ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the 'fitness' of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth

Induction of the oxidative stress response has been described under many physiological conditions in Saccharomyces cerevisiae, including industrial fermentation for wine yeast biomass production where cells are grown through several batch and fed-batch cultures on molasses. Here, we investigate the influence of aeration on the expression changes of different gene markers for oxidative stress and compare the induction profiles to the accumulation of several intracellular metabolites in order to correlate the molecular response to physiological and metabolic changes. We also demonstrate that this specific oxidative response is relevant for wine yeast performance by construction of a genetically engineered wine yeast strain overexpressing the TRX2 gene that codifies a thioredoxin, one of the most important cellular defenses against oxidative damage. This modified strain displays an improved fermentative capacity and lower levels of oxidative cellular damages than its parental strain after dry biomass production.     

Zymogram profiling of superoxide dismutase and catalase activities allows Saccharomyces and non-Saccharomyces species differentiation and correlates to their fermentation performance

Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Catalase modifies yeast Saccharomyces cerevisiae response towards S-nitrosoglutathione-induced stress

Abstract

Nitric oxide is known to be a messenger in animals and plants. Catalase may regulate the concentration of intracellular ^?NO. In this study, yeast Saccharomyces cerevisiae cells were treated with 1–20 mM S-nitrosoglutathione (GSNO), a nitric oxide donor, which decreased yeast survival in a concentration-dependent manner. In the wild-type strain (YPH250), 20 mM GSNO reduced survival by 32%. The strain defective in peroxisomal catalase behaved like the wild-type strain, while a mutant defective in cytosolic catalase showed 10% lower survival. Surprisingly, survival of the double catalase mutant was significantly higher than that of the other strains used. Incubation of yeast with GSNO increased the activities of both superoxide dismutase (SOD) and catalase. Pre-incubation with cycloheximide prevented the activation of catalase, but not SOD. The concentrations of oxidized glutathione increased in the wild-type strain, as well as in the mutants defective in peroxisomal catalase and an acatalasaemic strain; it failed to do this in the mutant defective in cytosolic catalase. The activity of aconitase was reduced after GSNO treatment in all strains studied, except for the mutant defective in peroxisomal catalase. The content of protein carbonyls and activities of glutathione reductase and S-nitrosoglutathione reductase were unchanged following GSNO treatment. The increase in catalase activity due to incubation with GSNO was not found in a strain defective in Yap1p, a master regulator of yeast adaptive response to oxidative stress. The obtained data demonstrate that exposure of yeast cells to the ^?NO-donor S-nitrosoglutathione induced mild oxidative/nitrosative stress and Yap1p may co-ordinate the up-regulation of antioxidant enzymes under these conditions.     

Enhanced fermentative capacity of yeasts engineered in storage carbohydrate metabolism

During yeast biomass production, cells are grown through several batch and fed‐batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench‐top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic capacity of yeast which can be an interesting application for the food industry. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:20–24, 2015     

Biotechnological impact of stress response on wine yeast

Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.     

Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress

In this paper, we evaluate the extent to which flavonoids in red wine (catechin, epicatechin, quercetin and procyanidins) protect against hydrogen peroxide-induced oxidative stress in Fao cells. When cells were exposed to H(2)O(2), malondialdehyde (MDA) levels, oxidized glutathione (GSSG) levels and lactate dehydrogenase (LDH) release increased, indicating membrane damage and oxidative stress. All the flavonoids studied, and in particular epicatechin and quercetin, protected the plasma membrane. Only procyanidins lowered MDA levels and LDH leakage, maintained a higher reduced/oxidized glutathione ratio, and increased catalase/superoxide dismutase and glutathione peroxidase/superoxide dismutase ratios, and glutathione reductase and glutathione transferase activities. These results show that the procyanidin mixture has a greater antioxidant effect than the individual flavonoids studied, probably due to its oligomer content and/or the additive/synergistic effect of its compounds. This suggests that the mixture of flavonoids found in wine has a greater effect than individual phenols, which may explain many of the healthy effects attributed to wine.     

Improved tenderness of beef from bulls supplemented with active dry yeast is related to matrix metalloproteinases and reduced oxidative stress

Supplementing diets with active dry yeast (ADY, Saccharomyces cerevisiae) improves the carcass quality grade of beef cattle and the tenderness of beef. The relevant mechanisms have not been fully elucidated, but may be related to the effect of ADY on oxidative stress and the activity of matrix metalloproteinases (MMPs). To provide further insight into these mechanisms, this study evaluated the influence of ADY supplementation on growth performance, carcass traits, meat quality, concentrations of MMPs in serum (MMP-2, MMP-9 and MMP-13), oxidative stress indices and antioxidant capacity indices in beef cattle. Forty-six crossbred Simmental × Yanbian bulls (～18 months of age, BW 436 ± 35 kg) participated in a 145-day finishing trial. ADY supplementation significantly improved marbling deposition, intramuscular fat content, and beef tenderness (P < 0.05); altered individual fatty acid proportions in the beef and increased saturated fatty acids while decreasing polyunsaturated fatty acids (P < 0.05); significantly decreased the abundance of reactive oxygen species in serum and meat; significantly increased the level of superoxide dismutase in meat (P < 0.05); tended to increase the level of catalase (P = 0.075) in serum and glutathione reductase (P = 0.066) in meat; and increased the secretion of MMPs. The improvement of beef tenderness following ADY supplementation of finishing bulls is related to the effects of ADY on the secretion of MMPs and the lowering of oxidative stress.     

Influence of red wine fermentation oenological additives on inoculated strain implantation

Pure selected cultures of Saccharomyces cerevisiae starters are regularly used in the wine industry. A survey of S. cerevisiae populations during red wine fermentations was performed in order to evaluate the influence of oenological additives on the implantation of the inoculated strain. Pilot scale fermentations (500 L) were conducted with active dry yeast (ADY) and other commercial oenological additives, namely two commercial fermentation activators and two commercial tannins. Six microsatellite markers were used to type S. cerevisiae strains. The methodology proved to be very discriminating as a great diversity of wild strains (48 genotypes) was detected. Statistical analysis confirmed a high detection of the inoculated commercial strain, and for half the samples an effective implantation of ADY (over 80 %) was achieved. At late fermentation time, ADY strain implantation in fermentations conducted with commercial additives was lower than in the control. These results question the efficacy of ADY addition in the presence of other additives, indicating that further studies are needed to improve knowledge on oenological additives’ use.     

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts

Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.     

<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Monitoring Stress-Related Genes during the Process of Biomass Propagation of Saccharomyces cerevisiae Strains Used for Wine Making

Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.     

Effect of vitamin A treatment on superoxide dismutase-deficient yeast strains

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Δ) and CuZnSOD/MnSOD (sod1Δsod2Δ). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.