海洋破囊壶菌△^5-延长酶和△^4-脱饱和酶在酿酒酵母中的共表达  

Co-expression of Thraustochytrium sp. FJN-10 △~5-elongase and △~4-desaturase in Saccharomyces cerevisiae

作　　者：刘艳如[1] 江贤章[1] 高媛媛[1] 田宝玉[1] 陈晓峰[1] 陈金卿[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心,生命科学学院,福建省现代发酵技术工程研究中心,福州350108

出　　处：《应用与环境生物学报》2009年第6期851-855,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金(Nos.30370028;30970047);福建省自然科学基金重大项目(No.2003F005);福建省自然科学基金(No. 2008F3036);福建省发改委科技产业化项目(No.[2005]847);福建省科技平台(No.2005Q007)~~

摘　　要：二十二碳六烯酸(Docosahexaenoic acid,DHA C22:6n-3)是具有各种重要生理功能的高度不饱和脂肪酸.分别以质粒pYTFD5和pYFAD4为模板,扩增获得860bp的△5-延长酶基因(elo5)和1600bp的△4-脱饱和酶基因(fad4).利用重叠延伸PCR构建elo5-fad4融合基因,Hind Ⅲ/Sph Ⅰ双酶切后连接到经同样处理过的pYES2.0载体,构建重组表达质粒pYELO5-FAD4.转化酿酒酵母尿嘧啶缺陷型菌株INVScl,通过缺少尿嘧啶的选择性培养基筛选阳性克隆子.添加外源脂肪酸C20:5底物,半乳糖诱导表达.气相色谱-质谱分析表明,重组酵母总脂肪酸中出现了DHA(二十二碳六烯酸,C22:6n-3)新产物,融合基因elo5-fad4在酿酒酵母中得到了表达.Docosahexaenoic acid （DHA C22：6n-3）,a typical long chain polyunsaturated fatty acids （PUFAs） has many positive effects on diseases. △5-elongase gene（elo5,860 bp）and △4-desaturase gene （fad4,1 600 bp） were amplif ied by PCR using plasmid pYTFD5 and pYFAD4 as templates,respectively. elo5-fad4 fusion gene amplif ied by overlap extension PCR was digested by Hind Ⅲ and Sph Ⅰ and subcloned into the yeast-Escherichia coli shuttle vector pYES2.0. The recombinant plasmid pYELO5-FAD4 containing target gene was transformed into Saccharomyces cerevisiae strain INVScl and the recombinant yeast cells were selected on agar synthetic medium lacking uracil. Expression of the fusion gene in transformant was induced by the addition of galactose to 2% （w/V）. The yeast culture medium was supplemented with exogenous fatty acid substrate, eicosapentaenoic acid. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters were analyzed by GC detection. A novel peak corresponding to DHA （docosahexaenoic acid, C22：6n-3） methyl ester standards which was absent in the cell transformed with empty vector was detected with the same retention time and mass analysis. These results indicated that the protein encoded by elo5-fad4 could specifically catalyze EPA to DHA. Fig 7, Tab 1, Ref 18

关 键 词：破囊壶菌 二十二碳六烯酸 延长酶 脱饱和酶 共表达 

分 类 号：Q78[生物学—分子生物学]