经5 L发酵罐发酵,可合成63.64 g/L L-丙氨酸,整个发酵阶段体积生产强度达到1.91 g/L h、转化率达到62.89 g/100 g甘油,仅合成少量的乙酸(1.73 g/L)等副产物。实现了以甘油为唯一碳源高效合成L-丙氨酸,为工业应用提供了重要参考。The feasibility of L-alanine fermentation using glycerol as a sole carbon source was investigated. The recombinant strain Escherichia coli B0016-050 with deletions of acetate, formate, ethanol, succinate, and lactate synthesis routes served as the parent strain. The λ pL promoter and L-alanine dehydrogenase（derived from Geobacillus stearothermophilus） controlled by the λ pL promoter were used to replace the dad X gene（encoding L-alanine racemase） on the chromosome of strain B0016-050 to generate the thermoregulated L-alanine fermentation strain B0016-060 BC. The fermentation by strain B0016-060 BC was a two-phase process（consisting of a cell growth phase and an L-alanine synthesis phase） with glycerol as the sole carbon source, indicating that L-alanine production in strain B0016-060 BC could be improved by initiating the production in the late exponential growth phase or by increasing air supplementation in the fermentation stage. Fermentation in a 5-L bioreactor yielded 63.64 g/L L-alanine, with overall productivity and yield of 1.91 g/L h and 62.89 g/100 g glycerol, respectively, and only a small amount of acetate（1.73 g/L） was produced as a byproduct. Therefore, the efficient production of L-alanine using glycerol as the sole carbon source is possible, providing an important reference for industrial applications.

关 键 词：L-丙氨酸 甘油 大肠杆菌 发酵 代谢工程 基因开关 

分 类 号：TQ922[轻工技术与工程—发酵工程]