转录因子MSN2基因过表达对酿酒酵母耐受性的影响

Investigation on Stress Tolerance of Saccharomyces cerevisiae of Overexpression of Transcription Factor MSN2 Gene

摘要:

cAMP信号通路在调控酵母细胞代谢、增殖、分化及压力抗性的获得过程中具有重要的作用。工业应用中对酵母的耐受性有很高的要求，在发酵过程中，胁迫环境如高温、高渗透压、营养饥饿和高浓度酒精毒性等不可避免，故而提高酵母菌种的耐受性，可以提高菌种的发酵性能，降低发酵过程中的能量消耗。本文构建的突变株在工业应用方面具有重要的意义。以实验室现有菌种AY12a为出发菌株，URA3基因作筛选标记，利用胞内重组，在MSN2基因的N端加上强启动子PGK1p以实现基因的过表达，最终通过PCR验证，成功构建突变株AY12a-msn2。对酵母进行耐受性的测定，发现AY12a-msn2不具有一定的耐高温性能。同时将突变株与AY12a进行玉米高温浓醪发酵，并测定发酵完成后的酒度、残糖、48 h细胞存活率、CO2失重及发酵时间。结果发现突变株AY12a-msn2酒度下降，残糖含量上升，48 h细胞存活率上升，发酵时间较长。

Abstract:

For the yeast Saccharomyces cerevisiae, the cAMP signal transduction pathway plays an important role in the control of metabolism, proliferation, differentiation and acquisition of stress resistance. During the fermentation process, stress environments such as high temperature, high osmotic pressure, nutrient starvation, high alcohol toxicity, etc. are unavoidable. There is a high requirement for yeast tolerance in industrial applications. It can increase the fermentation performance of strains and reduce the energy consumption during fermentation. The constructed mutant has great signification in industrial application. Based on the existing strain AY12a in the laboratory, the strong promoter PGK1p was added to the N-terminus of MSN2 gene to achieve overexpression of the gene by using the intracellular recombination method, using URA3 gene as the screening marker. The mutant AY12a-msn2 was successfully constructed by PCR verification. The results showed that the mutant had no temperature tolerance. The mutant and AY12a were tested for very high gravity (VHG) fermentation, and the alcohol content, residual sugar, 48 h cell survival rate, CO2 weight loss and fermentation time were determined. The results showed that the alcohol content decrease, the residual sugar content and the survival rate of 48 h cells increased. The mutant had longer fermentation time.