Enhanced arginine biosynthesis and lower proteolytic profile as indicators of Saccharomyces cerevisiae stress in stationary phase during fermentation of high sugar grape must: A proteomic evidence.

A strain of Saccharomyces (S) cerevisiae (ISE19), which displayed an initial good adaptation to a high sugar medium with increased acetate and glycerol production but weak overall growth/fermentation performances, was selected during the alcoholic fermentation of Cortese grape must. To obtain insights into the metabolic changes that occur in the must during growth in particular conditions (high ethanol, high residual sugars and low nitrogen availability) leading to a sluggish fermentation or even fermentation arrest, comparative in-gel proteomic analyses were performed on cells grown in media containing 200g/L and 260g/L of glucose, respectively, while the YAN (Yeast Assimilable Nitrogen) concentration was maintained as it was. Two post-translationally different arginine synthases (pIs 5.6 and 5.8) were found in higher abundances in the high glucose-grown cells, together with an increased abundance of a glycosyltransferase involved in cell-wall mannans synthesis, and of two regulatory proteins (K7_Bmh1p and K7_Bmh2p) that control membrane transport. In parallel, a proteinase K-like proteolytic enzyme and three other protein fragments (Indolepyruvate decarboxylase 1, Fba1p and Eno1p) were present in lower abundances in the high glucose condition, where oxidative stress and cell cycle involved enzymes were also found to be less abundant. The overall results suggest that in stationary phase stress conditions, leading to stuck fermentation, S. cerevisiae ISE19 decreases cell replication, oxidative stress responses and proteolytic activity, while induces other metabolic modifications that are mainly based on cell-wall renewal, regulation of the solute transport across the cell membrane and de novo arginine synthesis.

Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis.

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains.

Identification of reference genes suitable for normalization of RT-qPCR expression data in Saccharomyces cerevisiae during alcoholic fermentation.

Expression data from RT-qPCR (reverse transcription quantitative PCR) needs to be normalized to account for experimental variability among samples caused by differential yields of the transcripts in RNA extraction or in the reverse transcription. The most common method is to normalize against one or more reference genes (RG). We have selected RGs suitable for normalization of RT-qPCR raw data in Saccharomyces cerevisiae during alcoholic fermentation. The RGs were evaluated by three different statistical methods. The suitability of the selected RG sets was compared with ACT1, a commonly used non-validated single RG, by normalizing the expression of two target genes. Expression profiles of the target genes revealed the risk of misleading interpretation of expression data due to an unreliable RG.

An RT-qPCR approach to study the expression of genes responsible for sugar assimilation during rehydration of active dry yeast.

A short reactivation period in aqueous media is required for active dry yeast (ADY) to be utilised in winemaking. Rehydration restores the active metabolic conditions necessary for good fermentative and competitive abilities. We used a reverse transcription-quantitative PCR (RT-qPCR) method with relative quantification to investigate the expression of seven hexose transporter genes (HXT1-7) and one invertase-encoding gene (SUC2) during ADY rehydration in water with or without sucrose. For this, seven candidate reference genes were evaluated, and the three most stably expressed genes were selected and used for mRNA normalisation. The results show that, during the rehydration in the presence of sucrose, yeast cells are able to immediately hydrolyse this sugar into glucose and fructose as soon as they are introduced in the medium. Subsequently, differential glucose/fructose uptake occurs, which is mediated by hexose transporters. At the transcriptomic level, there is a strong induction of the high-affinity transporters, HXT2 and HXT4, and the low-affinity transporters, HXT3 and HXT1, when ADY is rehydrated with sucrose, while HXT5 and HXT6/7 are expressed at high levels with a moderate tendency to decrease. In water, the HXT2 gene was the only one of the transporter genes studied that showed significant variations. These results suggest that during rehydration, expression is not simply regulated by the affinity to hexose but is also controlled by other mechanisms that allow the cell to bypass glucose control. Moreover, the expression of SUC2 showed little variation in media with sucrose, suggesting that other invertases and/or posttranscriptional controls exist.