木糖异构酶在酿酒酵母中高效转化木糖的适配性分子机制研究

项目名称： 木糖异构酶在酿酒酵母中高效转化木糖的适配性分子机制研究

项目编号： No.31270151

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 鲍晓明

作者单位： 山东大学

项目金额： 82万元

中文摘要： 木糖异构酶(XI)直接异构木糖为木酮糖，无需辅酶参与，是建立木糖转化的首选途径，但一直以来的结果显示XI基因(xylA)在酿酒酵母中高活性表达困难,令人困惑。多年努力下，课题组得到具有自主知识产权的，在酿酒酵母中高活性的Ru-xylA(201110042170.2;PCT/CN 2011/001014)及低活性的Sc-xylA（200810138563.1）；同时得到XI的酶活性明显提高、而引入的xylA表达框(启动子-ORF-终止子)序列没有发生任何突变的进化菌株。在此基础上，本项目通过不同表达系统的纯酶活性及结构差异分析，研究木糖异构酶ORF序列与酿酒酵母中高酶活性表达的适配机制；对进化前后菌株基因组、转录组学差异特征的分析，结合XI的结构特征，系统揭示酿酒酵母活性表达异源XI与木糖高效转化的适配机制。研究成果将有力推动酿酒酵母作为利用木质纤维素为原料的细胞工厂的改良及相关技术进步。

中文关键词： 木糖异构酶；结构与功能；蛋白与细胞适配机制；木糖代谢；酿酒酵母

英文摘要： Xylose isomerase (XI) directly isomerizes xylose to xylulose without assistance of coenzymes so that to build the XI pathway in Saccharomyces cerevisiae is seen as the key to efficient xylose metabolism. However, it is a puzzle that the various XIs were cloned and expressed in S. cerevisiae by many research group, but detected only negligible or no XI activities. After years of efforts, several XIs,with our own proprietary intellectual property rights, were cloned and functional expressed in S.cerevisiae,respectively.Thereinto, not only low ( such as the Sc-xylA form Rumen Sorangium cellulosum, CN.200810138563.1) but also high ( such as the Ru-xylA from rumen metagenome , CN.201110042170.2; PCT/CN2011/001014) activities were gotten in recombinant S.cerevisiae. Further adaptive evolution doubled the Ru-XI activities in the multiple modified S. cerevisiae strain, however,there is no any DNA sequence mutation in the XI's expression cassettes (promoter-ORF-terminator). Base on these previous results , in the present work, the protein-host matching molecular mechanism for high XI activity and effective xylose metabolism will be investigated systematacially, including the structure characteristic of XIs, the genetic characteristic of host S. cerevisiae strains for the higher XI activities and for high efficiency xylos

英文关键词： xylose isomerase；structure and function；protein-host matching mechanism；xylose metabolism；Saccharomyces cerevisiae