大肠杆菌ptsHIcrr操纵子的快速敲除及敲除菌生长性能测定

One-step Knockout of the ptsHIcrr Operon in Escherichia coli and Characterization of the Mutant

摘要:

敲除大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统(简称PTS系统) ptsHIcrr操纵子, 考察敲除菌株生长特性并将其与ptsG敲除菌进行比较。利用I-SceⅠ特异性切割和Red同源重组方法成功构建了大肠杆菌DH5α△ptsHIcrr敲除菌。在LB培养基中, DH5α△ptsHIcrr的生长行为与DH5α和DH5α△ptsG明显不同, 其最高菌密度是DH5α和DH5α△ptsG的近2倍, 而DH5α△ptsG生长行为与DH5α无明显差异。但在含1%葡萄糖的LB中, DH5α△ptsHIcrr 和DH5α△ptsG均表现出生长优势, 最高菌密度依次是DH5α的2.8和2倍; 培养液中最终乙酸含量分别是DH5α的12.2%、47%。在M9修饰培养基中, DH5α△ptsHIcrr比生长速率(1/h)和比葡萄糖消耗速率[g/(g·h)]明显低于DH5α, 并略低于DH5α△ptsG。结果说明, ptsHIcrr操纵子敲除菌改变了葡萄糖的代谢速率, 并呈现与ptsG基因敲除菌不同的代谢特点。

Abstract:

The ptsHIcrr operon of phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Escherichia coli DH5α was deleted to generate DH5α△ptsHIcrr strain, and its characteristics of growth and metabolism were compared with that of DH5α△ptsG. DH5α△ptsHIcrr and DH5α△ptsG were successfully constructed by using a one-step markerless deletion method that is a modified Red homologous recombination combined with specific cutting of I-SceⅠ. In LB medium, DH5α△ptsHIcrr has a significant different characteristic of growth with DH5α and DH5α△ptsG, and the maximum biomass of DH5α△ptsHIcrr is about two times of that of DH5α or DH5α△ptsG. However, there was no difference between the growth of DH5α and that of DH5α△ptsG. In LB medium supplemented with 1% glucose, DH5α△ptsHIcrr and DH5α△ptsG both grew better than DH5α, and the maximal cell densities were approximately 2 times and 2.8 times of that of DH5α respectively. In the end of culture, the concentrations of acetic acid of DH5α△ptsHIcrr, DH5α△ptsG were12.2%, 47% of that of DH5α. In a modified M9 medium, the specific growth rate (1/h) and the specific glucose consumption rate [g/(g·h)] of DH5α△ptsHIcrr were much slower than that of DH5α and a little slower than that of DH5α△ptsG. Altogether, the ptsHIcrr operon-deleted mutant changed the specific glucose consumption rate and showed many different characteristics of growth and metabolism from that of the △ptsG-deleted mutant.