大肠杆菌LG101生物合成L-丙氨酸特征及作为细胞增殖调控节点的研究  

Biosynthetic pathways of L-alanine and their role in the regulation of cell proliferation in Escherichia coli LG101

作　　者：韩梦圆 王萌 高明亮 张濛 牛丹丹 王正祥 HAN Mengyuan;WANG Meng;GAO Mingliang;ZHANG Meng;NIU Dandan;WANG Zhengxiang(College of Chemical Engineering and Materials Science,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Key Laboratory of Industrial Microbiology,Tianjin 300457,China)

机构地区：[1]天津科技大学化工与材料学院,天津300457 [2]天津市工业微生物重点实验室,天津300457

出　　处：《微生物学通报》2025年第3期980-991,共12页Microbiology China

基　　金：天津市杰出人才计划(JC20200309);国家重点研发计划政府间国际科技创新合作重点专项项目(2018YFE0100400)。

摘　　要：【背景】细胞增殖可控是实现发酵自动控制的重要策略之一,通过对关键营养物可控供给可方便实施细胞增殖调控。【目的】以L-乳酸单体生产菌株大肠杆菌(Escherichia coli)LG101为起始菌株,系统解析其L-丙氨酸生物合成的特征及作为细胞增殖调控节点的可行性。【方法】在全面分析菌株L-丙氨酸生物合成途径组成特征的基础上,采用基因重组技术敲除L-丙氨酸合成途径基因(avtA、alaA、alaC)获得系列突变株,通过摇瓶和发酵罐发酵检验菌体代谢与生理变化。【结果】大肠杆菌基因组中同时存在至少3条L-丙氨酸生物合成途径,其中2条合成途径以同样是乳酸单体合成前体的丙酮酸为前体物。对相关编码基因进行删除突变,获得了突变株LG101A(LG101 ΔavtA)、LG102(LG101 ΔalaA ΔalaC)和LG103(LG101 ΔalaA ΔalaC ΔavtA)。摇瓶培养下,突变株LG101A和LG102的细胞增殖特征与出发菌株无明显差异,突变株LG103则表现为L-丙氨酸营养缺陷型,并且其细胞增殖与L-丙氨酸补加量间呈现剂量依赖性正相关关系。进一步在5 L发酵罐评估菌株LG103的生理与代谢性能。菌株LG103可在补加L-丙氨酸后恢复细胞增殖,并且发酵结束时的发酵液中L-丙氨酸水平(0.12 g/L)显著低于出发菌株(0.92 g/L)。【结论】大肠杆菌LG101过量合成和积累L-丙氨酸,主要通过以谷氨酸和缬氨酸为氨基供体的还原反应进行生物合成,相应合成途径去除后的突变菌株,呈现L-丙氨酸营养缺陷型并呈现剂量依赖关系,可作为大肠杆菌发酵生产主要化合物如乳酸单体时高效控制细胞增殖的营养开关。[Background]Controllable cell proliferation is one of the important strategies for achieving automatic control of fermentation.It can be easily implemented by the controlled supply of key nutrients.[Objective]To analyze the characteristics of L-alanine biosynthesis in the L-lactic acid-producing strain Escherichia coli LG101 and explore the feasibility of taking L-alanine biosynthesis as a node to regulate cell proliferation.[Methods]We employed site-specific recombination to obtain mutants by deleting the corresponding genes(avtA,alaA,alaC)of the L-alanine biosynthesis pathway on the basis of comprehensively analyzing the L-alanine biosynthesis pathway.Shake-flask and bioreactor experiments were carried out to examine the physiological and metabolic differences between the original strain and the mutants.[Results]There were at least three L-alanine biosynthesis pathways in the genome of E.coli,two of which utilized pyruvate(also the precursor for the synthesis of lactic acid)as a precursor.The relevant coding genes were deleted,and mutants LG101A(LG101 ΔavtA),LG102(LG101 ΔalaA ΔalaC)and LG103(LG101 ΔalaA ΔalaC ΔavtA)were obtained.In shake-flask culture,the cell proliferation of the mutants LG101A and LG102 was not significantly different from that of the original strain.However,the mutant LG103 was L-alanine auxotrophic,and its proliferation presented a positive dose-dependent correlation with L-alanine supplementation.The physiological and metabolic properties of strain LG103 were further analyzed and evaluated in a 5 L fermenter.The strain LG103 restored cell proliferation after supplementation of L-alanine,and the accumulation of L-alanine(0.12 g/L)in the fermentation broth at the end of fermentation was significantly lower than that(0.92 g/L)of the original strain.[Conclusion]E.coli LG101 over-synthesizes and accumulates L-alanine through the reduction reaction with glutamate and valine as amino donors.The mutants with removal of the corresponding synthetic pathways became L-alanine auxotrophic and the

关 键 词：大肠杆菌 L-丙氨酸合成 细胞增殖控制 乳酸单体 

分 类 号：Q93[生物学—微生物学]