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

作　　者：周军智[1,2] 邹永康[1,2] 戴红梅[2] 李树龙[2] 蔡亚非[1] 方宏清[2] 

机构地区：[1]安徽师范大学生命科学学院,安徽芜湖241000 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《微生物学通报》2010年第8期1146-1152,共7页Microbiology China

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。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 wasno 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.

关 键 词：RED同源重组 PTS系统 ptsHIcrr操纵子 大肠杆菌 

分 类 号：Q933[生物学—微生物学]