Screening Of Saccharomyces Cerevisiae For Greengage Wine And Its Mechanism Of Acid Tolerance

Greengages?Prunus mume Sieb.Et Zucc?belong to the Rosaceae family and they are grown mainly in China,Japan,and Korea.It contains amino acids,phenols,minerals,and organic acids,together with specific ingredients?anthocyanins,terpenes,and phytosterols?with potentially beneficial effects for human health.Greengage wine is a popular alcoholic beverage in Asia because of its favorable taste,unique flavor,nutritional value,and functional properties.However,those strains on the market fail to solve the problems with the fermentation of high acidic fruits.The low p H environment during the production of fruit wines badly inhibits the microbial activities,possibly leading to a longer fermentation time and poor taste,and eventually affecting the overall wine quality.Therefore,screening of brewing yeast with acid resistance is the fundamental method to solve the problem in greengage wine.In the present study,we used a combined strategy comprising the atmospheric pressure room temperature plasma?ARTP?mutagenesis technology,a considerable time-saving high-throughput screening?HTS?,as well as the adaptive laboratory evolution?ALE?strategy to improve the fermentation performances of yeasts at low p H in greengage wine production.Whole genome re-sequencing and RNA-seq analysis of the two strains revealed the metabolic regulation mechanism suggesting a significant divergence in adaptation to low p H,and the important genes related to acid tolerance were identified and knockouted.We also confirmed the role of cell membranes of S.cerevisiae in acid tolerance.Finally,the evolved strain was applied to the production of greengage wine.The study contributes to the winery by providing theoretical basis and practical guidance for high-quality greengage wine production.The main results were described as follows:?1?Saccharomyces cerevisiae?S.cerevisiae?ET008-c54 was successfully selected as the target strain from the mutant library through the combination of the ARTP,the HTS,and the ALE.Compared with the parental strain ET008,ET008-c54 displayed great excellent performances in survival rate,fermentation time,aroma profile,and genetic stability.More specifically,the survival rate of ET008-c54 at p H 2.5 reached as high as 95.8%compared with 9.4%for the parental strain ET008.And this 10.2 times improvement is the highest reported to date.The fermentation time of greengage wine was reduced by 45.5%?from 22days to 12 days?,and the content of main aroma compounds and benzaldehyde were respectively increased by 51.9%(from 17.8 mg·L^-1 to 27.0 mg·L^-1)and 54.3%(from 1.8mg·L^-1 to 2.8 mg·L^-1),which solved the problem of lengthened fermentation and poor flavor of greengage wine.In addition,the morphological characterization,reactive oxygen species?ROS?,catalase?CAT?and superoxide dismutase?SOD?activities,and the relative expression of the corresponding genes of ET008-c54 were further characterized the excellent fermentation performance,and the findings also point to the fact that these outstanding performances are pivotal for S.cerevisiae ET008-c54 to be a promising industrial strain.?2?To uncover the genomic alterations responsible for the altered phenotypes,the parental strain ET008 and the evolved strain ET008-c54 were subjected to whole genome re-sequencing.A total of 2310 mutations including 1671 single nucleotide polymorphisms?SNP?and 639 insertion-deletions?In Del?were found,and the mutation sites related to acid tolerance and flavor were analyzed and mined by combining the references.To further investigate the tolerance mechanism of the intracellular processes,we performed transcriptome analysis.Based on the results,688 differentially expressed genes comprising364 up-regulated and 324 down-regulated genes were identified.We combined the analysis of whole-genome resequencing,transcriptome,and the annotation results of bioinformatics.Genes involved in cell membrane components,iron metabolism,and steroids biosynthesis played an important role in adapting to acidic environments.Genes related to the biosynthesis of carbon metabolism,secondary metabolites,and fatty acid metabolism,had an important role in the formation of the main flavor compounds.Specifically,the genes of?i.e.,ERG5,FET3,FIT2,FRE1,GCN1,INO1,IZH4,and OPT2?related to ergosterol synthesis and ferrous iron uptake in ET008-c54 may be beneficial for the tolerance mechanism against low p H.Also,we showed that the biosynthesis genes?i.e.,ARO7,ATF1,BAT1,EEB1,EHT1,and OLE1?of fatty acid ethyl esters and aromatic amino acid responsible for aroma production were important in ET008-c54.Moreover,an exciting finding was that the gene IZH4 was highly possibly participating in cellular responses underlying acid tolerance,which provided more possible operating targets for the construction of other strains.?3?Here we reported the use of sg RNA-guided Cas9 nucleases to generate knockout mutants of acid-resistant genes,and also verified that the growth of the knockout strain ET008-c54?Izh4?abbreviated as?Izh4?was inhibited at p H 2.5.The stress responses between the ET008,ET008-c54,and?Izh4 during acid stress were compared based on the micro-environment and cell membrane analysis.Compared to ET008-c54,the intracellular p H?p H_i?of?Izh4 and plasma membrane H⁺-ATPase slightly decreased,indicating that IZH4 had little effect on intracellular p H_i and plasma membrane H⁺-ATPase.The strain?Izh4 maintains p H_i homeostasis by utilizing the H⁺-ATPase to expel H⁺at the expense of ATP consumption.At the level of cell membranes,the content of unsaturated fatty acids and ergosterol in?Izh4decreased by 38.8%and 34.0%,respectively.In addition,the permeability of?Izh4 cell membrane enhanced by 10.8%,and its fluidity and integrity reduced by 9.3%and 46.5%,respectively.The above results show that the gene IZH4 plays an important role in the acid telerance of cell membrane,and prove that IZH4 regulates the integrity of cell membrane by affecting the synthesis of unsaturated fatty acids and ergosterol.?4?ET008-c54 was used for the fermentation of greengage wine.The relevant parameters of greengage wine were optimized using central composite design?CCD?experiments after one-factor experimental design by studying the individual and interactive effects of three variables,including fermentation temperature,material-liquid ratio,and sugar content.The optimal conditions for fermented greengage wine were determined to be a fermentation temperature of 20?,a material-liquid ratio of 1:2.0,and a sugar content of 200g?L^-1.After 12 days,greengage wine?abbreviated as FT?had the typical almond aroma and desired taste.Taste-active compounds of fermented greengage wines,including amino acid,organic acid,phenolic acid,and sugar,were characterized using high performance liquid chromatography?HPLC?.It was found that the concentration of each taste-active compounds in fermented greengage wines was quite different,and 11 taste-active compounds were identified as the key contributors in greengage wine using dose-over-threshold?Do T?.Gas chromatography-mass spectrometry and gas chromatography-olfactometry?GC-MS?was used to compare and characterized the differences between FT and commercially available greengage wine.It was confirmed that the contents and kinds of flavor compounds in FT were significantly higher than other greengage wines,and 13 flavor compounds were identified as the key contributors in greengage wine using odor activity value?OAV?.In order to further improve the quality of greengage wine,partial least squares regression?PLSR?was used to analyze the contribution of taste and aroma compounds of greengage wine to sensory attributes.It was found that citric acid,succinic acid,tartaric acid,glucose,fructose,sucrose,and tannic acid had important contributions to the taste of greengage wine,while ethyl acetate,ethyl butanoate,ethyl hexanoate,ethyl octanoate,ethyl decanoate,3-methylbutanol,linalol,and benzaldehyde had important contributions to the aroma of greengage wine.Here,the 200L pilot-scale results show that the physicalchemical characterization,taste-active compounds,aroma-active compounds,and microbiological indicators of fermented greengage wine with ET008-c54 as the fermentation strain are good,which meets the general technical requirements in GB2758-2012 and QB/T 5476-2020 for fruit wine.