Locating, Screening And Preliminary Verification Of The Gene For Acetic Acid Tolerance In Saccharomyces Cerevisiae

As a green bioenergy,bioethanol has great developing potential.However,cellulose hydrolyzate is used to be substrate for Saccharomyces cerevisiae in industrial production.The composition of the cellulose hydrolyzate is complex,many of which could inhibit fermentation.Those inhibitors contain acetic acid,phenol,furfural.Acetic acid is a major inhibitor among them,which can cause chromatin agglutination along the nuclear membrane,DNA fragmentation,ROS response increasing,mitochondrial membrane depolarization.That leads to growth inhabitation,even program cell death in yeast.Although there are some omics researches on acetic acid tolerance and some improved strains by overexpressing or deleting identified genes,the explicit mechanisms of acetic acid tolerance on Saccharomyces cerevisiae is not clear,and some of them even are inconsistent.Therefore,a method is required directly reflects the essential changes of yeast to conduct in-depth analysis of the acetic acid tolerance of Saccharomyces cerevisiae.Our study analyzed acetic acid tolerance at the genome-scale level in S.cerevisiae.This method proposed by our research can also be used for the study on other stress response in yeast.Our experimental objects were two haploid strains with significant phenotypic differences of acetic acid tolerance.They were a strain with high acetic acid tolerance(namely YHA)and a strain with low acetic acid tolerance(namely YLA).We obtained three quantitative trait loci(QTL)about acetic acid tolerance by sporulation in the preliminary study.In this study,we screened the simple sequence repeats(SSR)in the three QTLs.Then we obtained 41 SSR which performed polymorphism in the two strains.After that,we analyzed the distribution of those SSRs in haploid groups.It only shrunk down a QTL in XII chromes.But failed to shrink down the rest QTLs.The results showed that QTL couldn't be fine-divided by SSR.We obtained the gene information of the two strains(YHA and YLA)by whole genome sequencing.Afterwards,we sorted the SNP information in the three QTL.Through comparing the SNP between two strains,we obtained 78 and 45 SNP in the candidate loci of two strains.Then we conducted a genetic function classification and a protein mutation prediction.Finally,we obtained ten candidate genes,namely ECI1,FRE1,HOP1,IRC20,MAM33,PRK1,THI7,UTP25,YIR007 W and YSH1.The results showed that QTL could be fine-divided by SNP.Then single gene knockout of these ten genes in YHA strain was performed to preliminary verification.Our results showed that the tolerance of single gene deletion strain ?fre1 was decreased 10 mM compared with haploid strain YHA.In contrast,the acetic acid tolerance of strain ?prk1 had increased 10 mM.FRE1 gene encodes a ferric reductase and cupric reductase,which can reduce siderophore-bound iron and oxidized copper prior to uptake by transporters;PRK1 gene codes a protein serine/threonine kinase,which regulates the organization and function of the actin cytoskeleton and reduces endocytic ability of cell through the phosphorylation of the Pan1p-Sla1p-End3 p protein complex.Combined SSR with SNP,we could identify genes about acetic acid tolerance in S.cerevisiae.And this method is suitable for the study of yeast response to other stress mechanisms.