Investigation On Stress Tolerance Of Saccharomyces Cerevisiae Through Regulation Of Cyclic Adenosine Monophosphate Signal Pathway

Saccharomyces cerevisiae characteristic with excellent growth and fermentation capabilities has been widely used in ethanol industries.Yeasts are confronted with several environmental stresses such as hyper-osmotic stress,high temperature,which can negatively affect yeast growth rate and fermentation performance.This will cause some economic losses in related fermentation industry.In 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.Our study changes the key genes in the cAMP signal pathway and its branch to construct strains with elevated stress resistance as well as normal fermentation.The constructed mutants with good tolerance have great signification in industrial application.The main research contents and results were as follows:(1)Based on the existing strain AY 12a in the laboratory,the RAS2 gene knockout and PDE2 gene overexpression were achieved by using the long primer knockout method and the intracellular recombination method,respectively,using the URA3 gene as the selectable marker.The mutant AY12a-ras2? knocked out RAS2 gene and the mutant strain AY12a-pde2,which was overexpressed,were successfully constructed by PCR verification.The resistance of the mutant was measured and it was found that AY12a-pde2 had some tolerance to temperature.At the same time,the mutant and AY 12a 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 of mutant strain AY12a-pde2 was increased by 7.05%,while that of mutant strain AY12a-ras2? was lower than AY 12a.(2)Based on the existing strain AY 12a in the laboratory,the strong promoter PGK1p was added to the N-terminus of MSN2?MSN4 and GIS1 gene to achieve overexpression of the gene by using the intracellular recombination method,using URA3 gene as the screening marker.The mutants AY12a-msn2?AY12a-msn4 and AY12a-gis1 were successfully constructed by PCR verification.The results showed that the mutant had some temperature tolerance except AY12a-msn2.At the same time,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 survival rates of AY12a-gis1 and AY12a-msn4 were increased by 5.13%and 3.85%respectively,the survival rate of 48 h cells was increased,the residual sugar content decreased,while the AY12a-msn2 was decreased and the fermentation time was the longest.(3)Based on the existing strain AY12a in the laboratory,SCH9 and TOR1 were knocked out by long primer knockout method using URA3 gene as screening marker.The mutants AY12a-sch9? and AY12a-tor1? knocked out SCH9 and TOR1 genes were successfully constructed by PCR verification.The resistance of the yeast was measured,and it was found that the mutant strain AY12a-sch9? had a certain temperature resistance.At the same time,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 content of AY12a-sch9? ethanol was increased by 8.33%,the survival rate of 48 h was increased,and the content of residual sugar decreased and the fermentation time was prolonged,while the alcohol content of mutant AY12a-tor1? decreased compared with AY12a.