木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。

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

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.