Construction Of Xylulose Kinase Gene Expression Vector And Its Overexpression In

The xylulokinase catalyze xylulose phosphorylation to form 5- phosphoric acid xyluloses, This reaction is the node position in the xylose metabolite before entering the phosphoric acid pentose way. The xylulose phosphorylation is one of the xylose metabolism rate-limited steps of the recombination Saccharomyces cerevisiae which is able to ferment xylose to produce ethanol. Saccharomyces cerevisiae has its own xylulokinase, but its activity is low and limits the xylose metabolism of xylose metabolic engineering strain to flow to the phosphoric acid pentose way, then influences ethanol production. The ultra expression of Saccharomyces cerevisiae own xylulokinase gene XKS1 may accelerate the xylose metabolism, and enhances the xylose utilization rate and the ethanol yield of Saccharomyces cerevisiae.Primers KanR-up and KanR-down, ADH1T-up and ADH1T-down were designed and plasmid pKT0150 was used as template to amplify the KanR gene and ADH terminator gene; Primers XKS1-up and XKS1-down , rDNA-up and rDNA-down were used and industrial Saccharomyces cerevisiae strain W5 genome was taken as a template to amplify the XKS1 gene and rDNA gene. The results showed that 4 fragments were successful amplified, which were the dominant select marker G418 resistance gene KanR (1477bp); ADH1 terminator fragment(301bp), which was used for constructing the strong ADH1 promoter - terminator terminator sequences for gene expression; Target gene- Saccharomyces cerevisiae W5 own xylulokinase gene XKS1(1831bp); and a Saccharomyces cerevisiae chromosome specific 2.2kb rDNA fragment as plasmid vector’s high copy homologous recombination site.Took p406ADH1 plasmid as a construction framework, used 5 kind of restriction endonuclease NdeⅠ, AatⅡ, SpeⅠ, SalⅠand Eco52Ⅰ(EagⅠ), through the way of cutting and connecting with enzyme, the 4 element fragments were constructed into p406ADH1, then obtained the 9.5kb fragment length high-copy integration expression vector pCXS-RKTr, which was suitable for industrial Saccharomyces cerevisiae strains. After recombinant plasmid vector pCXS-RKTr linear transformation industrial Saccharomyces cerevisiae W5, by increasing the concentration of G418 to 1000ug/mL, screening obtained the 5 high copy recombinant strains XKS1-1, XKS1-2, XKS1-3, XKS1 -4, XKS1-5, the xylulokinase activity respectively was 141.20 U/mg、134.70 U/mg、346.76 U/mg、341.00 U/mg、374.40 U/mg. The xylulokinase activity of 5 recombinant strains all were higher than Initial strain W5(130.60 U/mg), XKS1-1, XKS1-2 were slightly higher than W5, but XKS1-3, XKS1-4 and XKS1-5 respectively were W5’s 2.66 times, 2.61 times and 2.87 times, and the obtained recombinant strains had the high heredity stability, this indicated that after recombinant plasmid vector transformation Saccharomyces cerevisiae W5, successfully realized the xylulokinase high level to express stably.The construction of high-copy homologous recombination vector pCXS-RKTr made xylulokinase gene XKS1 high-copy integrate into Saccharomyces cerevisiae chromosome genome, realized a highly efficient and stable expression of XKS1, increased the xylulokinase activity of recombinant strain. The pCXS-RKTr was a ideal vector which was applicable to industrial Saccharomyces cerevisiae strains for genetic engineering transformation. This study laid the foundation for the dredging of xylose metabolic flux, improving the xylose utilization rate and reducing the principal by-product xylitol production, the improvement of xylose metabolism downstream way impeded question of Saccharomyces cerevisiae engineered strain which to utilize xylose to product ethanol,as well as laid the foundation for construction of yeast strain which has a high-yield ethanol production using xylose.