【作者】 丁明珠；

【导师】 元英进；

【作者基本信息】 天津大学 ， 制药工程， 2011， 博士

【摘要】 纤维素预处理过程中产生的呋喃类、弱酸类和酚类化合物等对酵母的纤维素乙醇生产产生明显的协同抑制。发育能够耐受多种抑制剂的菌种,并深入剖析菌株与抑制剂之间复杂的相互作用关系,对优化纤维素乙醇工业生产是一个挑战。本研究通过紫外诱变结合驯化的方法获得一株能够同时耐受三种抑制剂的酵母菌株;并从转录、蛋白和代谢水平系统地比较分析原始酿酒酵母和耐受酵母对复合抑制剂响应机制的差异,简化复合抑制剂与酵母细胞之间的复杂关系。以酿酒酵母为出发菌种,通过紫外诱变结合驯化的方法筛选出的耐受菌株,在含1.3 g/L糠醛,0.5 g/L苯酚和5.3 g/L乙酸的高糖培养基中发酵培养,与原始酵母相比,延滞期由48 h缩短至4 h,生长速率提高了8.7倍、葡萄糖消耗速率和乙醇生产速率均提高了3.3倍,糠醛转化时间由40 h减少到6 h,乙醇产量没有显著变化,为48.28 g/L,表现出很强的耐受能力。通过对三种抑制剂对细胞协同作用的代谢水平分析,发现乙酸对酵母细胞的胁迫作用最明显,而糠醛、苯酚则增强了乙酸对细胞的胁迫;耐受酵母在代谢方面表现出较强的耐受性,主要表现为氨基酸和碳代谢等受复合抑制剂的影响较小。蛋白水平研究表明复合抑制剂对酵母产生氧化胁迫和未折叠蛋白胁迫等,但原始和耐受酵母的应答机制不同;耐受酵母能通过胁迫响应和脱毒来自我保护,且通过较低的氨基酸和核苷酸代谢和较高能量代谢,使其本身具有更强的耐受能力,而原始酵母需要依赖糖酵解和糖异生产生更多能量以抵抗抑制剂的胁迫作用。在蛋白水平和代谢水平的研究都发现,复合抑制剂使酿酒酵母的蛋白降解增加,且诱发氧化胁迫,从而抑制细胞的生长和生产。对原始和耐受酵母在转录水平对复合抑制剂的响应进行分析,发现复合抑制剂使原始和耐受酵母中与RNA和蛋白合成相关的基因显著下调,参与电子传递和能量合成相关基因显著上调,说明复合抑制剂影响酵母的生物合成和能量代谢,细胞需要合成更多能量抵抗抑制剂的胁迫;另外,复合抑制剂导致原始酵母蛋白降解,诱发氧化脱毒,使细胞产生自噬。通过研究细胞膜磷脂对复合抑制剂的响应机制,发现磷脂酰胆碱(PC)和磷脂酰肌醇(PI)链长的变化是酵母细胞对复合抑制剂响应的一个重要方面;磷脂酰乙醇胺(PE)和磷脂酰丝氨酸(PS)在原始和耐受酵母中对复合抑制剂的响应差异最大。转录和磷脂水平的研究结果表明,复合抑制剂使酵母细胞PE转化为PC的步骤严重受阻,酵母细胞膜曲率增加、细胞膜孔径增大,使抑制剂更易进入胞内,对细胞产生抑制作用。更多还原

【Abstract】 Inhibitors produced during lignocellulose pretreatment including furan derivatives, weak acids and phenolic compounds impose severely synergistic effects on Saccharomyces cerevisiae during ethanol fermentation. Obtaining a stain that could tolerate combined inhibitors and deeply investigating the relationship between strain and inhibitors were challenges for lignocellulosic fermentation. In this study, a tolerant yeast which could exhibit better characteristics in the presence of combined-inhibitors (furfural, acetic acid and phenol) was obtained. The complex relationship between combined inhibitors and yeast cells was analyzed systematically on mRNA, protein and metabolite levels.The tolerant yeast which was obtained by UV mutagenesis and domestication exhibited better characteristics in the presence of combined-inhibitors (1.3 g/L furfural, 5.3 g/L acetic acid and 0.5 g/L phenol). The lag phase of tolerant yeast shortened from 48 h to 4 h. The rates of growth, glucose consumption, and ethanol production increased 8.7, 3.3 and 3.3 fold, respectively. Furfural conversion time shortened from 40 h to 6 h. And ethanol production was 48.28 g/L, which did not change significantly compared to parental yeast.Analysis on metabolite levels indicated that acetic acid played the most important role in the combined inhibitors. And its influence on the growth and metabolism of yeast cells was enhanced by the presence of phenol and furfural. Tolerant yeast exhibited better tolerance to inhibitors, reflecting on less affected amino acid metabolism and carbon central metabolism. Study on protein levels suggested that oxidative, osmotic and unfolded protein stress responses were induced in yeast by combined inhibitors, but the mechanisms of stress response in parental and tolerant yeasts were different. Higher stress response and detoxification related proteins were important for tolerant yeast to protect itself. And lower levels of amino acid and nucleotide metabolism related proteins and higher levels of energy related proteins were necessary for tolerant yeast to defend the inhibitors stresses. Glycolysis and gluconeogenesis related proteins were upregulated in parental yeas to produce more energy to resist the inhibitors stresses. Metabolomic and proteomic results indicated that protein degradation was increased by combined inhibitors, inducing oxidative stress and inhibiting growth and production of yeast.Study on transcriptional level found that RNA and protein synthesis related genes were repressed, while electron transport and membrane-associated energy conservation related genes were upregulated by combined inhibitors. It indicated that biosynthesis in yeast was inhibited, and more energy was needed to defend the inhibitors stresses. In addition, inhibitors caused protein degradation in cells, resulting in oxidative detoxification and autophagy. Research on phospholipids of cell membrane revealed that the variation of chain length in phosphatidylcholine (PC) and phosphatidylinositol (PI) was induced by inhibitors. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) were significantly different for parental and tolerant yeasts in response to combined inhibitors. Transcriptomic and lipidomic results indicated that PC synthesized was inhibited by inhibitors, resulting in increased membrane curvature and pore size, which facilitated the import of inhibitors to cells to affect normal metabolism.更多还原