Cloning Of Key Enzymes Of Xylitol Metabolism And Construction Of Recombinant Yeast

Along with the rapidly development of global economy,varieties of non-renewable mineral resource are decreasing, especially petroleum. It will threaten the sustainable development of society. There is no denying the fact that the only way to solve this problem is to exploit renewable resources. Fuel ethanol is regarded as one of the best renewable energy sources which is benefit to the environmental protection and social development. Utilizing lignocelluloses to product bioethanol can not only reduce the cost of production but also play an important role both in environmental protection and refuse disposal.Xylitol is able to be oxidated to xylulose by xylitol dehydrogenase. This reaction is the node position in the xylose metabolism. Saccharomyces cerevisiae does not have its own xylitol dehydrogenase and it can not ferment xylose. We can insert xyl2 into the genome of Saccharomyces cerevisiae in order to consummate xylose metabolic flux, and enhance xylose utilization rate as well as the ethanol yield by Saccharomyces cerevisiae.In this paper, target gene xyl2, ADH terminator gene, resistance gene of G418-Kan_R as well as 2.2kb rDNA fragment can be cloned by PCR. Xyl2 is from the genome of Candida shehatae(code 20335). ADH terminator gene and resistance gene of G418-Kan_R derive from the commercializatived plasmid named pKT0150 purchased from Addgene. Specific 2.2kb rDNA fragment come from Saccharomyces cerevisiae chromosome. Choose the plasmid p406ADH1 as the construction framework. Using restriction endonuclease XhoⅠand KpnⅠto digest both the backbone and ADH terminator gene in order to replace CYC1 terminator by ADH terminator gene. And then insert xyl2 to the whole ADH1 promoter - terminator sequences for the high expression of xylitol dehydrogenase gene. G418 resistance gene have been introduced into the recombinant as a dominant select marker. Specific 2.2kb rDNA of Saccharomyces cerevisiae chromosome have been inserted into the recombinant vector’s as a high copy homologous recombination site. The constructed recombinant plasmid is a high-copy homologous recombination vector named pLX-AGRX which can highly enhance the stability and expression of xyl2. pLX-AGRX can be linear transformated into Saccharomyces cerevisiae W5. The vector has integrate into Saccharomyces cerevisiae chromosome genome which was verificated by PCR and high density of G418. The highest activity of XDH is 65.9574 U/mg, and successful realized the high and stable expression of xyl2. The stability of recombinant strain LX-4 which is the highest activity of XDH is 99.67% after transfer 60 culture. It is showed that the high-copy recombination vector pLX-AGRX is integrated into Saccharomyces cerevisiae chromosome genome. This study laid the foundation for the dredging of xylose metabolic flux, improving the xylose utilization rate and reducing the production of principal by-product xylitol in Saccharomyces cerevisiae engineered strain which utilize xylose to product ethanol,as well as laid the foundation for the construction of the yeast strain which has high-yield ethanol production when it use xylose to ferment.