Construction of integrative vector for xylulokinase gene and its overexpression in Saccharomyces cerevisiae

Abstract:

Abstract: [Objective] An integrative vector of Saccharomyces cerevisiae for xylulokinase gene expression was constructed to overexpress xylulokinase activity. [Methods] On the basis of plasmid p406ADH1, 4 components were integrated, which were KanR gene as G418 resistant marker, ADH1 terminator fragment, xylulokinase gene from Saccharomyces cerevisiae W5 and 18S rDNA sequence for homologous recombination. After enzyme digestion and ligation, high copy recombinant expression vector pCXS-RKTr was constructed. pCXS-RKTr was linearized and transferred into Saccharomyces cerevisiae W5 , then xylulokinase activity was detected to determine the expression of pCXS-RKTr. [Results] Xylulokinase gene located on pCXS-RKTr was highly expressed in W5. The xylulokinase activity was 2.87 times of the original strain. [Conclusion] An integrative vector of industry strain Saccharomyces cerevisiae is successfully constructed and xylulokinase gene of Saccharomyces cerevisiae itself was over expressed by this vector. This intergrative vector can efficiently raise the xylulokinase activity of Saccharomyces cerevisiae. This system laid a foundation for the construction of gene engineering Saccharomyces cerevisiae strain which can ferment xylose to ethanol.

Key words:Keywords：Saccharomyces cerevisiae;Homologous recombination; Vector construction; Integrative expression; Xylulokinase