代谢工程手段改造酿酒酵母PP途径提高木糖发酵能力-生物化工专业论文.docx

PAGE PAGE VI and used to construct the recombinant strains with different combination of the overexpressed PPP genes. Fermentation performance was comparatively studied, results showed that yeasts with the PPP genes overproduced performed much better on xylose condition, and their ethanol tolerance was also improved to a large extent. but xylose utilization of the engineered strains was still severely damaged when glucose and xylose were used. where, however, we found that at the range of ethanol produced during the glucose fermentation, there was no significant inhibition of xylose utilization, although their xylose consumption rate was a little bit lowered, all the recombinants can still use up 5% xylose within the 48h to 72h. Put these all together we can see that the inhibitory effect of ethanol can only partially account for the lower xylose utilization in glucose and xylose condition, and there must be some other factors available in the overall process that influencing the xylose fermentation. Key words：Saccharomyces cerevisiae; Xylose; Ethanol; 6-phosphate glucose isomerase; Non-oxidative pentose phosphate pathway 目录 摘要I ABSTRATEIII 第一章 文献综述 1 1.1 概述 1 1.1.1 国外生物质乙醇的发展及现状 1 1.1.2 我国生物质乙醇的发展及现状 1 1.1.3 生物质乙醇工业化发展的瓶颈 1 1.2 木糖代谢重组酿酒酵母的构建2 1.2.1 木糖的异构化途径3 1.2.2 木糖运输5 1.2.3 氧化还原辅酶不平衡6 1.2.4 木酮糖磷酸化7 1.2.5 非氧化磷酸戊糖（PP）途径 8 1.3 本课题的研究背景及依据9 第二章 材料与方法 11 2.1 实验材料 11 2.1.1 质粒、引物及菌株11 2.1.2 实验仪器与设备16 2.1.3 主要试剂16 2.1.4 培养基 18 2.1.5 溶液 19 2.2 实验方法 19 2.2.1 感受态细胞的制备与大肠杆菌转化 19 2.2.2 小规模提取大肠杆菌 E.coil 质粒（CTAB 法） 20 2.2.3 酵母染色体的快速提取20 2.2.4 PCR 扩增反应 21 2.2.6 DNA 限制性内切酶消化 22 2.2.7 DNA 连接反应 22 2.2.8 DNA 琼脂糖凝胶电泳 23 2.2.9 DNA 片段的纯化 23 2.2.10 酿酒酵母细胞的转化23 2.2.11 一步基因置换 24 2.2.12 两步基因置换25 2.2.13 酿酒酵母总 RNA 的提取 26 2.2.14 cDNA 的合成和 RT-QPCR. 27 2.3 发酵过程的准备及主要分析方法27 2.3.1 发酵培养基制备27 2.3.2 发酵液各组分的测定28 第三章 木糖专一化重组酿酒酵母菌株的构建 29 3.1 概述 29 VII VII 3.2 缺失质粒 YIp-pPGI1-URA3-tPGI1 的构建 29 3.3 木糖专一化菌株 AYHNEW2-pgi1 的构建 30 HYPERLINK \l _TOC_250005 3.4 菌株 AYHNEW2-pgi1 的木糖发酵性能研究 30 3.5 本章小结 32 第四