【作者】 张颖；

【导师】 林敏；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 充分利用天然可再生的纤维素资源,扩大产乙醇微生物的底物利用范围、提高乙醇产率和对环境因子的耐受能力是世界各国研究者关注的重点。本研究构建木糖代谢重组运动发酵单胞菌,分析了耐辐射调控蛋白增强细胞对环境因子耐受能力。并利用基因芯片开展了调控因子(σs,rpoS)对模式菌株大肠杆菌在乙醇胁迫条件下的全基因组转录水平的分析。本研究从大肠杆菌(Escherichia coli)基因组DNA中克隆了木糖代谢途径中木糖异构酶基因(xylA)、木酮糖激酶(xylB)、转酮醇酶基因(talB)及转酮醛酶基因(tktA),以及运动发酵单胞菌(Zymomonas mobilis)的两个组成型的强启动子(eno和gap),构建了木糖代谢基因的的融合质粒。经电击转化获得能利用木糖发酵的重组运动发酵单胞菌。酶活分析显示引入的四种酶(木糖异构酶、木酮糖激酶、转酮醇酶及转酮醛酶)的酶活分别为80U/mg蛋白、53U/mg蛋白、1105U/mg蛋白和180U/mg蛋白,葡萄糖和木糖生成乙醇的产率分别达到理论值的81.2%和63.1%。利用纤维素水解产物进行乙醇发酵过程中,乙醇、高糖、高盐、酸等是影响乙醇产率的重要环境因子。本研究从耐辐射异常球菌(Denococcus radiodurans)基因组DNA中克隆了耐辐射关键调控基因irrE,构建了含irrE基因表达质粒的重组运动发酵单胞菌,乙醇、酸、高渗和NaCl的冲击实验结果表明irrE基因可以明显提高该菌株对这些胁迫因子的耐受能力,其细胞存活率和乙醇产率均比对照菌株显著提高。为了进一步研究环境胁迫因子对产乙醇菌株乙醇发酵的影响,本研究以模式菌株大肠杆菌作为研究对象,构建了调控因子rpoS(σs)缺失突变株,利用基因芯片技术,对大肠杆菌BW35113(rpoS-)进行了乙醇冲击下的基因组表达谱的研究分析。与野生型菌株相比,对数期rpoS缺失突变株中772个基因表达差异显著(变化倍数大于2),其中467个基因表达显著上调;305个基因的表达受到显著抑制;稳定期细胞表达差异显著的基因有1422个,其中465个基因表达显著上调, 943个基因的表达受到显著抑制。按照COG库的标准分类,发现对数期和稳定期细胞中1)与细菌遗传信息的保存和处理(DNA的复制、重复、修复及基因的转录、表达等)有关的基因分别有90和209个基因;2)参与能量合成与转化的基因有111和130个;3)参与物质代谢(糖类、氨基酸、核酸、辅酶、酯类)及无机离子的转运与代谢的基因有218和376个;4)16和32个基因参与了信号的传递过程, 5)7和16个基因与细胞运动相关,6)29和57个基因涉及蛋白的转录后修饰及蛋白折叠等过程。对差异表达基因进行分析,发现在乙醇胁迫条件下,RpoS缺失分别导致了海藻糖磷酸化酶基因、6-磷酸海藻糖合成酶基因、高渗保护蛋白及甜菜碱运输系统基因下调,引起海藻糖和甜菜碱在细胞内的减少;同时调控谷氨酸型抗酸体系基因gadABC蛋白基因下调,这些基因表达通常在E. coli胁迫反应中发挥重要的作用。表明RpoS转录因子在增强细胞对乙醇胁迫抗性上起到重要作用。更多还原

【Abstract】 Lignocellulosic biomass has long been recognized as a potential sustainable source of mixed sugars. Recent research has focused on the development of recombinant strain for efficient production of ethanol from lignocellulosic hydrolysates, together with high stress tolerance. In this study, we constructed the recombinant xylose-metabolizing strains of Zymomonas mobilis and demonstrated that expression of IrrE, a global regulator for extreme radiation resistance from Deinococcus radiodurans, confers significantly enhanced to tolerance to the environmental stresses in Z. mobilis. Futhermore, enthanol stress-responsive gene expression profiles of Escherichia coli strain and its rpoS mutant strain were analyzed using DNA microarrys.Four genes encoding xylose assimilation and pentose phosphate pathway enzymes (xylA/xylB, talB/tktA) from Escherichia coli and two promoters ( eno and gap) from Z. mobilis were constructed into the shuttle plasmid (pBBR1MCS-1) and transferred into Z. mobilis. The resulting recombinant strain PZ. mobilis fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic biomass to ethanol. Enzymatic analyses of PZM strain grown in RM medium containing xylose of 40g/L showed the presence of XI ( 80U/mg proteins), XK(53U/mg proteins), TAL (1105U/mg proteins) and TKT (180U/mg proteins). The productivity of ethanol from glucose is 81.2% of theory value and 63.1% from xylose.The components (such as acetic acid) in lignocellulosic hydrolysates and the accumulation of ethanol during the fermentation can inhibit the growth and ethanol production of ethanologenic strains. To improve growth and ethanol production of Z. mobilis under the stresses, the D. radiodurans gene irrE were cloned and transferred into Z. mobilis to generate the recombinant strain. The results showed that the expression of IrrE regulator resulted in a significant increase in cell viability and the tolerance to ethanol, acetic acid, salt stresses.To further understand the effect of environmental stresses on the ethanol production in ethanologenic strains, DNA microarrys was used to analyze the expression profiles of E. coli and its rpoS mutant strain under the ethanol stress. The results showed that there are 799 differentially expressed genes by at least twofold in the logarithmic phase cells after 15% (v/v) ethanol shock for 15 min. The expression of 467 genes were up-regulated, while 305 genes were down-regulated. In stationary phase cells, among 1422 differentially expressed genes, 465 genes were up-regulated expression and 943 genes were down-regulated. Six major groups might contribute in various ways to the enhanced stress tolerance: (i) transcription, replication, translation, ribosomal structure and biogenesis, recombination and repair; (ii) energy production and conversion; (iii) lipid transport and metabolism; inorganic ion transport and metabolism, secondary metabolites biosynthesis, transport and catabolism, carbohydrate transport and metabolism, amino acid transport and metabolism, nucleotide transport and metabolism; (iv) signal transduction mechanisms, cell wall/membrane/envelope biogenesis, coenzyme transport and metabolism; (v)cell cycle control, cell division, chromosome partitioning; defense mechanisms, cell motility; (vi) posttranslational modification, protein turnover, chaperones.Analysis of these differentially expressed genes showed. Two osmotregulated trehalose synthesis genes otsAB and the betaine transport gene yehZ, were down-regulated, which may lead to trehalose and betaine decrease in mutant. In response to ethanol shock, the glutamate decarboxylase and related genes gadABC were down–regulated in mutant, which are important for stress-tolerance in E. coli. These results indicated RpoS play a global regulator role in ethanol tolerance in E. coli.更多还原