树干毕赤酵母木糖醇脱氢酶基因表达载体构建及在酿酒酵母中的过表达

Construction of Vector for Pichia stipitis XYYL2 and Its Over Expression in Saccharomyces cerevisiae

针对树干毕赤酵母xyl2基因设计相应引物。以p-AKRX为骨架载体,通过酶切连接的方式构建1个酿酒酵母工业菌株的表达载体p-AKRX2-2。将该表达载体线性转化酿酒酵母后,测定转化子SUN-Ⅰ木糖醇脱氢酶(XDH)及其表达情况。与原始菌株相比,重组酿酒酵母中的XDH活力是原始菌株的3.65倍。该重组质粒载体的构建可有效弥补酿酒酵母缺少代谢木糖关键基因的缺陷,为利用木糖高产乙醇酿酒酵母基因工程菌株的构建奠定基础。

Based on primers of target gene Pichia stipitis xyl2 and framework plasmid p-AKRX a Saccharomyces cerevisiae industrial expression vector p-AKRX2-2 was constructed using the method of enzyme connection. The p-AKRX2-2 was linearized and transferred into S cerevisiae W5, then the XDH activities of transformant SUN-Ⅰ was detected to determine the expression of p-AKRX2-2. The xyl2 located in the p-AKRX2-2 was highly expressed in W5 the XDH activity was 3.65 times of the original strain. The construction of recombinant plasmid vector can make up for the gap that most yeast lake key gene which can metabolize xylose, this system laid a foundation for the construction of gene engineering Saccharomyces cerevisiae strain which can ferment xylose to ethanol.