多基因串联表达木糖发酵工程菌株的构建  

Construction of Engineered Strains for Multi-gene Tandem Expression of Xylose

作　　者：陆亮[1,2] 叶凯[2] 刘敏[3] 于孟斌[3] 陈高云[3] 涂振东[2] 

机构地区：[1]新疆农业大学食品科学与药学学院,新疆乌鲁木齐830052 [2]新疆农业科学院生物质能源研究所,新疆乌鲁木齐830091 [3]中国人民解放军防化学院三系生物防护教研室,北京102205

出　　处：《食品科学》2013年第13期180-185,共6页Food Science

基　　金：新疆维吾尔自治区科技支疆项目计划(指令性)项目(201091132);农业部引进"国际先进农业科学技术"项目(2010-Z46);农业部高粱产业技术体系建设项目(nycytx-12-03-01-01)

摘　　要：通过基因工程的手段,利用木糖发酵转化乙醇是近年来研究的热点。为引入木糖向木酮糖转化的途径,采用多重拼接PCR方法将木糖向木酮糖转化有关的木糖还原酶基因(xyl1)、木糖醇脱氢酶基因(xyl2)与转醛酶基因(tal1)进行拼接后克隆到表达载体pAUR123中,获得融合表达载体pAUR123-X12A;为超表达下游代谢途径的基因,将控制木酮糖向乙醇转化的木酮糖激酶基因(xks1)与转酮酶基因(tkl1)进行拼接后克隆到表达载体pAUR123中,获得融合表达载体pAUR123-SK。通过乙酸锂电击转化的方法分别将pAUR123-X12A与pAUR123-SK转入S.cerevisiae INVSc1中,得到S.cerevisiae INVSc1-X12A与S.cerevisiae INVSc1-SK重组子。对构建的重组子进行表达后,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和酶活性分析,目的基因获得表达。初步结果显示两株重组菌构建成功。In recent years,xylose fermentation to ethanol has become a research hotspot,and genetic engineering is a promising means to achieve this goal.In this study,multiple splicing PCR method was applied to transform xylose into xylulose,and relevant genes such as xylose reductase gene（xyl1）,xylitol off gene（xyl2）,turning aldehyde enzyme gene（tal1） were spliced and cloned into the expression vector pAUR123 to obtain the fusion expression vector pAUR123-X12A.In order to overexpress downstream genes in metabolic pathways,xylulosekinase gene（xks1） and ketonethe gene（tkl1）,which are responsible for controlling the conversion of xylulose into ethanol,were spliced and cloned into the expression vector pAUR123 to achieve the fusion expression vector pAUR123-SK.pAUR123-X12A and pAUR123-SK were transferred into S.cerevisiae INVSc1 by the lithium acetate method to obtain the recombinants of S.cerevisiae INVSc1-X12A and S.cerevisiae INVSc1-SK.The recombinants were expressed and then analyzed by polyacrylamide gel electrophoresis（SDS-PAGE） and enzyme activity to suggest successful expression of the target gene.Preliminary results from this study reveal that two recombinant strains have been built successfully.

关 键 词：多基因串联 木糖 表达 酿酒酵母 

分 类 号：TS261.11[轻工技术与工程—发酵工程]