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λ pL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（alaD）替换B0016-050菌株染色体上丙氨酸消旋酶基因（dadX），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 producing L-alanine via fermentation, using glycerol as the sole carbon source was investigated. The recombinant strain Escherichia coli B0016-050 lacking routes to metabolites like acetate, formate, ethanol, succinate, and lactate within its metabolic network served as the starting strain. The gene encoding L-alanine dehydrogenase, alaD, from Geobacillus stearothermophilus, which was controlled by the left promoter from phage λ (λ pL promoter), was used to replace the alanine racemase-encoding dadX gene in the genome of strain B0016-050 to generate the thermoregulatory L-alanine-producing strain B0016-060BC. Strain B0016-060BC carried out a two-phase fermentation (including a cell growth phase and an L-alanine-producing phase) with glycerol as the sole carbon source, and the results indicate that L-alanine production by strain B0016-060BC can be improved by initiating L-alanine production at the late exponential growth phase or by increasing the aeration rate during fermentation. After fermentation in a 5-L bioreactor, 63.64 g/L L-alanine was produced with an overall productivity and conversion rate of 1.91 g/L h and 62.89 g/100 g glycerol, respectively. Only small amounts of acetate (1.73 g/L) and other byproducts were synthesized. Therefore, efficient L-alanine production using glycerol as the sole carbon source was realized, providing an important reference for industrial application.

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