精氨酸代谢在工业酵母菌株热胁迫应答机制中的关键作用

The Saccharomyces cerevisiae yeast having been proved to be more robust than bacteria, to be more tolerant to ethanol and other inhibitors present in hydrolysates of lignocellulosic materials were used in bioethanol production. One of the problems associated with simultaneous saccharification and fermentation (SSF) of cellulose is the different optimum temperatures for sacchari?cation (45-50℃) and fermentation (25-35℃) by yeasts. Due to their potential applications, the development of thermotolerant yeasts at high temperature nearly 45-50℃ has been the object of numerous studies. Many researchers focused on the breeding and construction of thermotolerant yeasts domestic and abroad by traditional methods and genetic engineering, but the strains acquired could not reach the demands of industrial production. The genome sequence availability of this microorganism has allowed the development of DNA arrays in which special PCR products for each gene are used as targets for hybridization with cDNA obtained from mRNA isolated from cells exposed to different conditions. In previous studies, they usually used the haploid laboratory modal strains homegenetic to S288c, but diploid or polyploid strains are most oftenly used in production. In our previous study comparative transcriptome between the developing superior thermotolerant industrial strain T44-2 and its original strain CE6 at different temperatures were analysed. There were lots of differential expressing genes in amino acids metabolism in T44-2, especially the arginine synthesis genes whose transcription levels were obviously increased comparing to CE6. Arginine is found to have cryoprotective activity in yeast cells; and it could inhibit protein aggregation under high temperature invitro. So we suppose arginine synthesis is very important to high temperature growth of T44-2. The recombinants with knockouting or overexpression of arginine synthesis genes were constructed, and the inner relationship between cellular arginine concentration and high temperature growth of strains were discovered. Specially, the promoter sequences and functions were analysed to discover important transcription mechanism. Our study would elucidate mechanism responsible for thermotolerance in yeast in a new vision. The findings in our research may have more theoretical evidences to develop high temperature tolerant yeasts which are currently used in industrial fermentation for future bioethanol production.

酿酒酵母是生物乙醇生产的重点研究对象，为了使其能耐受同步糖化发酵过程酶解高温，耐高温菌株选育和热胁迫应答机制研究成为热点。在以往研究中大都以S288c同源的单倍体酵母模式菌株为研究对象，而工业菌株多为二倍体和多倍体，其热胁迫应答机制更复杂。我们前期分析了耐受高温的工业酵母重组株与其出发株的比较转录组学，发现了氨基酸代谢中有大量差异表达基因，特别是精氨酸合成基因转录水平发生了显著上调。研究报道精氨酸可以保护冷冻压力下的酵母细胞；在体外实验中精氨酸可以防止蛋白受热聚合，所以我们推测酵母加强精氨酸的合成是热胁迫应答的一个新机制。为证实这一假设，我们拟通过对精氨酸代谢基因进行敲除和过表达，揭示精氨酸浓度与菌株热耐受性的关系；分析精氨酸代谢和调控基因启动子序列建立新的热胁迫应答调控模式。本课题将从新的方面探索酵母的热胁迫应答机制，为指导工业酵母的热耐受性改造提供重要理论依据。

酿酒酵母作为重要的模式微生物，耐高温菌株选育和热胁迫应答机制成为研究热点。在本研究中，发现耐热性好的工业酵母重组菌株精氨酸合成基因转录水平提高，为了研究精氨酸对酵母热耐受性的作用，构建了一系列精氨酸代谢基因的敲除菌株和过表达菌株，通过菌株在各种压力胁迫下的表型差异分析，胞内氨基酸和多胺浓度测定，关键基因的表达水平分析，以及细胞形态观察，细胞膜渗透性分析，胞内氧化还原水平测定等方法，分析得到两个重要结论，其一发现精氨酸对提高酵母的乙醇抗性具有重要作用。精氨酸酶基因过表达菌株其胞内精氨酸浓度降低，对乙醇变得敏感，在乙醇压力下生长变弱；胞内精氨酸水平提高的重组菌株，对乙醇的抗性增加，在乙醇胁迫下存活率提高。胞内精氨酸对维持细胞膜稳定性，降低胞内活性氧水平，保护细胞器维持其形态和功能起着重要作用，这可能是精氨酸保护酵母细胞提高乙醇抗性的机制。另外，发现胞内成分-多胺特别是其中的亚精胺对提高酵母热胁迫抗性有重要贡献。酵母细胞中的三种多胺：腐胺，亚精胺和精胺是由精氨酸的分解产物鸟氨酸经过脱羧等步骤衍生而成。发现所构建的增强多胺合成的重组菌株中多胺浓度特别是亚精胺浓度与对照菌株相比大幅度提高，其在高温下的生长更好。有报道多胺合成缺陷的菌株对热敏感，回补亚精胺后表型回复。推测亚精胺在提高酵母的热胁迫抗性中起到关键作用。亚精胺是真核生物翻译起始因子elf5A成熟所必须，亚精胺的氨丁基在DYS1编码DHS酶的催化下转移到elf5A，然后经LIA1编码DOHH酶的催化下使elf5A成熟。elf5A在细胞中具有多种重要的生理功能。亚精胺浓度的提高激活了elf5A成熟所必须的两个酶基因DYS1和LIA1及海藻糖合成基因TPS1表达水平的提高，推测这可能是亚精胺的作用机制，今后将会提供更多的证据。