转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

Construction of Saccharomyces cerevisiae with xylose reductase gene XYL1 and its xylitol production ability

将人工合成的树干毕赤酵母(Pichia stipitis)的木糖还原酶基因XYL1插入酿酒酵母(Saccharomyces cerevisiae)表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达(13.68±2.37)g/L、(12.09±1.45)g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量(1.08±0.37)g/L(P<0.05),说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力(P<0.05)。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。

The xylose reductase gene XYL1derived from Pichia stipitis was artificially synthesized and inserted into Saccharomyces cerevisiae expression vector pYES2,and then the S.cerevisiae INVSc1/pYES2-XYL1with xylose reductase gene XYL1was constructed by introducing the recombinant plasmid pYES2-XYL1into S.cerevisiae INVSc1.The S.cerevisiae INVSc1/pYES2-XYL1with xylose reductase gene XYL1was screened using auxotroph selective medium and its ability to produce xylitol was detected.The results showed that2strains of S.cerevisiae with xylose reductase gene XYL1(INVSc1/pYES2-XYL1-01and INVSc1/pYES2-XYL1-02)were obtained,and the xylitol production was(13.68±2.37)g/L and(12.09±1.45)g/L after fermentation5d with xylose50g/L and galactose10g/L as carbon source,which was significantly higher than the xylitol production of non-transgenic S.cerevisiae INVSc1(P<0.05),indicating that the introduction of XYL1gene could significantly improve the xylitol production ability of S.cerevisiae INVSc1(P<0.05).The results provided a theoretical and technical base for preparation of edible xylitol using genetically engineered S.cerevisiae.