Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking.

BACKGROUND: Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC). RESULTS: In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out. CONCLUSIONS: Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.

The Yeast eIF2 Kinase Gcn2 Facilitates H2O2-Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production.

Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

Saccharomyces cerevisiae Cytosolic Thioredoxins Control Glycolysis, Lipid Metabolism, and Protein Biosynthesis under Wine-Making Conditions.

Thioredoxins are small proteins that regulate the cellular redox state, prevent oxidative damage, and play an active role in cell repair. Oxidative stress has proven to be of much relevance in biotechnological processes when the metabolism of Saccharomyces cerevisiae is mainly respiratory. During wine yeast starter production, active dry yeast cytosolic thioredoxin Trx2p is a key player in protecting metabolic enzymes from being oxidized by carbonylation. Less is known about the role of redox control during grape juice fermentation. A mutant strain that lacked both cytosolic thioredoxins, Trx1p and Trx2p, was tested for grape juice fermentation. Its growth and sugar consumption were greatly impaired, which indicates the system's relevance under fermentative conditions. A proteomic analysis indicated that deletion of the genes TRX1 and TRX2 caused a reduction in the ribosomal proteins and factors involved in translation elongation in addition to enzymes for glycolysis and amino acid biosynthesis. A metabolomic analysis of the trx1Δ trx2Δ mutant showed an increase in most proteogenic amino acids, phospholipids, and sphingolipids and higher fatty acid desaturase Ole1p content. Low glycolytic activity was behind the reduced growth and fermentative capacity of the thioredoxin deletion strain. All three hexokinases were downregulated in the mutant strain, but total hexokinase activity remained, probably due to posttranslational regulation. Pyruvate kinase Cdc19p presented an early level of aggregation in the trx1Δ trx2Δ mutant, which may contribute to a diminished hexose metabolism and trigger regulatory mechanisms that could influence the level of glycolytic enzymes.IMPORTANCE Oxidative stress is a common hazardous condition that cells have to face in their lifetime. Oxidative damage may diminish cell vitality and viability by reducing metabolism and eventually leading to aging and ultimate death. Wine yeast Saccharomyces cerevisiae also faces oxidative attack during its biotechnological uses. One of the main yeast antioxidant systems involves two small proteins called thioredoxins. When these two proteins are removed, wine yeast shows diminished growth, protein synthesis, and sugar metabolism under wine-making conditions, and amino acid and lipid metabolism are also affected. Altogether, our results indicate that proper redox regulation is a key factor for metabolic adaptations during grape juice fermentation.

Sch9p kinase and the Gcn4p transcription factor regulate glycerol production during winemaking.

Grape juice fermentation is a harsh environment with many stressful conditions, and Saccharomyces cerevisiae adapts its metabolism in response to those environmental challenges. Many nutrient-sensing pathways control this feature. The Tor/Sch9p pathway promotes growth and protein synthesis when nutrients are plenty, while the transcription factor Gcn4p is required for the activation of amino acid biosynthetic pathways. We previously showed that Sch9p impact on longevity depends on the nitrogen/carbon ratio. When nitrogen is limiting, SCH9 deletion shortens chronological life span, which is the case under winemaking conditions. Its deletion also increases glycerol during fermentation, so the impact of this pathway on metabolism under winemaking conditions was studied by transcriptomic and metabolomic approaches. SCH9 deletion causes the upregulation of many amino acid biosynthesis pathways. When Gcn4p was overexpressed during winemaking, increased glycerol production was also observed. Therefore, both pathways are related in terms of glycerol production. SCH9 deletion increased the amount of the limiting enzyme in glycerol biosynthesis, glycerol-3-P dehydrogenase Gpd1p at the protein level. The impact on the metabolome of SCH9 deletion and GCN4 overexpression differed, although both showed a downregulation of glycolysis. SCH9 deletion downregulated the amount of most proteinogenic amino acids and increased the amount of lipids, such as ergosterol.

RNA binding protein Pub1p regulates glycerol production and stress tolerance by controlling Gpd1p activity during winemaking.

Glycerol is a key yeast metabolite in winemaking because it contributes to improve the organoleptic properties of wine. It is also a cellular protective molecule that enhances the tolerance of yeasts to osmotic stress and promotes longevity. Thus, its production increases by genetic manipulation, which is of biotechnological and basic interest. Glycerol is produced by diverting glycolytic glyceraldehyde-3-phosphate through the action of glycerol-3-phosphate dehydrogenase (coded by genes GPD1 and GPD2). Here, we demonstrate that RNA-binding protein Pub1p regulates glycerol production by controlling Gpd1p activity. Its deletion does not alter GPD1 mRNA levels, but protein levels and enzymatic activity increase, which explains the higher intracellular glycerol concentration and greater tolerance to osmotic stress of the pub1∆ mutant. PUB1 deletion also enhances the activity of nicotinamidase, a longevity-promoting enzyme. Both enzymatic activities are partially located in peroxisomes, and we detected peroxisome formation during wine fermentation. The role of Pub1p in life span control depends on nutrient conditions and is related with the TOR pathway, and a major connection between RNA metabolism and the nutrient signaling response is established.

Up-regulation of Retrograde Response in yeast increases glycerol and reduces ethanol during wine fermentation.

Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.

Impact of Hydrogen Peroxide on Protein Synthesis in Yeast.

Cells must be able to respond and adapt to different stress conditions to maintain normal function. A common response to stress is the global inhibition of protein synthesis. Protein synthesis is an expensive process consuming much of the cell's energy. Consequently, it must be tightly regulated to conserve resources. One of these stress conditions is oxidative stress, resulting from the accumulation of reactive oxygen species (ROS) mainly produced by the mitochondria but also by other intracellular sources. Cells utilize a variety of antioxidant systems to protect against ROS, directing signaling and adaptation responses at lower levels and/or detoxification as levels increase to preclude the accumulation of damage. In this review, we focus on the role of hydrogen peroxide, H2O2, as a signaling molecule regulating protein synthesis at different levels, including transcription and various parts of the translation process, e.g., initiation, elongation, termination and ribosome recycling.

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 tsa1D 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.

A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to H2O2 and Is the Target of a Small Molecule.

Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2 The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2in vitro and significantly increased Wis1 activation by low levels of H2O2in vivo We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A.

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Yeast thioredoxin reductase Trr1p controls TORC1-regulated processes.

The thioredoxin system plays a predominant role in the control of cellular redox status. Thioredoxin reductase fuels the system with reducing power in the form of NADPH. The TORC1 complex promotes growth and protein synthesis when nutrients, particularly amino acids, are abundant. It also represses catabolic processes, like autophagy, which are activated during starvation. We analyzed the impact of yeast cytosolic thioredoxin reductase TRR1 deletion under different environmental conditions. It shortens chronological life span and reduces growth in grape juice fermentation. TRR1 deletion has a global impact on metabolism during fermentation. As expected, it reduces oxidative stress tolerance, but a compensatory response is triggered, with catalase and glutathione increasing. Unexpectedly, TRR1 deletion causes sensitivity to the inhibitors of the TORC1 pathway, such as rapamycin. This correlates with low Tor2p kinase levels and indicates a direct role of Trr1p in its stability. Markers of TORC1 activity, however, suggest increased TORC1 activity. The autophagy caused by nitrogen starvation is reduced in the trr1Δ mutant. Ribosomal protein Rsp6p is dephosphorylated in the presence of rapamycin. This dephosphorylation diminishes in the TRR1 deletion strain. These results show a complex network of interactions between thioredoxin reductase Trr1p and the processes controlled by TOR.

Herbicide glufosinate inhibits yeast growth and extends longevity during wine fermentation.

Glufosinate ammonium (GA) is a widely used herbicide that inhibits glutamine synthetase. This inhibition leads to internal amino acid starvation which, in turn, causes the activation of different nutrient sensing pathways. GA also inhibits the enzyme of the yeast Saccharomyces cerevisiae in such a way that, although it is not used as a fungicide, it may alter yeast performance in industrial processes like winemaking. We describe herein how GA indeed inhibits the yeast growth of a wine strain during the fermentation of grape juice. In turn, GA extends longevity in a variety of growth media. The biochemical analysis indicates that GA partially inhibits the nutrient sensing TORC1 pathway, which may explain these phenotypes. The GCN2 kinase mutant is hypersensitive to GA. Hence the control of translation and amino acid biosynthesis is required to also deal with the damaging effects of this pesticide. A global metabolomics analysis under winemaking conditions indicated that an increase in amino acid and in polyamines occurred. In conclusion, GA affects many different biochemical processes during winemaking, which provides us with some insights into both the effect of this herbicide on yeast physiology and into the relevance of the metabolic step for connecting nitrogen and carbon metabolism.

Interplay among Gcn5, Sch9 and mitochondria during chronological aging of wine yeast is dependent on growth conditions.

Saccharomyces cerevisiae chronological life span (CLS) is determined by a wide variety of environmental and genetic factors. Nutrient limitation without malnutrition, i.e. dietary restriction, expands CLS through the control of nutrient signaling pathways, of which TOR/Sch9 has proven to be the most relevant, particularly under nitrogen deprivation. The use of prototrophic wine yeast allows a better understanding of the role of nitrogen in longevity in natural and more demanding environments, such as grape juice fermentation. We previously showed that acetyltransferase Gcn5, a member of the SAGA complex, has opposite effects on CLS under laboratory and winemaking conditions, and is detrimental under the latter. Here we demonstrate that integrity of the SAGA complex is necessary for prolonged longevity, as its dismantling by SPT20 deletion causes a drop in CLS under both laboratory and winemaking conditions. The sch9Δ mutant is long-lived in synthetic SC medium, as expected, and the combined deletion of GCN5 partially suppresses this phenotype. However it is short-lived in grape juice, likely due to its low nitrogen/carbon ratio. Therefore, unbalance of nutrients can be more relevant for life span than total amounts of them. Deletion of RTG2, which codes for a protein associated with Gcn5 and is a component of the mitochondrial retrograde signal, and which communicates mitochondrial dysfunction to the nucleus, is detrimental under laboratory, but not under winemaking conditions, where respiration seems not so relevant for longevity. Transcription factor Rgm1 was found to be a novel CLS regulator Sch9-dependently.