Investigation On Stress Tolerance Of Saccharomyces Cerevisiae Through Promoter Modification Of CYR1

The yeast Saccharomyces cerevisiae characteristic with excellent growth and fermentation capabilities has been widely used in ethanol and food industries.Yeasts are confronted with several environmental stresses such as hyper-osmotic stress,high temperature,which individually or collectively can negatively affect yeast growth rate and fermentation performance.This will cause some economic losses in related fermentation industry.Therefore,it is important to construct stress-tolerant mutants.Ras-cAMP signaling pathway plays an important role in the regulation of stress resistance,and adenylate cyclase is the key enzyme of the signaling pathway To construct strains with elevated stress resistance as well as normal growth rates,the CYR1 promoter transformation was performed to finely modulate the expression of CYR1(CTR1-encoded adenylate cyclase).The main research contents and results were as follows:(1)The CYR1 promoter,which was serially truncated from the 3’end by 30 bp,was inserted into YIplac211,and five integration plasmids were contructed.A serial nucleotide sequences were truncated form the 3’end of CYR1 promoter of AY15a without any extraneous DNA residual by two-step integration,and URA3 acted as the selectable marker.The mRNA levels of CYR1 in yeasts AY15a-30,AY15a-60,AY15a-90,AY15a-120,and AY15a-150 were 89.1%,76.8%,60.1%,56.7%,and 41.3%of that of the parent strain AY15a,respectively.Stress-tolerance tests showed that AY15a-90 and AY15a-120 had more tolerance to heat shock than the parent strain.In addition,AY15a-90 and AY15a-120 also exhibited better tolerant to 8%(v/v)ethanol.The results of very high gravity(VHG)fermentation showed that the ethanol production rates of AY15a-90 and AY15a-120 were similar with AY15a in VHG fermentation at 30℃,whereas that of AY15a-90 and AY15a-120 was much higher than that of AY15a at 40℃.The ethanol yield for AY15a-90 and AY15a-120 were 14.0%and 14.6%higher,respectively,than that for AY15a at 40 ℃.(2)The 3’end of CYR1 promoter of BY-14a was precisely truncated 90 bp and 120 bp by two-step integration,and URA3 acted as the selectable marker.The mRNA levels of CYR1 in yeasts BY-14a-90 and BY-14a-120 were 43.1%and 20.3%of that of the parent strain BY-14a,respectively.The fermentation performance,the survival rate after freezing,and biomass of recombinant strains were investigated.The results showed that the survival rate of BY-14a-90 and BY-14α-120 after freezing were 71.8%and 97.0%higher,respectively,than that of parent strain.However,the recombinant strains displayed a little reduction of fermentation performance and biomass compared with BY-14α.This phenomenon may be caused by the low CYR1 transcription level,then the growth and fermentation performance of the mutants was negatively affected.