Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass
© Gómez-Pastor et al; licensee BioMed Central Ltd. 2010
Received: 20 November 2009
Accepted: 12 February 2010
Published: 12 February 2010
Wine Saccharomyces cerevisiae 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 TRX2 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.
Laboratory scale simulations of the industrial active dry biomass production process demonstrate that TRX2 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.
We report several beneficial effects of overexpressing the thioredoxin gene TRX2 in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in TRX2 overexpressing strain can be of special interest for several industrial applications.
The production of Saccharomyces cerevisiae biomass has become a powerful industry in the last years due to the increasing demand for modern winemaking practices, bread-manufacturing processes and also to its consumption as a dietary complement. Many selected natural yeast strains are now produced and commercialized as active dry yeast to be used as starters for must fermentation . Several selection criteria for the choice of natural strains have been well established according to different aspects of the winemaking process, from the facilitation of specific stages to the improvement of wine organoleptic properties . The search for wine yeast strains with innovative characteristics has traditionally relied on the isolation and screening for new yeast strains from grape and wine samples [3, 4]. However, numerous research laboratories worldwide have succeeded in the generation, by genetic manipulation, of strains capable of improving processing efficiency, fermentative performance, and wine's sensory quality [1, 5]. The commercial viability of genetically modified wine yeast strains has already been discussed .
During the last years, several studies have been carried out in order to analyze the complexity of the industrial biomass production process to use this knowledge as a tool for wine yeast strains improvement [7, 8]. Previous stress gene marker analysis during bench-top trials of wine yeast biomass propagation demonstrated the induction of specific stress-related genes and enabled us to determine the environmental disturbances to which cells are dynamically exposed . The data indicated that osmotic and, specially, oxidative stresses are the main two adverse conditions that Saccharomyces cerevisiae strains sense during the process. The relevance of oxidative stress for industrial yeasts performance including wine and brewing strains, and also different technological processes as brewing, wine making and biomass propagation has been pointed out in several studies [8–10]. The specific induction observed for the TRX2 gene in early stages of the biomass propagation process, especially under aeration conditions, suggested its involvement in the oxidative stress response. TRX2 gene codes for the yeast cytoplasmic thioredoxin 2, one of the most important redox controls together with glutathione/glutaredoxin system . There are evidences pointing that both antioxidant systems are linked in the response against oxidative stress during specific biomass propagation, revealing novel overlapping roles between these two antioxidant systems [11, 12]. Overexpression of the TRX2 gene in a wine yeast strain (TTRX2) produced an increase in the fermentative capacity in the biomass obtained at the end of the process . The technological advantage of this improvement in the wine yeast properties encourage us to go far on the study of the oxidative stress response by different technical approaches. Despite the large accessible bibliography about oxidative stress mutants in laboratory yeast strains , very little is published about the effects of overexpression of oxidative stress genes , particularly in industrial yeasts.
The cellular response to reactive oxygen species involves a very complex network of biochemical mechanisms, from transcriptional control of gene expression to enzymatic repair of damaged cellular structures . The transcriptional response affects a large number of genes participating in the different redox control and defense systems. In addition to the general stress response factors Msn2/4p, two specific transcriptional factors are mainly involved in reprogramming gene expression in response to oxidative injury, Yap1p and Skn7p . Although they partially cooperate, the Skn7p factor controls only a subset of genes involved in the thioredoxin system, whereas the Yap1p factor is required for the induction of all the oxidative responsive genes . Furthermore, other proteins, as Trx2p, have been implicated in the Yap1p-related oxidative response pathway .
Many genes induced under oxidative challenge code for antioxidant enzymatic activities which play important roles in cellular protection, both as ROS detoxifiers and regulators of the protein redox state . Catalases, superoxide dismutases, and peroxidases are crucial to reduce the presence of ROS and several differentially regulated activities can be detected. In addition to these enzymatic detoxifiers, other proteins, such as thioredoxins and glutaredoxins, participate in the protection of protein activity against oxidative damage by repairing chemically modified proteins or by modulating the redox state of protein sulphydryl groups . Several isoforms of these two types of thiol oxidoreductases are present in different subcellular compartments and act coordinately to maintain and recover full protein functioning. A complex interplay exists between these two main protein redox regulatory systems through the major redox buffer in eukaryotic cells, glutathione . The redox potential of the GSH/GSSG couple determines the redox cellular state and is greatly influenced by ROS generation, both by addition of external oxidants or by metabolic leakage of electrons. The role of GSH in the connection of the two oxidoreductase systems has been pointed by the behavior of many different mutants and particularly by the increased GSH concentration and redox potential of the couple GSH/GSSG in a double trx1trx2 mutant [13, 19].
Despite the presence of adaptative responses to oxidative stress, ROS accumulation can exceed the preventing and scavenging capacity of antioxidant defenses and cause damages on structural and functional cell components, such as nucleic acids, carbohydrates, lipids or proteins . The cell membrane is a critical target for free radical attack, as lipid peroxidation can lead to cell leakage and death. High level of lipid peroxidation has been described as a common consequence of ROS accumulation, and also it has been related to the protective action of several antioxidant molecules . Proteins are also major targets for ROS and different protein modification can lead to unfolding or alteration of protein structure . Carbonylation is a well characterized, irreversible, and non-enzymatic modification of proteins which is most widely used as biomarker for oxidative damage of proteins .
In this work we found that the enhanced fermentative capacity produced by overexpression of thioredoxin 2 in a wine yeast strain correlated to an increased induction of several oxidative response genes, and also to increased activity of several ROS scavenging enzymes. Additionally, both total glutathione and the GSH/GSSG ratio were higher in the modified strain. In accordance to these effects on the protection mechanisms against oxidative stress, the TTRX2 strain displayed lower levels of molecular damage, and both lipid peroxidation and protein carbonylation were diminished. The improved response to oxidative stress caused by thioredoxin overexpression can explain the beneficial effects on fermentative performance, both in lab conditions and in microvinification experiments on natural musts, and does not affect negatively the analyzed oenological parameters of the produced wine.
Improved performance of the TTRX2 strain during biomass propagation and winemaking
The winemaking performance of T73 and TTRX2 strains after ADY biomass production was assessed in controlled microvinification experiments on natural musts. As can be observed in Figure 1C, both strains presented similar sugar consumption profiles, especially at the end of microvinification, when sugar exhaustion occurred at the same time. However, the profiles are different at the beginning of the fermentation, when TTRX2 strain showed a significant reduction in the lag phase, starting the fermentation at least 4 hours before T73 strain. This phenotype was observed in three independent experiments, where no growth differences were observed between different precultures.
Wine parameters and final aroma concentration
Mean ± SD (g/l)
9.85 ± 0.151
9.6 ± 0.2
0.55 ± 0.01
0.58 ± 0.02
16.5 ± 0.43
20.37 ± 3.17
117.9 ± 10.2
94.32 ± 6.85
Secondary aroma compounds
Mean ± SD (mg/l)
60.96 ± 7.03
22.83 ± 9.53
59.76 ± 1.63
49.12 ± 5.71
180.66 ± 6.56
79.97 ± 1.56
26.57 ± 6.16
44.75 ± 9.55
0.045 ± 0.002
0.33 ± 0.06
0.19 ± 0.04
1.14 ± 0.16
1.6 ± 0.02
0.16 ± 0.007
0.231 ± 0.006
0.016 ± 0.003
0.028 ± 0.001
0.069 ± 0.012
1.07 ± 0.027
0.495 ± 0.028
0.81 ± 0.073
0.27 ± 0.068
0.49 ± 0.025
TTRX2 strain displays increased expression of oxidative stress related genes during the biomass production process
Increased activity of ROS scavenging enzymes in TTRX2 strain
TTRX2 strain overaccumulates reduced glutathione during the fed-batch phase
TRX2 gene overexpression causes diminished lipid peroxidation
Determination of lipid oxidation has been widely used to evaluate negative effects of ROS . Lipid oxidation was measured during the biomass propagation experiments for the parental T73 and the genetically modified TTRX2 and TGSH1 strains (Figure 6C). The wild type industrial strain T73 showed a main peak of oxidation during the batch phase, when cells start to consume the ethanol produced during the initial fermentative phase. Strain TGSH1 showed a similar profile but lipid peroxidation was higher during the fed-batch phase, where catalase activity was lower than for T73. TTRX2 strain was more uniform and it did not show increased lipid peroxidation. This result supports the idea that TTRX2 strain has a better defense against the endogenous oxidative stress generated during the batch phase of the biomass propagation process.
The decrease in total glutathione levels in wild type T73 strain (Figure 6A) was coincident with the increase in lipid oxidation (Figure 6C). In fact, diminished glutathione level was already described in biomass propagation conditions for brewing yeasts . Interestingly, TTRX2 strain displayed low glutathione levels from the beginning, probably due to a redox compensation as a consequence of the increased TRX2 gene levels. In contrast, it was more protected against lipid oxidation.
Protein oxidation damage is reduced by TRX2 gene overexpression
Studies by our group and others have found that oxidative stress response is critical during industrial yeast biomass propagation [7–9]. Therefore we designed several strategies of genetic modification in order to enhance the response of the commercial wine strain T73 against oxidative damage. Here we present the most promising one: the TTRX2 strain, carrying an overexpression of the thioredoxin-encoding gene TRX2, which shows a dual improvement. This strain is able to increase the produced biomass yield during cell propagation experiments and to increase the fermentative capacity after ADY biomass production that is observed as a "fast start" winemaking phenotype. Our results show that TTRX2 strain has an enhanced molecular oxidative stress response, and then it suffers a lower degree of oxidative damage on macromolecular components, such as lipids and proteins.
Optimized industrial yeast propagation processes are designed to produce the highest biomass yield by forcing the oxidative catabolism of molasses sugars. In the case of wine yeasts, the produced biomass is dehydrated to preserve the starter as ADY, due to its seasonal use for musts inoculation after grape harvest. The whole industrial process has critical impact on the quality of the produced biomass affecting viability, vitality and fermentation rate, and these parameters are determinant for yeast performance in winemaking. Previous results pointed out that the oxidative stress response of yeast cells is important during the biomass production process , probably because the respiratory metabolism and even fermentation  generates endogenous ROS. Furthermore, ROS generation during dehydration can also damage cell structures . Our hypothesis was that a modified yeast strain showing enhanced oxidative stress defenses would improve their performance during the ADY biomass production process and also afterwards, in winemaking. Several modifications in genes related to the glutathione and thioredoxin redox systems were carried out in the commercial wine yeast strain T73 and the overexpression of TRX2 gene was selected for further studies. Simulations of the industrial biomass propagation process were developed at laboratory scale and they showed that this modified strain displays higher fermentative capacity during batch and fed-batch stages, and also that it produces higher biomass yield at the end of the process. These properties of the strain TTRX2 can have high economical impact for yeast biomass producers. Furthermore, when ADY biomass was used as starter for microvinification experiments in natural must, the lag phase of fermentations conducted by TTRX2 strain was significantly reduced compared to T73. This "fast starting" phenotype is a valuable character because it can help rapid microbial stabilization at the beginning of grape must fermentation and ensure the domination of the selected strain over natural strains. Furthermore, it presents increased content of aroma compounds, probably due to a better performance of less oxidized enzymes implicated in their synthesis.
The beneficial effect of thioredoxin increased dosage on oxidative stress defense has been extensively described in different organisms and circumstances. Overexpression of human TRX1 in transgenic mice (TRX1-Tg) has been reported to provide a protective activity against oxidative stress-related disorders such as post-ischaemic reperfusion injury in the brain  kidney , diabetic nephropathy  and adriamycin-induced cardiotoxicity . Beneficial effects related to oxidative response has been also described in plants . In yeasts, TRX2 was found to be essential for a proper oxidative response against hydroperoxides . The oral administration of thioredoxin has been proposed in clinical and functional foods applications due to its antioxidant, anti-inflammatory and antiallergenic properties [33, 34]. From this point of view, the strain TTRX2 will be valuable for the industrial production of thioredoxin and also for production of functional foods.
An effort to understand the physiology of the TTRX2 strain at molecular level and explain its efficiency improvement has been made. Experimental data obtained in laboratory strains adding external oxidants support that TRX2 is involved in Yap1p reduction and inactivation . But this mechanism is not completely understood because trx- mutant strains present normal inactivation after diamide treatment. Accordingly to our results in industrial strains, overexpression of TRX2 causes increased responses in oxidative defenses in the industrial modified strain TTRX2. In fact, Yap1-regulated genes, and also other oxidative stress related genes, showed increased mRNA levels in TTRX2 strain. These results can be explained in several ways. First, our experiments were developed with industrial strains that show important differences in gene expression compared with laboratory strains . Second, Yap1-mediated regulation was studied in laboratory conditions as a response to external oxidants. In contrast, the transcriptional responses observed in this study were likely due to internal production of ROS derived from respiratory metabolism. A possible explanation for the behavior of the TTRX2 strain is that increased levels of Trx2p alter the NADPH/NADP+ ratio, as these molecules participate in the maintenance of the thioredoxin redox state. Furthermore, increased GSH/GSSG ratio can alter NADPH/NADP+ balance since glutathione reductase enzyme need NADPH to reduce GSSG to GSH. Changes in this redox buffer would be sensed as an oxidative alteration and it would promote stronger oxidative stress responses.
Several data support that TTRX2 strain has enhanced defense against oxidative stress. It shows increased mRNA levels of oxidative stress related genes, and also increased glutathione content, increased GSH/GSSG ratio and increased activities of antioxidant enzymes, such as superoxide dismutases and catalase. All these elements would produce an enhanced protection against ROS-mediated oxidation. In fact, here we present consistent data supporting that TTRX2 strain displays lower oxidation damages than the control strain. Lipid oxidation is drastically increased in the control strain during ethanol respiration in the batch phase (20-30 h), probably due to oxidative metabolism and endogenous ROS generation. However, the TTRX2 strain presents much lower level of lipid oxidation. Interestingly, glutathione levels change during biomass production process. A possible explanation for the glutathione decrease in the wild type strain T73 can be its use in glutathionylation reactions. This protein modification it's important in different organisms for the correct function of many proteins, including glycolytic enzymes, in response to oxidative stress conditions . The low glutathione levels in the TTRX2 strain during first hours of fermentation could be due to a higher degree of glutathionylation events. This effect would be in agreement with the increased expression of oxidative stress genes, supporting that TTRX2 overexpression generates a cellular signal that activates responses against oxidation.
The level of cellular oxidation was also followed by determination of carbonyl groups in proteins. Strain TTRX2 presents much lower level of protein carbonylation during the biomass propagation process than the control T73 strain, corroborating that thioredoxin overaccumulation protects against oxidation events. Protein oxidation leads to altered protein structure and function  so negative effects on cell physiology are expected when cells suffer ROS attacks. The diminished protein oxidation observed for the TTRX2 strain is then consistent with its better performance in biomass propagation trials and could be related to a better preserved activity of specific metabolic or detoxifying enzymes. Also, our previous work showed that specific proteins involved in sugar fermentation were heavily damaged in oxidative conditions like chronological or replicative aging .
In this study we have shown the beneficial effects of enhancing stress redox response by overexpressing a cytosolic thioredoxin in a wine yeast strain. The strain TTRX2 that overexpresses the TRX2 gene produces higher biomass in an ADY biomass industrial production process modelization at laboratory scale. Furthermore, TTRX2 yeast biomass presents a better quality as indicates its increased fermentative capacity and decreased lag phase. It's also able to produce wines with enhanced aroma compounds in microvinification experiences.
Enhanced performance of TTRX2 strain is a consequence of its increased defense against redox stresses. In fact, it presents increased levels of antioxidant glutathione and reduced levels of proteins and lipids oxidation and increased levels of ROS scavenging enzymes Sod1p and Sod2p. The increased defense against redox stress of TTRX2 can be just a consequence of the increased content of cytosolic thioredoxin due to its antioxidant properties. Our data points to other side effects derived from the implication of TRX2 in gene transcription regulation since TTRX2 strain presented increased transcription of redox genes. However, the fact that this regulation is not completely understood and the complexity of the relations between thioredoxin and glutathione systems make difficult to establish the specific role of TRX2 in TTRX2 strain.
We think TTRX2 strain can be very valuable to be used as a cell factory for added-value products or for several industrial applications as biomass production and production of flavored wines.
Materials and methods
Yeast strains, plasmids and cultivation conditions
S. cerevisiae industrial strain T73 (CECT1894) is a natural diploid strain isolated from Alicante (Spain) musts  and has been commercialized by Lallemand Inc. (Montreal, Canada). This strain has been previously used in several studies [7, 8, 35, 40, 41] and has proven to be a good wine yeast model.
The YEp-TRX2 plasmid was obtained by subcloning a 0.7 kb Eco RI fragment containing the yeast TRX2 gene into the episomal yeast plasmid YEp352 vector. TTRX2 strain  is a genetically modified T73 strain obtained by transformation of a ura- T73 derivate  with YEp-TRX2 following the lithium acetate procedure as modified by . The empty YEp352 vector was also introduced in the T73 ura- strain as a control of its influence in growth conditions and oxidative stress experiments. The TGSH1 strain was obtained transforming T73 ura- with a YEp352-GSH1 plasmid, kindly provided by Dr A.K. Bachhawat.
Precultures for industrial biomass propagation experiments were prepared in YPD liquid medium (1% Yeast extract, 2% Peptone, 2% Glucose) and were incubated at 30°C with shaking (250 rpm) during 12 h. YPD plates (1% Yeast extract, 2% Peptone, 2% Glucose, 2% Agar) were used for growth analysis in laboratory conditions. The liquid medium YPGF (1% Yeast extract, 2% Peptone, 10% Glucose, 10% Fructose) was used to test the fermentative capacity of cells produced in industrial conditions.
Molasses medium (diluted to 60 g of sucrose liter-1 for batch phase or 100 g of sucrose liter-1 for fed-batch phase) was supplemented with 7.5 ml of (NH4)2SO2 liter-1, 3.5 g of KH2PO4 liter-1, 0.75 g of MgSO47H2O liter-1, 10 ml of vitamin solution liter-1, and 1 ml of antifoam 204 (Sigma, St. Louis, Mo.) liter-1. Molasses and mineral solutions were autoclaved separately. The vitamin solution containing 50 mg of D-biotin liter-1, 1 g of calcium pantothenate liter-1, and 1 g of thiamine hydrochloride liter-1 was filter sterilized (0.2-μm pore size) prior to use in the molasses medium.
Industrial propagation conditions
Biomass propagation experiments were designed with two growth stages, batch and fed-batch, in a bioreactor BIOFLO III (NBS, New Jersey, USA), and technical parameters (agitation, pH and fed rate) were established as previously described [7, 8]. The bioreactor containing 2 liter of sterilized molasses medium at pH 4.5 was inoculated to an initial optical density of 0.05 (OD600 = 0.05) from overnight YPD precultures. During batch phase cells consumed all the sucrose present into the medium using a fermentative metabolism. When sucrose was finished (12-15 h), cells changed its metabolism to a respiratory metabolism allowing the consumption of the ethanol produced, until approximately 40 h of the process. During this phase pH was allowed to freely vary between 4 and 5. When the ethanol was finished, the fed-batch phase started feeding the reactor continuously with molasses medium by a type 501 peristaltic pump (Watson-Marlow, Falmouth, United Kingdom) at the desired flow rate, avoiding fermentative metabolism. During the fed-batch phase, the reactor pH was maintained at 4.5 by the automatic addition of 42.5% H3PO3 or 1 M NaOH. Dissolved oxygen, measured with an electrode (Mettler-Toledo), was maintained above 20% by a PID control system that allowed the automatic modification of the agitation speed between the range limits of 300 to 500 rpm. Four independent experiments were carried out for each T73 and TTRX2 strains.
Analysis and quantification of mRNA
Genes and primers used for the amplification of DNA probes
Probe length (bp)
Biomass dehydration and measurement of fermentative capacity
Yeast biomass was dehydrated under air flux in an oven at 30°C until approximately 8% relative humidity and kept at room temp for few days. For determination of fermentative capacity, dehydrated cells were inoculated (107 cells/ml) in YPGF and incubated at 30°C and 65 rpm. CO2 production (in ml) was measured every 20 min for 6 h in a Chittick instrument (American association of cereal Chemist, 12-10). After sample normalization to cell number, fermentative capacity is expressed as ml of CO2 produced per 107cells.
Determination of glutathione
Glutathione was determined as described previously . Collected cells (100 mg) were washed twice with phosphate-buffer saline (PBS, pH 7.4) and suspended in 1 ml ice-cold 8 mM HCl, 1.3% (w/v) 5-sulphosalicylic acid. Cells were broken by vortexing at 4°C with 0.5 g of glass beads in four series of 1 min alternated with 1 min incubation on ice. Cell debris and proteins were pelleted in a microcentrifuge for 15 min (13000 rpm at 4°C), and supernatants were used for glutathione determination. For total GSH determination, supernatant was used directly in 200 μl of total volume reaction while GSSG determination was carried out in the same volume reaction with 2 μl of 1 M 2-vinyl-piridine for 1 h at room temperature. Reduced GSH values were obtained as the difference between total glutathione and oxidized glutathione. Glutathione levels are expressed as nmol per mg of cells.
Quantification of lipid peroxidation
Quantification of lipid peroxidation was carried out by reaction of thiobarbituric acid with the malondialdehide (MDA) product of oxidized fatty acid breakage  Cells (50 mg) were collected, washed twice with distilled water and then extracted by vortexing with 0.3 g glass beads in 0.5 ml of 50 mM sodium phosphate buffer, pH 6.0, 10% trichloroacetic acid (TCA), in three series of 1 min alternated with 1 min incubation on ice. After centrifugation at 13000 rpm for 10 min, 300 μl of supernatants were mixed to 100 μl of 0.1 M EDTA and 600 μl 1% thiobarbituric acid in 0.05 M NaOH, and then incubated at 100°C 15 min. After cooling on ice and centrifugation to eliminate precipitates, malondialdehide was measured by the absorbance at 535 nm. The molar absorptivity of MDA (1.56 × 105 M-1 cm-1) was used to express lipid peroxidation levels as pmoles of MDA per mg of cells.
Quantification of protein carbonylation
Cells were collected, washed twice in distilled water and then extracted by vortexing with 0.4 g glass beads in 0.5 ml of extraction buffer (25 mM imidazol, 2 mM EDTA pH 7.0) and a mix of protease inhibitors (200 μM phenylmethylsulfonyl fluoride (PMSF), 20 μM TPcK, 200 μM pepstatin A). Protein concentration was determined by the Bradford assay  and all samples were diluted in SDS 6% using the Bio-Rad Protein Assay following manufacturer instructions. Protein carbonylation in crude extracts was measured by dinitrophenilhydrazine (DNPH) derivatization and western immunodetection of protein-bound 2,4-dinitrophenylhydrazones . The anti-2,4-dinitrophenol antibody (DAKO Ref. V0401) was used at a 1/4000 dilution and the secondary antibody (goat anti-rabbit HRP conjugated, Amersham) was used at a 1/25000 dilution. Signals in blots were visualized using Lumigen TMA-6 (Amersham) and images were analyzed using Bio-Rad Lumicapt software.
Determination of antioxidant enzyme activities
Cell extracts for enzymatic determinations were prepared using glass beads and assayed as described for catalase  and superoxide dismutase  activities. Mn-SOD and CuZn-SOD activities were analyzed after separating cell lysates in native acrylamide gel electrophoresis. Activity stained bands were measured with a densitometer and the relative density calculated by the Quantity One software (Bio-Rad). Data were normalized to total loaded protein from Coomassie blue stained gels and to the amount of Sod1p and Sod2p proteins from western blot assays. Western Blots were performed in triplicate. To analyze the amounts of Sod1p and Sod2p, cell extracts were separated in SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Antibodies against superoxide dismutases were purchased from Stress-Gene (SOD-111, Mn-SOD) and Chemicon (AB1237, CuZn-SOD). A secondary HRP-conjugated anti-rabbit antibody was used for detection. Image acquisition was performed in a ChemiDoc CCD camera (Bio-Rad). Three independent experiments were carried out.
For microvinification experiments, natural must from Bobal red grapes (Requena, Spain) was sterilized with 0.2% (vol/vol) dimetyl dicarbonate for 48 h at 4°C to allow decomposition. 0.2 liter glass bottles were completely filled and capped to reproduce the anaerobic conditions of wine fermentation. Musts were inoculated (5 × 106 cells/ml) of rehydrated air-dried biomass (24 h, 30°C) from 24 h grown yeast cultures on molasses. Bottles were incubated at 28°C with gentle shaking (125 rpm) without aeration. Culture growth was followed by measuring optical density at 600 nm (OD600).
Determination of reducing sugar concentration, ethanol, and enological parameters
Reaction with DNS (10 g liter-1 3,5-dinitrosalicylate, 16 g liter-1 sodium hydroxide, 300 g liter-1 potassium sodium tartrate 4-hydrate), and measurement of the developed color (as OD at 540 nm) was used for quantification of reducing sugars, with a glucose standard curve between 0 and 2 g liter-1. Alcoholic grade (g liter-1) was determined by the alcohol dehydrogenase (ADH) enzymatic assay, with ethanol and NAD+ as substrates, by measuring the NADH produced (OD at 340 nm) and using a standard curve 0 and 0.15 mM ethanol. Other enological parameters (acetic acid, glycerol and acetaldehyde) were quantified by automated enzymatic determination (ECHO; Logotech).
The gas chromatographic analysis of volatile compounds was carried out using a Hewlett-Packard capillary gas chromatograph, model 5890 series II, controlled with a 3365 Chemstation (flame ionization detector; Supelcowax 10 [Supelco, Inc., Bellafonte, Pa.] fused-silica capillary column [30 m by 0.25 mm, 0.25 μm film]). Final microvinification samples (1.5 ml) were analyzed using 0.005% (w/v) 2-heptanone as internal standard. Each volatile compound was quantified within each sample as a dimensionless number corresponding to the area of the chromatographic peak divided by the area of the internal standard.
Sample averages were compared to measure significance of the difference using Fisher Exact t-Test online tool SISA (Simple Interactive Statistical Analysis). P-values below 0.05, 0.01 or 0.001 (labeled as *, ** or *** respectively) were used.
Reduced glutathione form
Oxidized glutathione form
Phosphate buffered saline
Radical oxygen species
N-tosyl-L-phenylalanylchloromethyl ketone dinitrophenilhydrazine.
We are grateful with Dr. A.K. Bachhawat that kindly donate us YEp352-GSH1 plasmid. This work was supported by grants AGL 2005-00508 and AGL 2008-00060 from "Ministerio de Educación y Ciencia" (MEC), Spain. R.P.T was a predoctoral fellow of Generalitat Valenciana and R.G-P. is a predoctoral fellow of the I3P program of "Consejo Superior de Investigaciones Científicas" (Spain).
- Pretorius IS: Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast. 2000, 16: 675-729. 10.1002/1097-0061(20000615)16:8<675::AID-YEA585>3.0.CO;2-B.View Article
- Dequin S: The potential of genetic engineering for improving brewing, wine-making and baking yeasts. Appl Microbiol Biotechnol. 2001, 56: 577-588. 10.1007/s002530100700.View Article
- Querol A, Barrio E, Ramon D: Population dynamics of natural Saccharomyces strains during wine fermentation. Int J Food Microbiol. 1994, 21: 315-323. 10.1016/0168-1605(94)90061-2.View Article
- Manzanares P, Ramon D, Querol A: Screening of non-Saccharomyces wine yeasts for the production of beta-D-xylosidase activity. Int J Food Microbiol. 1999, 46: 105-112. 10.1016/S0168-1605(98)00186-X.View Article
- Schuller D, Casal M: The use of genetically modified Saccharomyces cerevisiae strains in the wine industry. Appl Microbiol Biotechnol. 2005, 68: 292-304. 10.1007/s00253-005-1994-2.View Article
- Cebollero E, Gonzalez-Ramos D, Tabera L, Gonzalez R: Transgenic wine yeast technology comes of age: is it time for transgenic wine?. Biotechnol Lett. 2007, 29: 191-200. 10.1007/s10529-006-9236-y.View Article
- Perez-Torrado R, Bruno-Barcena JM, Matallana E: Monitoring stress-related genes during the process of biomass propagation of Saccharomyces cerevisiae strains used for wine making. Appl Environ Microbiol. 2005, 71: 6831-6837. 10.1128/AEM.71.11.6831-6837.2005.View Article
- Perez-Torrado R, Gomez-Pastor R, Larsson C, Matallana E: Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth. Appl Microbiol Biotechnol. 2009, 81: 951-960. 10.1007/s00253-008-1722-9.View Article
- Gibson BR, Lawrence SJ, Boulton CA, Box WG, Graham NS, Linforth RS, Smart KA: The oxidative stress response of a lager brewing yeast strain during industrial propagation and fermentation. FEMS Yeast Res. 2008, 8: 574-585. 10.1111/j.1567-1364.2008.00371.x.View Article
- Landolfo S, Politi H, Angelozzi D, Mannazzu I: ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochim Biophys Acta. 2008, 1780: 892-898.View Article
- Grant CM: Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol Microbiol. 2001, 39: 533-541. 10.1046/j.1365-2958.2001.02283.x.View Article
- Herrero E, Ros J, Belli G, Cabiscol E: Redox control and oxidative stress in yeast cells. Biochim Biophys Acta. 2008, 1780: 1217-1235.View Article
- Drakulic T, Temple MD, Guido R, Jarolim S, Breitenbach M, Attfield PV, Dawes IW: Involvement of oxidative stress response genes in redox homeostasis, the level of reactive oxygen species, and ageing in Saccharomyces cerevisiae. FEMS Yeast Res. 2005, 5: 1215-1228. 10.1016/j.femsyr.2005.06.001.View Article
- Tarrio N, Garcia-Leiro A, Cerdan ME, Gonzalez-Siso MI: The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis. FEMS Yeast Res. 2008, 8: 597-606. 10.1111/j.1567-1364.2008.00366.x.View Article
- Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO: Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 2000, 11: 4241-4257.View Article
- Lee J, Godon C, Lagniel G, Spector D, Garin J, Labarre J, Toledano MB: Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem. 1999, 274: 16040-16046. 10.1074/jbc.274.23.16040.View Article
- Delaunay A, Pflieger D, Barrault MB, Vinh J, Toledano MB: A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell. 2002, 111: 471-481. 10.1016/S0092-8674(02)01048-6.View Article
- Grant CM, Maciver FH, Dawes IW: Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. Curr Genet. 1996, 29: 511-515. 10.1007/BF02426954.View Article
- Garrido EO, Grant CM: Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides. Mol Microbiol. 2002, 43: 993-1003. 10.1046/j.1365-2958.2002.02795.x.View Article
- Toledano MB, Delaunay A, Biteau D, Spector D, Azevedo D: Oxidative stress responses in yeasts. Yeast Stress Responses. Edited by: Hohmann S, Mager WH. 2003, Berlin: Springer Verlag, 241-287. full_text.View Article
- Dani C, Bonatto D, Salvador M, Pereira MD, Henriques JA, Eleutherio E: Antioxidant protection of resveratrol and catechin in Saccharomyces cerevisiae. J Agric Food Chem. 2008, 56: 4268-4272. 10.1021/jf800752s.View Article
- Cabiscol E, Ros J: Oxidative damage to proteins: structural modifications and consequences in cell function. Redox proteomics: from protein modification to cellular dysfunction and disease. Edited by: Dalle-Donne I, Scaloni A, Butterfiled DA. 2006, Hoboken: John Wiley and Sons, 399-471.View Article
- Levine RL, Stadtman ER: Carbonylated proteins and their implications in physiology and pathology. Redox proteomics: from protein modification to cellular dysfunction and disease. Edited by: Dalle-Donne I, Scaloni A, Butterfiled DA. 2006, Hoboken: John Wiley and Sons, 563-603.
- Costa VM, Amorim MA, Quintanilha A, Moradas-Ferreira P: Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radic Biol Med. 2002, 33: 1507-15. 10.1016/S0891-5849(02)01086-9.View Article
- Ohtake Y, Yabuuchi S: Molecular cloning of the gamma-glutamylcysteine synthetase gene of Saccharomyces cerevisiae. Yeast. 1991, 7: 953-961. 10.1002/yea.320070907.View Article
- Franca MB, Panek AD, Eleutherio EC: Oxidative stress and its effects during dehydration. Comp Biochem Physiol A Mol Integr Physiol. 2007, 146: 621-631. 10.1016/j.cbpa.2006.02.030.View Article
- Takagi Y, Mitsui A, Nishiyama A, Nozaki K, Sono H, Gon Y, Hashimoto N, Yodoi J: Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage. Proc Natl Acad Sci. 1999, 96: 4131-4136. 10.1073/pnas.96.7.4131.View Article
- Kasuno K, Nakamura H, Ono T, Muso E, Yodoi J: Protective roles of thioredoxin, a redox-regulating protein, in renal ischemia/reperfusion injury. Kidney Int. 2003, 64: 1273-1282. 10.1046/j.1523-1755.2003.00224.x.View Article
- Hamada Y, Miyata S, Nii-Kono T, Kitazawa R, Kitazawa S, Higo S, Fukunaga M, Ueyama S, Nakamura H, Yodoi J, Fukagawa M, Kasuga M: Overexpression of thioredoxin1 in transgenic mice suppresses development of diabetic nephropathy. Nephrol Dial Transplant. 2007, 22: 1547-1557. 10.1093/ndt/gfm099.View Article
- Shioji K, Kishimoto C, Nakamura H, Masutani H, Yuan Z, Oka S: Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity. Circulation. 2002, 106: 1403-1409. 10.1161/01.CIR.0000027817.55925.B4.View Article
- Wong JH, Kim YB, Ren PH, Cai N, Cho MJ, Hedden P, Lemaux PG, Buchanan BB: Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone. Proc Natl Acad Sci USA. 2002, 99: 16325-16330. 10.1073/pnas.212641999.View Article
- Kuge S, Jones N: YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J. 1994, 13: 655-664.
- Takatsume Y, Maeta K, Izawa S, Inoue Y: Enrichment of yeast thioredoxin by green tea extract through activation of Yap1 transcription factor in Saccharomyces cerevisiae. J Agric Food Chem. 2005, 53: 332-337. 10.1021/jf048818h.View Article
- Inoue Y, Nomura W, Takeuchi Y, Ohdate T, Tamasu S, Kitaoka A, Kiyokawa Y, Masutani H, Murata K, Wakai Y, Izawa S, Yodoi J: Efficient extraction of thioreodoxin from Saccharomyces cerevisiae by ethanol. Applied and Environmental Microbiology. 2007, 73: 1672-1675. 10.1128/AEM.02597-06.View Article
- Kuge S, Arita M, Murayama A, Maeta K, Izawa S, Inoue Y, Nomoto A: Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation. Mol Cell Biol. 2001, 21: 6139-6150. 10.1128/MCB.21.18.6139-6150.2001.View Article
- Puig S, Perez-Ortin JE: Expression levels and patterns of glycolytic yeast genes during wine fermentation. Syst Appl Microbiol. 2000, 23: 300-303.View Article
- Casagrande S, Bonetto V, Fratelli M, Gianazza E, Eberini I, Massignan T, Salmona M, Chang G, Holmgren A, Ghezzi P: Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems. Proc Natl Acad Sci USA. 2002, 99: 9745-9749. 10.1073/pnas.152168599.View Article
- Reverter-Branch , Cabiscol E, Tamarit J, Sorolla MA, ngeles de la TM, Ros J: Chronological and replicative life-span extension in Saccharomyces cerevisiae by increased dosage of alcohol dehydrogenase 1. Microbiology. 2007, 153: 3667-3676. 10.1099/mic.0.2007/009340-0.View Article
- Querol A, Barrio E, Huerta T, Ramon D: Molecular Monitoring of Wine Fermentations Conducted by Active Dry Yeast Strains. Appl Environ Microbiol. 1992, 58: 2948-2953.
- Perez-Torrado R, Carrasco P, Aranda A, Gimeno-Alcaniz J, Perez-Ortin JE, Matallana E, del Olmo ML: Study of the first hours of microvinification by the use of osmotic stress-response genes as probes. Syst Appl Microbiol. 2002, 25: 153-161. 10.1078/0723-2020-00087.View Article
- Perez-Torrado R, Gimeno-Alcaniz JV, Matallana E: Wine yeast strains engineered for glycogen overproduction display enhanced viability under glucose deprivation conditions. Appl Environ Microbiol. 2002, 68: 3339-3344. 10.1128/AEM.68.7.3339-3344.2002.View Article
- Puig S, Ramon D, Perez-Ortin JE: Optimized method to obtain stable food-safe recombinant wine yeast strains. Journal of Agricultural and Food Chemistry. 1998, 46: 1689-1693. 10.1021/jf9706538.View Article
- Gietz RD, Schiestl RH, Willems AR, Woods RA: Studies on the Transformation of Intact Yeast-Cells by the Liac/S-Dna/Peg Procedure. Yeast. 1995, 11: 355-360. 10.1002/yea.320110408.View Article
- Kohrer K, Domdey H: Preparation of High-Molecular-Weight Rna. Methods in Enzymology. 1991, 194: 398-405. full_text.View Article
- Griffith OW: Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980, 106: 207-212. 10.1016/0003-2697(80)90139-6.View Article
- Espindola AS, Gomes D, Panek AD, Araujo EC: The role of glutathione in yeast dehydration tolerance. Cryobiology. 2003, 47: 236-241. 10.1016/j.cryobiol.2003.10.003.View Article
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.View Article
- Levine RL, Williams JA, Stadtman ER, Shacter E: Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 1994, 233: 346-357. full_text.View Article
- Jakubowski W, Bilinski T, Bartosz G: Oxidative stress during aging of stationary cultures of the yeast Saccharomyces cerevisiae. Free Radic Biol Med. 2000, 28: 659-664. 10.1016/S0891-5849(99)00266-X.View Article
- Luk EE, Culotta VC: Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p. J Biol Chem. 2001, 276: 47556-47562. 10.1074/jbc.M108923200.View Article
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