利用同源重组的方法提高大肠杆菌W3110天冬氨酸的积累  

Accumulation of Aspartic Acid in Escherichia coli W3110 is Improved by Homologous Recombination

作　　者：马怀远[1] 黄非[1] 白林含[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064

出　　处：《中国生物工程杂志》2014年第6期61-67,共7页China Biotechnology

基　　金：国家自然科学基金(30970043);四川省科技厅支撑计划省院科技合作项目(2013JZ0009)资助项目

摘　　要：大肠杆菌中存在3种天冬氨酸激酶,分别为LysC,MetL,ThrA,使天冬氨酸磷酸化后分别进入Lys、Met、Thr的合成途径.因此大肠杆菌菌体中无法积累大量天冬氨酸.以大肠杆菌W3110为出发菌株,利用Red同源重组系统分别构建了LysC、ThrA和MetL单基因缺陷株和LysC-ThrA和LysC-MetL双基因缺陷株.采用高效液相色谱法测定L-天冬氨酸积累量.发现除MetL单基因突变株外,其余突变株均积累了比野生型更多的L-天冬氨酸,这为代谢工程改造菌株并通过发酵法生产天冬氨酸奠定了基础.There are three kinds of aspartate kinase in the metabolic pathway of Escherichia coli, including LysC, MetL and ThrA. Lysine, methionine and threonine are synthesized after aspartic acid phosphorylation pathway caused by aspartate kinase so that aspartic acid is not able to be accumulated to a high concentration in E. coll. Aspartie acid phosphorylation could partly inhibit by gene knock-out. Single gene mutants which lack LysC, ThrA and MetL respectively, are constructed from W3110. After chloroamphenicol resistance removed by pCP20, double gene mutants which lack LysC-ThrA and LysC-MetL respectively are constructed. All mutants are finally confirmed by check primer. These constructions are based on Red recombination system. Concentration of aspartic acid is determined by high performance liquid chromatography. The results show that all mutants except MetL single gene mutants are able to accumulate more L-aspartic acid than wild type. This will lay a foundation for strain improvement by metabolic engineering and contribute to production of aspartic acid by fermentation.

关 键 词：大肠杆菌W3110 RED同源重组 基因敲除 高效液相色谱 L-天冬氨酸 

分 类 号：Q784[生物学—分子生物学]