运动发酵单胞菌RecET重组系统的构建及应用

Construction and Application of RecET Recombination System in Zymomonas mobilis

摘要:

运动发酵单胞菌（Zymomonas mobilis）是一种极具潜力的工业乙醇生产菌株，通过遗传改造可以进一步克服该菌底物利用范围窄等缺点和提高乙醇生产效率. 为了提高外源基因整合到染色体的效率，在已构建的运动发酵单胞菌-大肠杆菌穿梭载体基础上，构建了RecET表达质粒pSUZM3a-RecET. 选取乙醇脱氢酶I（adhA）基因作为靶基因，四环素抗性基因（Tcr）作为筛选标记基因，检测RecET重组系统在Z. mobilis中的进行基因重组的可行性. 将两端带有60bp adhA基因同源臂的四环素抗性基因片段电转化含有RecET重组系统表达质粒的运动发酵单胞菌ZM4，获得adhA基因缺失突变菌株. 对突变菌株adhA基因的PCR产物进行测序发现，adhA基因已被置换为四环素抗性基因. 上述结果表明：RecET重组系统在运动发酵单胞菌中具有高效、便捷和可操作性，只需60bp同源臂即可完成同源重组.

Abstract:

Zymomonas mobilis is a Gram-negative bacterium with excellent ethanol-producing capabilities. The ability of ethanol yield and substrate utilization can be further improved through genetic manipulations. In this study, in order to increase the recombination efficiency of foreign genes into chromosome of Z. mobilis, the RecET genes were cloned into the E. coli-Z. mobilis shuttle expression vector pSUZM3a, resulted in pSUZM3a-RecET. The adhA gene encoding the alcohol dehydrogenase and tetracycline resistant gene were used as the target and the selection marker genes, respectively. The PCR fragments of tetracycline resistant marker with 60bp flanking sequences homologous to adhA were electroporated directly into Z. mobilis ZM4 cells which harbored pSUZM3a-RecET. After the PCR analysis and DNA sequencing, it was found that the RecET-mediated recombination reaction resulted in adhA gene replaced by tetracycline resistant gene. The result showed that RecET system could make efficient, rapid targeted gene knock-out with only 60bp homologous arm in Z. mobilis.