Construction Of Wine Yeast With Enhanced Glycerol And Glutathione Production

Glycerol is a major by-product of alcoholic fermentation and is reported to have positiveeffects on the sensory properties of wines. Glutathione which served as an oxidative buffercan inhibit the decrease of aromatic esters and terpene alcohols during the storage of whitewines, and is responsible for preventing the deterioration of wines. To improve wine taste andflavor stability, a novel indigenous yeast with enhanced glycerol and glutathione (GSH)production for winemaking was constructed.In this study, the gene ALD6encoding one of aldehyde dehydrogenases (ALDH) of theindigenous wine yeast LFE1225was replaced by the GPD1and CUP1gene cassette, whichare responsible for glycerol-3-phosphatase dehydrogenase (GPDH) and copper resistancerespectively. The constructed GA expression cassette (ald6Δ::GPD1-CUP1) was inserted intothe locus of the ALD6gene in the S. cerevisiae strain LFE1225by homologous recombinationtechnology. The recombinant strains were screened on YEPD plates containing8mmol/LCuSO4and the positive recombinant was designated as RA. The colony PCR test showed thatthe GA expression cassette have integrated into the locus of ALD6of the chromosome of RA,and the enzyme activities test showed that the GPDH activity of the recombined strain RA wassignificantly increased by33.7%(P<0.05), while aldehyde dehydrogenases was decreased by22.53%compared to LFE1225.Furthermore, the α-acetohydroxyacid synthase gene ILV2of the recombinant strain RAwas disrupted by integration of GSH1gene which encoding γ-glutamylcysteine synthetaseand CUP1gene cassette. The positive transformant was screened on YEPD plates containing9mmol/L CuSO4and was designated as RIA. The colony PCR test exhibited that the GIexpression cassette have integrated into the locus of ILV2of the chromosome of RIA. TheGSH content of RIA after fermentation in YEPD medium was enhanced by17.82%and19.04%when compared with S. cerevisiae LFE1225and recombinant RA, respectively.The fermentation capacity of the recombinant was similar to that of the wild-type strain,with an increase of20.6%and18.7%in glycerol and GSH content compared with thewild-type strain, reaching to7.1±0.2g/L and43.9±0.1mg/L respectively. The final aceticacid and ethanol contents in the model wine made with RIA were0.7±0.3g/L and81±1g/Lrespectively,5.4%and8.5%lower compared with LFE1225. No heterologous DNA was harbored in the recombinant, therefore, it may be more applicable for the commercial use.