稳定高效表达木糖还原酶基因工业酿酒酵母的构建及木糖醇发酵的初步研究

将树干毕赤氏酵母(Pichia stipitis)木糖还原酶基因XYL1连接到适用于酿酒酵母工业菌株的多拷贝整合载体pYMIKP中,构建得到表达质粒pYMIKP-XYL1,转化酿酒酵母工业菌株Saccharomyces cerevisiae6508。在G418平板上筛选转化子,得到含高拷贝木糖还原酶基因的酿酒酵母重组菌株XGH2,,该菌株的木糖还原酶比活力为0.8 U/mg(蛋白),比出发菌株提高了80倍以上,表明外源基因在工业菌株中实现了高效表达。摇瓶发酵结果显示,重组菌株XGH2木糖消耗为27.9 g/L,木糖消耗率为51%;木糖醇产量为30.2 g/L,木糖醇的转化率大于1.0 g/g木糖。

Construction of the Industrial Saccharomyces cerevisiae Strain Expressing Xylose Reductase Gene Efficiently and Primary Study on It''''s Xylitol Fermentation

Integration vector pYMIKP-XYL1 was constructed by cloning xylose reductase gene(XYL1),which originated from Pichia stipitis,into multi-copy integration vector pYMIKP.The plasmid containing XYL1 gene was transformed into the industrial strain of Saccharomyces cerevisiae 6508 and the G418 resistance gene KanMX acted as a dominant selectable marker.The xylose reductase specific activity of recombinant strain XGH2 is 0.8U/mg protein,which is more than 80 times as much as the parent strain,meanwhile the consumption of xylose and yield of xylitol are 27.9g/L and 30.2g/L respectively.The conversion efficiency of xylose into xylitol is 1.0g/g xylose.