Study Of The Homologous Recombination Methods And Inducible Expression System In Z. Mobilis

Zymomonas mobilis, a facultative anaerobic gram-negative bacterium, has been considered as a possible candidate for industrial ethanol production. However, the efforts to broaden its substrate have been limited in the lack of genetic manipulation technique and inducible expression system. The purpose of this study is to construct a recombination method based on native homologous recombination system in Z. mobilis, and testify the probability of applying L-arabinose-inducible expression system of E. coli in Z. mobilis.A plasmid with cml gene and long homologous arms of Z.mobilis ldh gene has been constructed. This plasmid has been transformed into Z.mobilis ZM4, the positive transformants has been screened successfully, and the efficiency of homologous recombination is 1/4.5μgDNA.A shuttle plasmid with L-arabinose-inducible expression system from E. coli and lacZ gene as a reportor has been constructed and transformed into Z.mobilis CP4. LacZ enzyme activities of a transformant are 0.259 U/OD₆₀₀.ml with L-arabinose- induced and 0.021U/OD₆₀₀.ml without L-arabinose, but in E. coli DH5αthey are 27.818 and 0.217 U/OD₆₀₀.ml. It is indicated that the E. coli L-arabinose-inducible expression system can operate in Z. mobilis, but lacZ expression is much lower than E.coli counterpart.The effects of inducer concentration and induced time on lacZ expression level have been studied by variance analysis. It is indicated that inducer concentration and induce time have an obvious effect on expression level, but they don't interact with each other. The optimized inducing condition is final inducer concentration 15mM, and the inducer is added at the very beginning of the culture.