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 (T TRX2) 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 T TRX2 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.