Cloning And Expressing Of The Xylose Isomerase And Xylulose Kinase Genes From Escherichia Coli

With the graduate recongization for the global energy crisis and environmentalprotection, it is urgent to find an appropriate substitute to replace the petroleum.Bio-ethanol,being made from renewable resources, could be used as fuel andchemicals and is such a substitute. Screening or constructing a valuable microbialstrain, which converts biomass feedstocks to bio-ethanol by fermentationeffenciently,is an important job in bio-ethanol industry.Under such circumstances, the project of constructing hybrid strains whichmetabolite pentose for bio-ethanol production has been set up, with the purpose toalternate the trait of Zymomonas mobilis by means of genetic manipulation. The workin this paper, cloning Escherichia coli K12 xylAB and expressing them in Zymomonasmobilis CP4, is a portion of the whole project.First, the wildtype strain E.coli CSH25-1 was irradiated by UV. After twiceampicillin enrichment, xylose auxotraph mutants were screened onX-EMB(xylose-eosin methylene blue) selective plates and identified by enzymeactivity. Second, xylAB gene was obtained by means of PCR,and introduced intoxylose deficient strain 787 while pBSKS(+) was used as a vector.The XI,XK activitydetermination and the sequence analysis revealed the transformant having the rightgenes we wanted. Finally, the xylAB promoter of E.coli was replaced with gappromoter from Zymomonas mobilis CP4 by PCR-mediated overlap extension.The XI and XK activity determined were shown as follows: the XI and XKactivity levels of DH5α(pZB1-xylAB) were about 1 and 99 folds higher than DH5α(pZB1)'s,and the XI and XK activity levels of DH5α(pZB1-Pgap-xylAB) wereabout 2 and 823 folds higher than DH5α(pZB1)'s.These results suggested that thegap promoter from Zymomonas mobilis CP4 could be well recognized by Escherichiacoli,and conducted xylAB expressing in Escherichia coli.