Construction and Application of RecET Recombination System in Zymomonas mobilis

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.