【作者】 郭亭；

【导师】 鲍晓明；

【作者基本信息】 山东大学 ， 微生物学， 2006， 硕士

【摘要】 燃料乙醇是最有发展前景的新型可再生能源，其生产和应用因在经济发展和战略安全上的重大意义，越来越受到各国政府的重视。自然界中含有丰富的植物纤维资源，而目前只有3-4％的植物纤维资源被有效地利用。木糖是木质纤维素水解物中含量仅次于葡萄糖的一种单糖，实现木糖转化乙醇是纤维素乙醇生产需要解决的难题之一。 酿酒酵母(Saccharomyces cerevisiae)是传统上食品及化工工业的最佳生产菌株，在工业生产中有上百年的酒类发酵应用历史，具有乙醇产量高、抗逆性强等许多优良特性，是乙醇工业生产的首选菌株。然而酿酒酵母菌缺乏转化木糖生成木酮糖的酶而不能利用木糖，但能利用木糖的异构体形式——木酮糖。 根据代谢途径工程理论，采用代谢流扩增和底物谱拓展的所谓“加法战略”，重组表达木糖向木酮糖转化所需的相关酶基因，是在酿酒酵母中建立木糖代谢途径的有效措施。木糖异构酶(xylose isomerase Ⅺ)能直接转化木糖生成木酮糖，不需要任何辅因子，被认为是在酿酒酵母建立木糖代谢的首选途径。但迄今为止只有三种来源的木糖异构酶基因xylA在酿酒酵母中得到活性表达，由于它们在酵母发酵温度30℃条件下的酶活性相对比较低，还不能适应乙醇发酵的要求。 本论文主要包括两个方面，一方面对已经在酿酒酵母中得到活性表达的嗜热细菌Thermus thermophilus木糖异构酶Ⅺ进行分子改造，以提高其在酵母发酵温度下的酶活性；另一方面，克隆中温型嗜纤维素粘细菌纤维堆囊菌Sorangium cellulosum的木糖异构酶基因xylA并在酿酒酵母中得到活性表达。 在相关文献研究结果的基础上，对一系列木糖异构酶氨基酸序列进行比对，同时分析了T．thermophilus木糖异构酶的三维结构；选择两个氨基酸残基位点Pro137和Asp247突变成Gly。通过定点突变获得三个突变体Ⅺ137(Pro137Gly)、Ⅺ247(Asp247Gly)、Ⅺ137247(Pro137Gly，Asp247Gly)，并构建表达载体XM204-Ⅺ137、XM204-Ⅺ247和XM204-Ⅺ137247，转化实验室酿酒酵母H158，得到重组菌株H158-Ⅺ137、H158-Ⅺ247和H158-Ⅺ137247，然后对重组菌株进行酶活性分析。结果显示：相对于野生型的Ⅺ，突变体Ⅺ137、Ⅺ247和Ⅺ137247中温条件下的酶活性有所提高，特别30℃的酶活比原初Ⅺ提高一倍；最适pH范围有很大的拓展，在pH6.0-9.0之间都能表现出很高的酶活性；但是，突变体更多还原

【Abstract】 The production and application of fuel ethanol which is the most promising new renewable energy is paid much attention by many governments for the importance of economic development and strategic significance. There are abundance of lignocellulosic materials in nature, however only 3-4% of lignocellulosic materials can be utilized. D-xylose is the most abundant monosaccharide in lignocellulose hydrolysates after glucose.Utilizing xylose to produce ethanol adequately is one of the bottlenecks to ethanolic fementation of lignocellulosic materials.Saccharomyces cerevisiae has been traditionally used in ethanol producing, which has acquired qualities such as high ethanol productivity, tolerance to process hardiness, tolerance to fermentation by-products and is, therefore, preferred for ethanol production from crops. Although a few xylose-fermenting yeasts were found, the preferred organism for industrial ethanol production is the yeast S. cerevisiae which can not utilize D-xylose, only its isomer D-xylulose.Metabolic engineering can be used to extend the substrate range for growth and product formation of an organism. It is a useful way to establish the xylose metabolic pathway by expressing correlative gene in the S. cerevisiae. Xylose isomerase (XI) does not require any cofactors that can transform xylose to xylulose directly,which is regarded as the first way to establish the xylose metabolic pathway in the S. cerevisiae. Previous attempts to express xylose-isomerase gene from a variety of bacteria and Archaea in S. cerevisia have failed. Anotable exception involved xylose isomerases from thermophiles. How ever, exhibite only very low activities at temperatures that are permissive yeast to grow. We do molecular evolution to xylA from T. thermophiles and clone other xylose-isomerase gene which can be actively expression in S. cerevisia.This paper main work is the expectation that establish effective XI xylose metabolism pathway in S. cerevisiae.On the one hand, XI from Thermus thermophilus was mutanted by molecular evolution , on the other hand, the S.cellulosum xylA gene was cloned and active expressed in Saccharomyces cerevisiae.We choose two amino acid Pro137 and Asp247 for mutation to Gly basd on the更多还原