木糖发酵酵母Pichia stipitis木糖还原酶基因XYL1在酿酒酵母中的表达

Expression of Pichia stipitis Xylose Reductase Gene XYL1 in Saccharomyces cerevisiae

通过PCR方法克隆得到树干毕赤氏酵母木糖还原酶 (XR)基因XYL1。将该基因连入酵母表达载体pYX2 12的强启动子磷酸丙糖异构酶 (TPI)启动子下 ,得到融合表达载体pYX XYL1。通过电转化方法将 pYX XYL1转入酿酒酵母SaccharonmycescerevisiaeW 30 3-1A中 ,酶活测定表明 ,在酿酒酵母中树干毕赤氏酵母木糖还原酶 (XR)基因XYL1得到活性表达 ,2酿酒酵母转化子粗酶液中木糖还原酶活分别为 0 .89U/mg(蛋白 )和 0 .83U/mg(蛋白 ) ,为供体菌的 1 5倍多。与基因供体菌不同 ,木糖还原酶基因在酿酒酵母中表达不需木糖诱导 ,为组成型表达。树干毕赤氏酵母木糖还原酶 (XR)

Pichia stipitis Xylose Reductase (XR) Gene XYL1 was amplified by PCR. The XYL1 gene were placed under the triose phosphate isomerase (TPI) promoter in yeast vector pYX212 to produce the fusion expression vector pYX XYL1. pYX XYL1 was transformed into Saccharomyces cerevisiae W303-1A by electroporation. Transformants of S.cerevisiae containing XYL1 of P.stipitis synthesized an active XR. Specific XR activities of two transformants were 0.89U/mg protein and 0.83U/mg protein respectively, which were more than 1.5 times of that of P.stipits CBS 5773 strain. Compared with P.stipitss CBS 5773, the XYL1 in S.cerevisiae transformants expressed constitutively and did not need xylose for induction.