Study On Weakening Ethanol Synthesis Capacity Of Saccharomyces Cerevisiae

With the global warming,the average alcohol of wine in the world has increased by 2%v/v in the past 30 years.Excessive alcohol in wines exerts a variety of bad influences on the wine sensory quality.Therefore,it is urgent to reduce the synthesis of ethanol properly in winemaking process by reasonable methods.Using the Saccharomyces cerevisiae with weakened ethanol synthesis capacity for producing low-alcohol wines are the most simple and economical strategy.In this study,the screening of low-ethanol-yield S.cerevisiae was based on the S.cerevisiae YS59 mutant library,which was constructed with the random mutation of the transcription factor Spt15p.Transcriptomics and metabolomics methods were used to elucidate the physiological mechanism of the reduced ethanol synthesis capacity of S.cerevisiae from the overall level.The main results and conclusions are as followed:（1）A S.cerevisiae strain with 5 amino acid mutation sites in SPT15 gene was obtained and the ethanol yield of the strain was reduced by 34.89%.Based on the yield of ethanol,the fermentation of SPT15 mutant strains were conducted in Triple M and a strain YS59-409 with low ethanol yield was gained.The ethanol yield of YS59-409 was 0.239 g/g sugar which was reduced by 34.89%compared with the control.Moreover,both biomass and CO₂ weight loss of YS59-409 were increased compared with the control.The results showed that this strain may use more sugar for biomass formation and CO₂ production,which led to the reducing of the carbon flow used for ethanol synthesis.Analysis of mutation points of transcription factor Spt15p showed that there were 5 sense amino acid mutation points（Ile 46 Met,Asp 56 Gly,Ser118 Pro,Tyr 195 His and Leu 205 Ser）in the YS59-409.However,the fermentation of the five recombinant strains indicated that ethanol yield of strains increased by 8.07%,10.99%,1.05%,3.12%,and 10.37%respectively.（2）Transcriptomic analysis of YS59-409 was performed and nine suspected key genes were obtained.The RNA-Seq technique was used to study the transcriptional characteristics of gene of the low-producing ethanol strain YS59-409 in the mid-fermentation stage,and the differentially expressed genes（DEGs）associated with ethanol synthesis were to be screened compared with the YS59-p Y16 strain.The key DEGs were excavated combined with differential gene interaction network analysis.The results demonstrated that there were 203significantly differentially expressed genes（101 down-regulated and 103 up-regulated genes respectively）.Through GO and KEGG pathway analysis,it was found that the low-yield ethanol of S.cerevisiae in the present study may be related to the shunting of carbon sources to other metabolic terminals（mainly glycerol）,the decreased absorption efficiency of hexose,and the stress response to ethanol of yeast.Combined with the analysis of differential gene interaction network,the 9 suspected key genes finally were discovered and they were RPL22B,REX4,IMA1,YNL195C,SOR2,YNL194C,HXT2,RGI2,and AQY3 respectively.（3）Metabolomics technique based on LC-MS was used to perform metabolomic analysis on the low-producing ethanol strain YS59-409 to find differential metabolites and metabolic pathway compared with the YS59-p Y16 strain.The results showed that a total of 42 differential metabolites were discovered in this study.Through metabolic pathway analysis,the most significant differential metabolites in the metabolic pathway were L-glutamine,L-glutamic acid,adenylosuccinate,pyruvate,succinate semialdehyde,and glutathione.Analysis showed that glutamate and glutamine metabolism may be related to the glycerol pathway,which may balance excess NADH in the glutamate and glutamine metabolic pathways through increasing glycerol production,resulting in reducing the carbon source for ethanol synthesis.The up-regulated of genes and metabolites associated with the kynurenine pathways participating in the de novo synthesis of NAD⁺are possibly due to an insufficient intracellular NAD⁺content（excess of NADH）,which was consistent with the inference that the excess of NADH was balanced by glycerol synthesis.This may be the main reason for low ethanol production of yeast.（4）RGI1/2 genes significantly affects the ethanol synthesis capacity of S.cerevisiae.The RGI1/RGI2 overexpression strains（BY4741 p Y26/RGI1 and BY4741 p Y26/RGI2）were constructed using the one step cloning method and the RGI1/RGI2 knockout strains（BY4741p Y26/ΔRGI1 and BY4741 p Y26/ΔRGI2）was constructed using the Cre-Loxp system respectively.Their fermentation characteristics were studied in Triple M.As the results indicated,compared with the control strain,the fermentation rate of the RGI1/2 gene overexpressing and knockout strains was slightly decreased and the ethanol yield was significantly reduced（20.43%,19.34%,21.36%,and 15.47%respectively）,which demonstrating that RGI1/2 played an important role in the ethanol metabolism of S.cerevisiae.