Breeding Of Ethanol Tolerant Mutant Saccharomyces Cerevisiae YN81 And Research Of Its Tolerance Mechanism

The unique flavor,mellow taste,and strongly aromatic fragrance of the high-alcoholic craft beer are gradually occupying the market of industrial beer produced on a large scale.However,the ethanol stress could inhibit cell growth,change cellular morphology and molecular function,and even cause cell death during the fermentation of high-alcoholic craft beer.The lack of ethanol-tolerant yeast and the unknown mechanism of ethanol tolerance has become the primary factors limiting the brewing of high-alcoholic craft beer.In this study,the ethanol tolerance mutant strain YN81 was bred by UV-DES mutagenesis using the Saccharomyces cerevisiae CS31 as the parental strain.The response mechanism of YN81 was investigated by compared with CS31 under 10% ethanol stress.At the same time,the molecular mechanism of ethanol tolerance of YN81 was illustrated by analyzing the DEGs before and after ethanol stress based on the transcriptome.The brewing process and beer quality of high-alcoholic craft beer brewing with mutant strain YN81 were analyzed.The study provides biological materials and process parameters for the brewing of high-alcoholic craft beer and provides a theoretical basis for the molecular mechanism of ethanol tolerance in S.cerevisiae YN81.The main experimental results of this study are as follows.1.The optimal mutagenesis conditions of UV and DES mutagenesis for S.cerevisiae CS31 were UV irradiation for 120 s and 0.05% DES mutagenesis for 30 min,respectively.UVDES compound mutagenesis of CS31 selected an ethanol highly tolerant mutant strain YN81,which could tolerate 10% ethanol,and its ethanol tolerance ability was increased by 10%compared with CS31.in addition,the cell viability of mutant strain YN81 was increased by144.04% compared with CS31 under 10%（v/v）ethanol stress.2.The stress of 10% ethanol induced a series of changes in YN81 and CS31 such as wrinkling,adhesion and cell smaller,enhanced cell membrane permeability,and increased intracellular trehalose content.The morphological changes of the mutant strain YN81 were significantly less than that of CS31 under 10% ethanol stress,while the relative cell membrane conductivity and intracellular nucleic acid exudation of the mutant strain YN81 were 32.8% and14.6% lower than that of CS31,respectively.In addition,the trehalose content of mutant strain YN81 and CS31 was increased by 96.1% and 159.8%,respectively,compared with the environment without ethanol stress,while there was no significant difference between mutant strain YN81 and CS31.Hence,the ethanol tolerance of YN81 and CS31 was correlated strongly with the cell membrane and intracellular trehalose content,but the high ethanol tolerance of mutant strain YN81 showed little correlation with trehalose content.3.DEGs between YN81 and CS31 before and after ethanol stress were 96 and 244 by transcriptomics,respectively,and it indicated that gene expression was more abundant in YN81 than CS31 under alcohol stress.The increase of 78 up-regulated genes and 12 down-regulated genes in DEGs about cell membrane and cell membrane fraction by GO functional annotation,it pointed out that mutant strain YN81 could promote the expression of a large number of membrane-related genes under ethanol stress.KEGG enrichment analysis showed that DEGs were mostly enriched in unsaturated fatty acid synthesis pathways and also involved in amino acid metabolic pathways,such as arginine biosynthesis,cysteine and methionine metabolism,glycine,serine and threonine metabolism,and alanine,aspartate and glutamate metabolism,etc.Clustering analysis of YN81 and CS31 trehalose genes showed that under ethanol stress,the expression of YN81 and the expression of trehalose synthesis genes was up-regulated and trehalase genes were down-regulated in YN81 and CS31 under ethanol stress.However,there was no significant difference in trehalose genes between YN81 and CS31.It was hypothesized that the high ethanol tolerance of YN81 was closely related to unsaturated fatty acid synthesis and amino acid metabolism genes.It is hypothesized that the high ethanol tolerance of YN81 is closely related to unsaturated fatty acid synthesis and amino acid metabolism genes.However,there was little relationship with trehalose genes,although trehalose genes are closely associated with ethanol tolerance in S.cerevisiae.4.High-alcohol craft beers were prepared using YN81 fermented with 25 °P wort and15 °P wort（containing 5% ethanol）with 10.78% and 10.02% alcohol,respectively,whereas that for CS31 was only 8.69% and 8.62%.The sensory analysis of the four prepared beer samples,YN81-25 °P,YN81-25 °P,CS31-25 °P,and CS31-15 °P,was evaluated according to the international standard ISO-11035.The results showed that YN81-25 °P had good visual characteristics,rich olfactory characteristics and pleasant taste performance,and the process of preparing high alcoholic craft beer by fermenting 25 °P wort with YN81 was good.