【作者】 刘哲；

【导师】 余世袁；

【作者基本信息】 南京林业大学 ， 生物化工， 2007， 硕士

【摘要】 酿酒酵母可以利用木酮糖发酵,但由于木酮糖代谢途径不畅和氧化还原不平衡等原因,含有毕赤树干酵母木糖还原酶和木糖醇脱氢酶编译基因的重组酿酒酵母在木糖培养基上出现木糖醇积累,从而导致木糖生产乙醇的产率较低。本研究将毕赤树干酵母的木糖还原酶基因、木糖醇脱氢酶基因和木酮糖激酶基因同时引入酿酒酵母并表达,从而达到疏通酿酒酵母木糖代谢途径,提高木糖利用率和乙醇产率的目的。采用Oligo和Primer PREMIER软件设计得到毕赤树干酵母木酮糖激酶基因xy13(gig100399,2942bp)的PCR上游引物5’-CCAACCAGCAGCGTGTG-3’和下游引物5’-TCTATCGTGATATTCGCACAT-3’,经过PCR反应由毕赤树干酵母基因组成功扩增出长度为2704bp木酮糖激酶基因X3片段,X3序列与xy13基因相似性达到98%,含有酿酒酵母基因转录所必需的区域。由起始密码子ATG开始转译合成的多肽链含有623个氨基酸残基,相对分子量为69324Da。X3序列与酿酒酵母木酮糖激酶基因xksl的相似性达到35.46%。蛋白序列相似性达到34.2%,蛋白序列存在高度保守区域。以酵母附加型质粒YEp24成功构建得到了重组质粒YEp24-X3,又以重组质粒YEp24-X3和含有木糖还原酶基因(X1)、木糖醇脱氢酶基因(X2)的质粒YEp24-X1X2构建了同时含有X1、X2和X3基因的重组质粒YEp24-X1X2X3。采用乙酸锂转化法转化营养缺陷型酿酒酵母菌株Saccharomyces cerevisiae 1949得到了重组酿酒酵母菌株S.c R5。重组酵母S.cR5能够利用木糖,利用分子生物学手段检测重组菌含有酵母表达质粒YEp24-X1X2X3,即成功得到了同时含有毕赤树干酵母木糖还原酶基因、木糖醇脱氢酶基因和木酮糖激酶基因的重组酿酒酵母菌株,为木糖代谢研究提供了种质资源。重组酵母菌株S.c R5在YCM培养基上利用木糖的能力较宿主菌有了较大幅度的提高。同时供氧水平可显著影响重组酵母菌株S.c R5的木糖代谢,在不同的供养条件下酵母代谢木糖的能力有明显差异。在好氧条件下重组酵母菌株S.c R5可以代谢发酵液中99.5%的木糖,乙醇浓度达到0.46g/l,在厌氧条件下S.c R5能够发酵木糖生成乙醇,得率达到理论得率的19%。这表明,转入的毕赤树干酵母木糖还原酶基因、木糖醇脱氢酶基因和木酮糖激酶基因可以对酿酒酵母木糖发酵乙醇起到促进作用。本研究已成功获得了能够发酵木糖生成乙醇的重组酿酒酵母菌株,达到了预期目的。更多还原

【Abstract】 Saccharymyces cerevisiae can ferment D-xylulose,however,because of the block of xylulose metabolic stream and infection of redox imbalance,recombinant Saccharymyces cerevisiae containing Pichia stipitis xylose reductase and xylitol dehydrogenase show xylitol accumulation phenomena which leads to the reduction of ethanol productivity.In this research, we clone Pichia stipitis xylose reductase gene,xylitol dehydrogenase gene and xylulokinase gene,express them in Saccharymyces cerevisiae to dredge xylose metabolic stream and rise ethanol productivity.Using Oligo and Primer PREMIER to design Pichia stipitis xylulokinase gene xy13 (gi8100399,2942bp)PCR primer.Upper primer is 5’-CCAACCAGCAGCGTGTG-3’,lower primer is 5’-TCTATCGTGATATTCGCACAT-3’.Gene xy13 coding xylulokinase was cloned from the genome DNA of Pichia stipitis with the length of 2704bp,namely X3.The similarity value between X3 and the gene sequence of xy13 in GeneBank was 98%.Analysis of this gene showed that there were promoters and terminators but no intron in xy13 which is essential to the expression in Saccharomyces cerevisiae.The gene X3 was coded and a 623AAR polypeptide (69324Daltons)was produced.The DNA similarity between X3 and Saccharymyces cerevisiae xylulokinase gene xksl is 35.46%,protein similarity is 34.2%,and they have highly conserved regions.From yeast episome plasmid YEp24,successfully construct YEp24-X3.With plasmid YEp24-X1X2 and YEP24-X3,expression vector YEp24-X1X2X3 contained genes X1,X2 and X3,was recombined.Recombinant of Saccharomyces cerevisiae,namely R5(with YEp24-X1X2X3)was constructed by transferring mutant Saccharomyces cerevisaie 1949 with LiAc reagent.Saccharomyces cerevisaie R5 could grow well in xylose medium,molecule test indicate that xylose reductase gene,xylitol dehydrogenase gene and xylulokinase gene were contained.Apparently different has shown in xylose fermentation by Saccharomyces cerevisaie R5 on YCM medium.Saccharomyces cerevisaie R5 could grow well in xylose medium under aerobic and anaerobic conditions,under aerobic condition R5 can consume 99.5%xylose in the medium and ethanol concentration is 0.46g/l.Under anaerobic condition,ethanol yield was 19%of theoretical yield.This shows that expression of Pichia stipitis xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene is helpful to xylose ferment ethanol. Recombinant xylose-fermenting yeast strains were constructed,which expands and substantiates the strains’ source and the theory for xylosc-metabolic engineering of microorganisms and the bioconversion of lignocellulosics to ethanol.更多还原