高效表达木糖醇脱氢酶基因酿酒酵母的构建及木酮糖发酵的初步研究  

Construction of Saccharomyces cerevisiae Strain Expressing Xylitol Dehydrogenase Gene Efficiently and Primary Study of Its Xylulose Fermentation

作　　者：陈高云[1] 刘敏[1] 叶凯[2] 张元忠[1] 涂振东[2] 于孟斌[1] 

机构地区：[1]防化指挥工程学院生物防护教研室,北京102205 [2]新疆农业科学院,新疆乌鲁木齐830091

出　　处：《酿酒科技》2011年第11期32-37,共6页Liquor-Making Science & Technology

基　　金：甜高粱秸秆五碳糖发酵菌株引进与基因工程改造(2010-Z46);自治区科技支疆项目计划(指令性)项目:甜高粱秸秆五碳糖发酵菌株工程基因改造(201091132);自治区新疆维吾尔自治区自然科学基金资助项目(200821175);现代高粱产业技术体系建设任务书(nycytx-12-03-01-01)

摘　　要：通过RT-PCR方法克隆得到Candida tropicalis木糖醇脱氢酶基因xyl2,将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下,构建表达质粒pYES2-xyl2;同时用从Pichia pastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因,构建含组成型启动子GAP基因的表达质粒pYES2-GAP-xyl2;通过电转化法将其依次转入酿酒酵母S.cerevisiae INVSc1,山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养,筛选出1株耐木糖醇浓度为20%的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15%的重组菌株YDX2。酶活测定表明,重组菌株ZCX4比酶活0.621 U/mg(蛋白),是YDX2比酶活的2.29倍。摇瓶发酵结果显示,重组菌株ZCX4木糖醇消耗76.46 g/L,木糖醇消耗率为76.46%,是重组菌株YDX2木糖醇消耗率的1.63倍,说明木糖醇脱氢酶实现了高效表达。Yeast expression vector pYES2-xyl2 was constructed by cloning xylitol dehydrogenase gene xyl2, which originated from Candida tropicalis and placed under the inducible promoter GALl of the vector. Meanwhile, the other yeast expression vector pYES2-GAP-xyI2 containing the constitutive strong promoter GAP gene instead ofGAL gene was constructed. The plasmids containing xyl2 gene were transformed into industrial strain of S.cerevisiae INVScl by electroporation. The recombinant transformants ZCX4 and YDX2 grew well on plates in condition of high-concentration xylitol. The xylitol dehydrogenase specific activity of recombinant strain ZCX4 was 0.621 U/mg protein, 2.39 times as much as the recombinant strain YDX2, In addition, flask-shaking fermentation results revealed that the consumption of xylitol for ZCX4 was 76.46 g/L, 1.63 times as much as the recombinant strain YDX2. The results demonstrated that the recombinant stain could utilize xylitol efficiently by xylulose fermentation.

关 键 词：木糖醇脱氢酶 组成型启动子 诱导型启动子 木酮糖 发酵 

分 类 号：TS261.1[轻工技术与工程—发酵工程] TQ920[轻工技术与工程—食品科学与工程]