Rearrangement Of Ring Chromosomes To Improve The Tolerance Of Inhibitors In Saccharomyces Cerevisiae

Biofuel is a very clean and environmentally friendly energy source,and cellulosic ethanol is the commonly used biofuels.Cellulosic ethanol is mainly produced from lignocellulose as the raw material by Saccharomyces cerevisiae in industry.However,during the pretreatment process,many inhibitors will appear as by-products that inhibit the growth of S.cerevisiae.Therefore,it is necessary to strengthen the tolerance of S.cerevisiae to inhibitors.In this study,the three most typical inhibitors(furfural,acetic acid,and phenol)in the pretreatment process were mixed as compound inhibitors(FAP)to explore the improvement of the tolerance of yeast.The starting strain contained ring synthetic chromosomes,and large-scale genome rearrangement of the strain was drived through the SCRaMbLE(Synthetic Chromosome Rearrangement and Modification by Lox Psym-mediated Evolution)system.Through multiple rounds of SCRaMbLE and adaptive domestication technology,five resistant strains with stable phenotypes were screened out,and the whole genome was sequenced.Through the comparison of the sequencing data,the structural variations on the synthetic chromosome X(syn X)and the synthetic chromosome V(syn V)were analyzed.It was found that there was deletion of 183,738-184,470 bp,deletion of631,427-637,506 bp,invertion of 276,636-585,463 bp,replication of 74,602-101,013 bp on syn X,deletion of 370,514-376,907 bp,invertion of 219,928-223,708 bp on syn V and some other variations.The 183,738-184,470 bp deletion on syn X was verified by RT-qPCR and yeast homologous recombination.It was found that overexpression of YJL120 W could improve the tolerance of strain to FAP.The cell doubling time was reduced to 9.91 h from 17.18 h in the strain overexpressing YJL120 W under the condition of 60% FAP,while the ethanol production rate was also increased from 0.05 g/L/h to 0.25 g/L/h and the time to degrade furfural was also shortened from 156 h to 24 h.Simultaneously,the gene expression levels of YNL241 C,YCR073W-A,YGR248 W,YNR034W,YGR256 W,and YHR183 W in the oxidation stage of the pentose phosphate pathway have increased,indicating that YJL120 W has a regulatory effect on the pentose phosphate pathway.In addition,the deletion of YJL078 C,YJR130C and YJR131 W could also improve the tolerance of yeast to FAP.In this study,strains tolerant to FAP were screened through SCRaMbLE.The structural variation in the rearranged strain was analyzed and the affected target genes were found.The tolerance mechanism of target genes was also analyzed.This study provides some ideas for the research on the tolerance of S.cerevisiae to FAP,and has certain reference value for improving the efficiency of cellulosic ethanol production.