絮凝酵母海藻糖合成酶基因TPS1启动子区的克隆和乙醇胁迫下启动子活性的变化

Cloning of the promoter region of the Trehalose-6-phosphate synthase gene TPS1 of the self-flocculating yeast and exploration of the promoter activity on ethanol stress

摘要:

提高生物能源生产菌株对各种胁迫因素的耐受性对于提高生产过程的经济性和高效生产生物能源具有重要的意义。对酿酒酵母乙醇耐性的分子机制的研究，可揭示影响其耐受性的关键基因，并通过代谢工程操作定向提高酵母菌的乙醇耐受性，从而提高燃料乙醇的生产效率。海藻糖对酵母菌在多种环境胁迫下的细胞活性具有保护作用，但其对乙醇耐性分子机制的研究还不够深入。克隆了自絮凝酵母Saccharomyces cerevisiae flo的海藻糖-6-磷酸合成酶基因TPS1的启动子区域，利用pYES2.0载体骨架，构建了PTPS1启动绿色荧光蛋白EGFP标记基因的报告载体，并转化酿酒酵母ATCC4126。对酵母转化子在含有7％和10％乙醇的生长培养基中的EGFP的表达情况进行相对荧光定量分析，发现PTPS1活性在7％乙醇存在下受到强烈诱导。EGFP表达量对高温和高糖胁迫无明显差别，显示了TPS1启动子对乙醇浓度的特异响应。研究结果表明，絮凝酵母海藻糖的合成是对乙醇胁迫的保护性反应。

Abstract:

Improving stress tolerance of the microbial producers is of great importance for the process economy and efficiency of bioenergy production. Key genes influencing ethanol tolerance of brewing yeast can be revealed by studies on the molecular mechanisms which can lead to the further metabolic engineering manipulations for the improvement of ethanol tolerance and ethanol productivity. Trahalose shows protective effect on the cell viability of yeast against multiple environmental stress factors, however, further research is needed for the exploration of the underlying molecular mechanisms. In this study, the promoter region of the trehalose-6-phosphate synthase gene TPS1 was cloned from the self-flocculating yeast Saccharomyces cerevisiae flo, and a reporter plasmid based on the expression vector pYES2.0 on which the green fluorescence protein EGFP was directed by the TPS1 promoter was constructed and transformed to industrial yeast strain Saccharomyces cerevisiae ATCC4126. Analysis of the EGFP expression of the yeast transformants in presence of 7% and 10% ethanol revealed that the PTPS1 activity was strongly induced by 7% ethanol, showing specific response to ethanol stress. The results of this study indicate that trehalose biosynthesis in self-flocculating yeast is a protective response against ethanol stress.