Wine yeast sirtuins and Gcn5p control aging and metabolism in a natural growth medium

Grape juice fermentation by wine yeast is an interesting model to understand aging under conditions closer to those in nature. Grape juice is rich in sugars and, unlike laboratory conditions, the limiting factor for yeast growth is nitrogen. We tested the effect of deleting sirtuins and several acetyltransferases to find that the role of many of these proteins during grape juice fermentation is the opposite to that under standard laboratory aging conditions using synthetic complete media. For instance, . SIR2 deletion extends maximum chronological lifespan in wine yeasts grown under laboratory conditions, but shortens it in winemaking. Deletions of sirtuin . HST2 and acetyltransferase . GCN5 have the opposite effect to . SIR2 mutation in both media. Acetic acid, a well known pro-aging compound in laboratory conditions, does not play a determinant role on aging during wine fermentation. We discovered that . gcn5Δ mutant strain displays strongly increased aldehyde dehydrogenase Ald6p activity, caused by blocking of Ald6p degradation by autophagy under nitrogen limitation conditions, leading to acetic acid accumulation. We describe how nitrogen limitation and TOR inhibition extend the chronological lifespan under winemaking conditions and how the TOR-dependent control of aging partially depends on the Gcn5p function. © 2012 Elsevier Ireland Ltd.This work was funded by grants from the Spanish Ministry of Science (AGL2008-00060) and CSIC (PIE 200970I028). H.O. is supported by a F.P.I. fellowship.Peer Reviewe

Saccharomyces cerevisiae nutrient signaling pathways show an unexpected early activation pattern during winemaking

[Background] accharomyces cerevisiae wine strains can develop stuck or sluggish fermentations when nutrients are scarce or suboptimal. Nutrient sensing and signaling pathways, such as PKA, TORC1 and Snf1, work coordinately to adapt growth and metabolism to the amount and balance of the different nutrients in the medium. This has been exhaustively studied in laboratory strains of S. cerevisiae and laboratory media, but much less under industrial conditions.[Results] Inhibitors of such pathways, like rapamycin or 2-deoxyglucose, failed to discriminate between commercial wine yeast strains with different nutritional requirements, but evidenced genetic variability among industrial isolates, and between laboratory and commercial strains. Most signaling pathways involve events of protein phosphorylation that can be followed as markers of their activity. The main pathway to promote growth in the presence of nitrogen, the TORC1 pathway, measured by the phosphorylation of Rps6 and Par32, proved active at the very start of fermentation, mainly on day 1, and ceased soon afterward, even before cellular growth stopped. Transcription factor Gln3, which activates genes subject to nitrogen catabolite repression, was also active for the first hours, even when ammonium and amino acids were still present in media. Snf1 kinase was activated only when glucose was exhausted under laboratory conditions, but was active from early fermentation stages. The same results were generally obtained when nitrogen was limiting, which indicates a unique pathway activation pattern in winemaking. As PKA remained active throughout fermentation, it could be the central pathway that controls others, provided sugars are present.[Conclusions] Wine fermentation is a distinct environmental situation from growth in laboratory media in molecular terms. The mechanisms involved in glucose and nitrogen repression respond differently under winemaking conditions.This work was funded by a grant from the Spanish Ministry of Economy and Competiveness (MINECO; AGL2017-83254-R) to EM and AA. BV was supported by an F.P.U. fellowship from the Spanish Ministry of Education.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking

BackgroundYeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS), while glycerol extends it.ResultsDifferent age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS.ConclusionsManipulation of regulators of gene expression at both transcriptional (i.e., sirtuins) and posttranscriptional (i.e., mRNA binding protein Pub1) levels allows to modulate yeast life span during its biotechnological use. Due to links between aging and metabolism, it also influences the production profile of metabolites of industrial relevance

Trx2p-dependent regulation of Saccharomyces cerevisiae oxidative stress response by the Skn7p transcription factor under respiring conditions

The whole genome analysis has demonstrated that wine yeasts undergo changes in promoter regions and variations in gene copy number, which make them different to lab strains and help them better adapt to stressful conditions during winemaking, where oxidative stress plays a critical role. Since cytoplasmic thioredoxin II, a small protein with thiol-disulphide oxidoreductase activity, has been seen to perform important functions under biomass propagation conditions of wine yeasts, we studied the involvement of Trx2p in the molecular regulation of the oxidative stress transcriptional response on these strains. In this study, we analyzed the expression levels of several oxidative stress-related genes regulated by either Yap1p or the co-operation between Yap1p and Skn7p. The results revealed a lowered expression for all the tested Skn7p dependent genes in a Trx2p-deficient strain and that Trx2p is essential for the oxidative stress response during respiratory metabolism in wine yeast. Additionally, activity of Yap1p and Skn7p dependent promoters by β-galactosidase assays clearly demonstrated that Skn7p-dependent promoter activation is affected by TRX2 gene deficiency. Finally we showed that deleting the TRX2 gene causes Skn7p hyperphosphorylation under oxidative stress conditions. We propose Trx2p to be a new positive efector in the regulation of the Skn7p transcription factor that controls phosphorylation events and, therefore, modulates the oxidative stress response in yeast. © 2013 Gómez-Pastor et al.This work has been supported by grants AGL 2008-00060 and AGL2011-24353 from the Spanish Ministry of Education and Science (MEC) to EM. EG was a predoctoral fellow of the FPI program from the MEC (Spanish Ministry of Education). RG-P was a predoctoral fellow of the I3P program from the CSIC (Spanish National Research Council). RP-T was a postdoctoral fellow of the JAEDOC program from the CSIC.Peer Reviewe

Sliding-end-labelling A method to avoid artifacts in nucleosome positioning

AbstractA method, termed ‘sliding-end-labelling’, has been devised to avoid a frequent artifact in nucleosome positioning by indirect end labelling, namely the appearing of DNA fragments originated by two nuclease cuts, one of them lying within the region covered by the probe. The method is applied to the nucleosome positioning in the yeast SUC2 gene for invertase

Wine yeast peroxiredoxin tsa1 plays a role in growth, stress response and trehalose metabolism in biomass propagation

Peroxiredoxins are a family of peroxide-degrading enzymes for challenging oxidative stress. They receive their reducing power from redox-controlling proteins called thioredoxins, and these, in turn, from thioredoxin reductase. The main cytosolic peroxiredoxin is Tsa1, a moonlighting protein that also acts as protein chaperone a redox switch controlling some metabolic events. Gene deletion of peroxiredoxins in wine yeasts indicate that TSA1, thioredoxins and thioredoxin reductase TRR1 are required for normal growth in medium with glucose and sucrose as carbon sources. TSA1 gene deletion also diminishes growth in molasses, both in flasks and bioreactors. The TSA1 mutation brings about an expected change in redox parameters but, interestingly, it also triggers a variety of metabolic changes. It influences trehalose accumulation, lowering it in first molasses growth stages, but increasing it at the end of batch growth, when respiratory metabolism is set up. Glycogen accumulation at the entry of the stationary phase also increases in the tsa1∆ mutant. The mutation reduces fermentative capacity in grape juice, but the vinification profile does not significantly change. However, acetic acid and acetaldehyde production decrease when TSA1 is absent. Hence, TSA1 plays a role in the regulation of metabolic reactions leading to the production of such relevant enological molecules

Correction: reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

AbstractFollowing publication of this work [Gomez-Pastor et al, Microbial Cell Factories 2010, 9:9] we have noticed a production error in the article. Figure 1 in the original version showed incorrect results, with graphs having been duplicated in error from another figure. The correct results for Figure 1 are shown below. Legend to Figure 1 Improved performance of TTRX2 strain in biomass production process. (A) Biomass produced (continuous line) and oxygen saturation (discontinuous line) along bench-top trials of biomass propagation for T73 (black diamond), TTRX2 (white square) and TGSH1 (white triangle) strains by measuring OD600 from diluted samples. Average of three independent experiments and standard deviations are shown. (B) Fermentative capacity of yeast biomass collected at the end of the batch and fed-batch stages of growth in bench-top trials of ADY production. Biomass from wild-type T73 (black bars) and TTRX2 (white bars) were dehydrated until 8% moisture before performing the analysis. Data were normalized to the fermentative capacity of the batch sample from T73 strain. Average of three independent experiments and standard deviations are shown. Significantly different values compared to the control (p < 0.001) were marked by asterisk. (C) Sugar consumption profiles during microvinification experiments using natural Bobal must for T73 (closed symbols) and TTRX2 (open symbols) strains. The start of must fermentation was followed in detail during the first 6 hours for both strains T73 (closed symbol) and TTRX2 (open symbol). Averages were obtained from two independent experiments with three technical replicates for each one.Peer Reviewe

Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

<p>Abstract</p> <p>Background</p> <p>Wine <it>Saccharomyces cerevisiae </it>strains, adapted to anaerobic must fermentations, suffer oxidative stress when they are grown under aerobic conditions for biomass propagation in the industrial process of active dry yeast production. Oxidative metabolism of sugars favors high biomass yields but also causes increased oxidation damage of cell components. The overexpression of the <it>TRX2 </it>gene, coding for a thioredoxin, enhances oxidative stress resistance in a wine yeast strain model. The thioredoxin and also the glutathione/glutaredoxin system constitute the most important defense against oxidation. Trx2p is also involved in the regulation of Yap1p-driven transcriptional response against some reactive oxygen species.</p> <p>Results</p> <p>Laboratory scale simulations of the industrial active dry biomass production process demonstrate that <it>TRX2 </it>overexpression increases the wine yeast final biomass yield and also its fermentative capacity both after the batch and fed-batch phases. Microvinifications carried out with the modified strain show a fast start phenotype derived from its enhanced fermentative capacity and also increased content of beneficial aroma compounds. The modified strain displays an increased transcriptional response of Yap1p regulated genes and other oxidative stress related genes. Activities of antioxidant enzymes like Sod1p, Sod2p and catalase are also enhanced. Consequently, diminished oxidation of lipids and proteins is observed in the modified strain, which can explain the improved performance of the thioredoxin overexpressing strain.</p> <p>Conclusions</p> <p>We report several beneficial effects of overexpressing the thioredoxin gene <it>TRX2 </it>in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in <it>TRX2 </it>overexpressing strain can be of special interest for several industrial applications.</p

Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation

<p>Abstract</p> <p>Background</p> <p>In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation.</p> <p>Results</p> <p>In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p). Bidimensional electrophoresis and carbonylated protein identification from <it>TRX</it>-deficient and <it>TRX</it>-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the <it>TRX2 </it>overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p) from carbonylation, decreased oligomer aggregates and increased its enzymatic activity.</p> <p>Conclusions</p> <p>The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.</p