木糖代谢基因表达水平对酿酒酵母重组菌株产物形成的影响

Effect on Product Formation in Recombinant Saccharomyces cerevisiae Strains

以Ｅ．ｃｏｌｉＳ．ｃｅｒｅｖｉｓｉａｅ穿梭质粒ＹＥｐ２４为骨架，将树干毕赤酵母（ＰｉｃｈｉａｓｔｉｐｉｔｉｓＣＢＳ６０５４）的木糖还原酶（ＸｙｌｏｓｅｒｅｄｕｃｔａｓｅＸＲ）基因ＸＹＬ１及木糖醇脱氢酶（ＸｙｌｏｌｉｔｏｌｄｅｈｙｄｒｏｇｅｎａｓｅＸＤＨ）基因ＸＹＬ２分别以不同的相对表达方向置于酿酒酵母的乙醇脱氢酶Ｉ（ＡＤＨ１）启动子和磷酸甘油激酶（ＰＧＫ）启动子下，构建不同ＸＹＬ１及ＸＹＬ２的重组质粒。这些重组质粒分别转化酿酒酵母（Ｓ．ｃｅｒｅｖｉｓｉａｅＨ１５８）受体菌。得到的重组菌株表现出不同的基因表达水平，ＸＲ与ＸＤＨ的酶比活力比值即ＸＲ／ＸＤＨ的变化范围在００６～１７５之间。当ＸＹＬ１或ＸＹＬ２受ＰＧＫ１启动子控制同时位于ＡＤＨ１启动子下游时，检测到最高的酶比活力。为了加强对木糖的利用，在ＸＹＬ１、ＸＹＬ２基因的酵母重组菌株中又引入磷酸戊糖途径中的转醛酶（Ｔｒａｎｓａｌｄｏｌａｓｅ）基因ＴＡＬ１及转酮酶（Ｔｒａｎｓｋｅｔｏｌａｓｅ）基因ＴＫＬ１，使酵母菌在原有水平的基础上超表达合成转酮酶及转醛酶。同时具有ＸＹＬ１和ＸＹＬ２以及超表达基因ＴＡＬ１、ＴＫＬ１的酵母重组菌株可以在木糖为唯一碳源平板?

Saccharomyces cerevisiae was transformed with the Pichia stipitis CBS6054 XYL1 and XYL2 genes respectlvely encoding xylose reductase(XR) and xylitol dehydrogenase (XDH).The XYL1 and XYL2 genes were placed respectively under the control of the alcohol dehydrogenase 1 (ADH1) and phosphoglycerate kinase (PGK) promoter and inserted into the yeast plasmid YEp24. Different recombiant S.cerevisiae were constructed resulting in different specific activities of XR and XDH.The highest XR or XDH activities were obtained when the expressed gene was controlled by the PGK promoter and located downstream of ADH1 promoter geng teminator sequence.The XR/XDH ratio (ratio of specific enzyme activities of XR and XDH) in those recombiant S.cerevisiae strains varied from 17.5 to 0.06.In order to enhance xylose utilisation in the XYL1、XYL2 containing S.cerevisiaes strains,the native TKL1 gene encoding transketolase and TAL1 gene encoding transaldolase were also overexpressed,which showed considerably good growth on xylose plate.Famentation of the recombinant S.cerevisiae strains containting XYL1、XYL2、TKL1 and TAL1 were studied in mixtures of glucose and xylose.The strain with an XR/XDH ratio of 0.06 consumed 3 25g/L xylose and formed no xylitol and less glycerol and acetic acid,but more ethanol compared with the strains with the higher XR/XDH ratio.