多基因串联发酵木糖产乙醇重组菌株构建初探  

Preliminary Study on Construction of Recombinant Strains of Polygenic Series Fermentable Xylose to Ethanol Production

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

机构地区：[1]新疆农业科学院生物质能源研究所,乌鲁木齐830091 [2]新疆农业大学食品科学与药学学院,乌鲁木齐830052 [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：《新疆农业科学》2013年第5期900-908,共9页Xinjiang Agricultural Sciences

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项目(nycytx-12-03-01-01)

摘　　要：【目的】解决酿酒酵母不能发酵或利用木糖的问题,以期选育出高效转化秸秆生产乙醇的菌株。【方法】将来自近平滑假丝酵母(Candida parapsilosis)的木糖还原酶基因(xyl1),热带假丝酵母(Candida tropicalis)的木糖醇脱氢酶基因(xyl2),树干毕赤酵母(Pichia stipitis)的木酮糖激酶基因(xks1),以及酿酒酵母内源的转醛酶基因(tal1)和转酮酶基因(tkl1),通过串联共表达的方法构建到表达载体pAUR123上,构建了一株重组酿酒酵母。【结果】经过酶活性分析和Western blot蛋白免疫实验,证明转化子pAUR123-XL成功导入酿酒酵母并得到表达。以木糖为唯一碳源进行限氧发酵,能稳定利用木糖,木糖发酵结果表明:木糖的利用率为79.5%,乙醇产率为31%。【结论】构建的重组菌株能较好的利用木糖,为进一步的乙醇发酵提供了依据。[ Objective ] This project aims to resolve the problem that Saccharomyces cerevisiae can＇t ferment or utilize xylose, with a view to the efficient conversion of culms to ethanol strains. [ Method] Xylose reductase gene （xyll） from Candida parapsilosis xylitol, dehydrogenase gene （xyl2） from Candida tropicalis and the xylitol dehydrogenase gene （xyl2） in Pichia stipitis from the trunk and its own xylose transaldolase gene in Saccharomyces cerevisiae （tall） on construction of expression vector pAUR123 by the method of concatenating co - expression, thus building a recombinant Saccharomyces cerevisiae strain. [ Result ] After analysis of enzyme activity and Western blot protein, the transformant pAUR123 -X12A ,was successfully imported into Saccharomyces cerevisiae INVScl. The bacteria use xylose as the sole carbon source to ferment in the limited oxygen, and it could used the xylose initially, the results showed that： The recombinant bacteria could metabolize xylose stably, the yield of ethanol was 31%, and the utilization of xylose reached 79. 5%. [ Conclusion] The recombinant strain can utilize xylose and provide the basis for further ethanol fermentation.

关 键 词：木糖还原酶 木糖醇脱氢酶 木酮糖激酶 转醛酶 转酮酶 

分 类 号：S188.4[农业科学—农业基础科学]