【作者】 池小琴；

【导师】 闵航； 吴雪昌；

【作者基本信息】 浙江大学 ， 微生物学， 2010， 博士

【摘要】 乙醇是已被大规模生产与应用的可再生生物能源之一。但乙醇作为一种新能源物质,现生产工艺的能耗与原料消耗高,能源与原料资源利用效率低,工艺废水量大质劣,环境治理面临严峻挑战,整体经济社会效益急待提高。因此,通过理论与技术创新,寻求高效清洁生产新工艺成该领域备受关注的热点。实现高浓度酒精发酵是提高乙醇生产效率效益的关键技术之一。但高浓度酒精发酵要求生产菌株酿酒酵母具有良好的抗逆性能。在酿酒酵母中,胞内海藻糖含量被认为是与其抗逆性能关系密切的一个重要指标。国内外研究显示,酵母细胞可以通过增加海藻糖的合成而抵御不良逆境的伤害。但关于菌株胞内海藻糖的积累对其高浓度酒精发酵性能影响的研究却鲜有报道。本研究用细胞与分子生物学及基因工程技术改造酿酒酵母海藻糖的代谢相关途径,增强菌株胞内海藻糖积累,构建适于工业生产的高抗逆酿酒酵母工程菌株。探讨海藻糖提高细胞抗逆性的机制。最后通过系统分析HOG (High Osmolarity Glycerol)途径的基因序列变异,阐释酵母菌渗透压耐受性差异的可能机制,为进一步深入开展提高酿酒酵母抗逆性能的研究奠定基础。主要研究结果如下：1、海藻糖代谢途径工程菌株构建从工业菌株D308中分离得到60株单倍体,并对这些菌株进行发酵性能及胞内海藻糖含量测定,结果显示二者呈显著正相关性。构建重组质粒改造海藻糖代谢途径,得到过表达编码海藻糖-6-磷酸合成酶基因(TPSl)菌株H11ptps1、H13ptps1;敲除编码海藻糖酸性水解酶基因(ATH1)菌株H11△ath1、H13△ath1;及过表达TPS1同时敲除ATH1基因菌株H11pT△A、H13pT△A。杂交实验获得二倍体工程菌株D309ptps1、D309△ath1及D309pTΔA。2、不同胁迫条件下工程菌株与亲株生长及发酵性能比较二倍体工程菌株D309pT△A不仅结合了优良单倍体的性状,同时增强了胞内的海藻糖积累,在高糖、高乙醇、低pH、高温等胁迫条件下其最大比生长速率比亲株D308均有显著提高,提高范围为4.92%-9.80%。在高糖、高温、低pH等条件下,工程菌株的发酵速率比亲株均有较大提高,胞内海藻糖含量也均比同一条件的亲株高。玉米糖化醪(254.906g/L葡萄糖)发酵时,工程菌株D309ptpsl、D309Aathl、D309pTΔA发酵82h的乙醇产量分别为117.171g/L、117.135g/L、117.555g/L,比亲株D308分别提高了4.71%、4.67%、5.05%；主酵期0～46h期间,以上二倍体工程菌株的乙醇发酵速率分别为2.253、2.243、2.264(g h-1L-1),分别比亲株提高了13.22%、12.20%、13.76%。3、海藻糖工程菌株抗逆性高于亲株的机制探讨从呼吸缺陷型形成比率、细胞膜完整性分析、胞内活性氧ROS水平、抗氧化性能及SOD酶(超氧化物歧化酶)活性等五个方面进行了研究分析。在乙醇胁迫下,工程菌株D309pTAA的呼吸缺陷型比率比亲株D308显著减少。D309pTAA在高渗、高温、低pH、高乙醇等条件胁迫下,细胞膜完整性均高于亲株(如高温50℃处理1h后,其细胞存活率可达48.98%,是亲株的1.71倍)；胞内ROS积累均比亲株低(仅为亲株的0.48～0.70倍)；SOD酶活性比亲株高9.98%～19.25%,同时5mmol/L H2O2胁迫下的最大比生长速率比亲株提高7.95%。因此,海藻糖提高酵母细胞在胁迫条件下的抗逆性机制可能是通过保护其细胞膜完整性,维持活性氧代谢过程中的关键酶活性,以增强菌株的抗氧化能力,进而提高工程菌株的整体抗逆性能。4、HOG途径基因变异与菌株高渗耐受力关系研究HOG途径基因在23株酿酒酵母(包括工业酿酒酵母H13、黄酒酵母WH1)中的基因多态性及其在10个酵母菌种间的序列突变,进化分析后发现HOG途径中的上游基因序列比下游基因序列更易发生突变,位于途径顶端的SLN1与MSB2基因碱基突变数量显著高于其它基因,且菌株H13、WH1的MSB2基因中含有一个大片段Indel区(缺失378bp)。这种现象可能是导致酵母菌株间高渗耐受力差异的分子机制之一。用通径分析等统计学方法分析发现,基因在途径中的位置、密码子偏好性、蛋白质长度、蛋白质相互作用及基因表达水平等对HOG途径基因的进化均有重要作用,为进一步从基因网络水平上阐明酵母菌在环境胁迫下耐受性差异的分子机制奠定了一定基础。更多还原

【Abstract】 Alcohol is one kind of most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of alcohol production, for example, high biomass-consuming, low efficiency of energy production, extremely large amount of wastewater produced, and arduous environmental management. So the novel technology with high efficiency and conservation has attracted more and more attentions. The achievement of fermentation under ultrahigh alcohol concentration opens a new and important avenue to increase the efficiency of alcohol production, and the basic requirement is the developing of Saccharomyces cerevisiae with highly potential ability of stress resistance during fermentation. The one of most important factors for the ability of stress resistance in Saccharomyces cerevisiae is the intracellular trehalose content. Researchers have found that Saccharomyces cerevisiae can increase the content of trehalose inside the cells to resist the potential damage in the extreme environment, but the study on the relationship between the accumulation of trehalose in Saccharomyces cerevisiae and their capability of alcoholic fermentation was rare.Herein, we reconstructed the correlation metabolism pathway of trehalose in yeasts to increase intracellular content of trehalose by cell and molecular biology technologies. The objective of this study is to establish one kind of Saccharomyces cerevisiae with excellent stress resistance and ability of high fermentation rate, which may have great potential in the applications of fuel industry. We also investigated the possible protection mechanism of trehalose and the evolution of HOG (High Osmolarity Glycerol) pathway in yeast to demonstrate the difference of stress resistance among yeasts. There are majorly four parts in this thesis:1. Construction of trehalose metabolic engineering strains. We separated 60 haploids from industrial strain D308. Then we analyzed and investigated the relationship between intracellular trhalose content and capability of alcoholic fermentation, and found there is the positive correlation of them. We reconstructed the metabolism process of trehalose by recombining the plasmid and produced two over-expressing trehalose-6-phosphate synthase gene (TPS1) strains: H11ptps1, H13ptpsl, two acid hydrolase gene (ATH1) knock-out strains:H11△athl, H13△athl, and two both over-expressing TPS1 and ATH1 knock-out strains:H11pTAA, H13pTAA. We further hybridized the above strains to produce three engineered diploid strains:D309ptpsl, D309Aathl, and D309pTAA.2. The comparison of fermentation performance between engineered strains and parental strains. The engineered diploid strain D309pTAA combined the excellent features of each haploid and increased the accumulation of trehalose inside cells. The maximal specific growth rate of D309pTAA was significantly higher than that of parental strain D308 under the same extreme stress environments such as high concentration of glucose, high concentration of alcohol, low pH value and high temperature. Moreover, the fermentation rate of D309pTAA was also higher than that of parental strain D308 under the same fermentation environments such as high concentration of glucose, high temperature, and low pH value. The intracellular trehalose content in engineered strain D309pTAA was higher than parental strain D308 over the duration of fermentation. Comparing with the parental yeast D308, the alcohol production rates from Oh to 46h of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 2.253,2.243, and 2.264 (g h-1L-1), which increased 13.22%, 12.20%, and 13.76%, respectively; moreover, the alcohol yields of engineered strains D309ptpsl, D309Aathl, and D309pTAA were 117.171g/L,117.135g/L,117.555g/L, and increased 4.71%,4.67%, and 5.05%, respectively than parental strain D308 (110.905g/L) after 82 h corn mash (containing 254.906g/L glucose) fermentation.3. The reasons about the high stress resistance of engineered strains. The existence ratio of respiratory deficient yeast, cell membrane integrality, the accumulation of ROS (reactive oxygen species), and the activity of SOD (superoxide dismutase) were investigated. Under the alcohol stress condition, we found that the existence ratio of respiratory deficient yeast in engineered yeast was dramatically lower than parental yeast. The analysis of cell membrane integrality indicated the cell viability in engineered yeast was much higher than parental yeast under the same stress conditions such as high osmolity, high temperature, low pH value, and high concentration of ethanol, especially in the case of engineered strain D309pTAA. For example, the cell viability of D309pTAA still can reach to about 48.98% after the treatment of 50℃high temperature incubation for 1 h, which was 0.7 times higher than that of parental strain. Such different stress conditions indeed induce the accumulation of ROS inside cells. The maximal specific growth rate of D309pTAA was 7.95% higher than that of parental strain D308 under the existence of H2O2.The level of reactive oxygen species in engineered yeast was significantly lower, while the SOD activity in engineered yeast was higher than parental strain under the same conditions from 9.98% to 19.25%. The data shown here indicated there are two possible reasons about the protection mechanism of trehalose in yeast under stress conditions, one is the protection of cell membrane integrality in order to insure the high cell viability, and the other one is the maintenance of ROS metabolism relative enzyme (e.g., SOD) structure to increase the oxidation resistance, so that the overall stress resistance of engineered yeast was evidently higher than that of parental strain.4. The relationship between gene mutation of HOG pathway and osmotic tolerance of yeast. The sequence mutation of HOG signaling pathway within 23 Saccharomyces cerevisiae (including industrial yeast H13 and yellow wine yeast WH1) and among 10 different yeast species was investigated. For example, the number of mutant in upstream genes SLN1 and MSB2 was significantly higher other genes, especially the MSB2 gene in H13 and WH1 yeasts containing the segment of Indel (378bp). These variations may induce the different mechanisms to environmental osmotic stress resistance in different yeasts. Meanwhile, we further analysised the evolution of HOG pathway based on the pathway analysis and other statistical analysis methods. We found that the location of gene in the pathway, codon usage, protein length, protein interaction, and gene expression all play important roles in the evolution of HOG pathway. This could be the theoretical basis to further study on stress resistance mechanism of different yeasts.更多还原