木糖醇脱氢酶基因高拷贝表达载体构建及在酿酒酵母中的表达

木糖醇脱氢酶（xylitol dehydrogenase, XDH）可以氧化木糖醇生成木酮糖，处于木糖代谢的节点位置。利用PCR方法克隆得到了休哈塔假丝酵母（Candida shehatae） 20335的木糖醇脱氢酶基因、质粒pKT0150的ADH1终止子序列和G418抗性基因（KanR），以及酿酒酵母(Saccharomyces cerevisiae) W5特定的2.2 kb的rDNA片段。以酿酒酵母整合载体p406ADH1为骨架，利用基因工程手段构建一个多拷贝整合表达载体pLX-AGRX。将重组载体pLX-AGRX线性化转入到酿酒酵母W5后，通过高浓度G418筛选和PCR双重鉴定，证实重组载体pLX-AGRX已整合到酿酒酵母W5基因组上，测定木糖醇脱氢酶酶活可达65.957 4 U/mg。

Construction of High Copying Vector of Xylitol Dehydrogenase Gene and Its Expression in Saccharomyces cerevisiae

Being in a node position in xylose metabolism, xylitol dehydrogenase (XDH) is able to oxidate xylitol into xylulose. In this experiment the XDH gene xyl2, ADH1 terminal subsequence and resistant gene G418 (KanR) of palsmid pKT0150 from the genome of Candida shehatae 20335 using PCR cloning, as well as specific 2.2 kb rDNA fragment of S. serevisiae W5 were obtained. The multicopying integrated expression vector pLX-AGRX was constructed using integrated vector p406ADH1 of S. serevisiae as a framework by means of genetic engineering. The recombinant vector pLX-AGRX was linearized and transferred into S. cerevisiae W5, then screened through high concentration G418 and PCR for double characterization, and proved that the recombinant vector pLX-AGRX was integrated into genome of S. serevisiae W5, it was tested that the activity of XDH was as high as 65.957 4 U/mg.