Screening Of Thermotolerant Saccharomyces Cerevisiae Through Promoter Directed Evolution Mutagenesis,Genome Shuffling And High Temperature Acclimation

Fuel ethanol has been recognized as an alternative fuel for gasoline in the world.With the rapid development of fuel ethanol,the technical research and development of its main production strain of yeast has been developed to a certain extent.In the fermentation of ethanol,it is necessary to use cooling water to cool down when the temperature of the fermentation mash exceeds 38℃,otherwise it will inhibit the activity of Saccharomyces cerevisiae,affect the yield of ethanol and reduce the utilization of reducing sugar,increase the cost of production.But at the same time,high temperature fermentation has the advantages of fasting fermentation speed,more suitable for simultaneous saccharification and fermentation process(SSF),shortening the fermentation cycle,saving the amount of cooling water,reducing the viscosity of fermentation materials,reducing productin costs and other advantages.Therefore,it is of great significance to breed high temperature resistant strains of S.cerevisiae in industrial application.In this study,the breeding of thermotolerant yeast was studied by means of molecular modification,ARTP mutation,genome shuffling and high temperature acclimation based on the background of AY 12 gene of S.cerevisiae.The main research contents and results are as follows:(1)According to the results of SSF at 35℃ and the cell survival rate of 48h,the yeast strain AY12-G isolated from the industrial strains was selected as the parent strain.AY 12a was selected as the starting strain for experimental study on gradient weakening CYR1 gene promoter after the isolation and the determination of the fermentation performance of haploid.We constructed a series of CYR1 gene promoter 3 ’partial deletion mutants,AY12a-60,AY12a-90,AY12a-120,and fused with the selected α-type haploid into diploids,AY12-60,AY12-90,AY12-120.The strains were measured growth curve at 40℃,SSF at 35℃ and the cell survival rate of 48h,and analysis results revealed,the gradient weakening CYR1 promoter had little effect on cell survival rate of 48h and fermentation performance of S.cerevisiae AY12.(2)The strain AY12-G isolated from the industrial strains was used as the starting strain,and ARTP was used to induce it,then a total of 150 strains with better growth were obtained in the condition of 37℃.Spore production and hybridization were carried out to 150 strains for genome shuffling at 30℃ and screened at 37℃,then 137 strains with good growth performance were obtained.The 137 strains were fermented by the hydrolysis of corn protein at the temperature of 35℃ and a total of 14 strains with good fermentation performance were obtained.The 14 strains were subjected to SSF at 35℃ and determined the cells survival rate of 48h.Finally,7 strains with high fermentation performance and high temperature resistance were selected.The 7 strains of yeast were subjected to high temperature acclimation at 41℃ and 42℃,a strain with good fermentation performance and high temperature resistance was obtained and named X-130.Because the fermentation period of X-130 is 24 h longer than AY12-G,we added acid protease to the fermentation medium to shorten its fermentation cycle.The results showed that by adding acid protease,the survival rate of X-130 was increased by 84.68%at 48h compared with the original strain AY12-G,and the fermentation performance of SSF did not change significantly at 35℃ and the fermentation cycle was shortened 12h.