[关键词]
[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。
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[Abstract]
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 dadX gene (encoding L-alanine racemase) on the chromosome of strain B0016-050 to generate the thermoregulated L-alanine fermentation strain B0016-060BC. The fermentation by strain B0016-060BC 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 B0016-060BC 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.
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[基金项目]
国家自然科学基金项目(31300087);江苏省自然科学基金项目(BK20130131)