Correlation of oxygen utilization and hydrogen peroxide accumulation with oxygen induced enzymes in Lactobacillus plantarum cultures

Two strains of Lactobacillus plantarum accumulated H₂O₂ when grown aerobically in a complex glucose based medium. The H₂O₂ accumulation did not occur immediately on exposure of the culture to O₂ but was delayed for a time which, in the case of one strain, was dependent on the amount of inoculum used to seed the culture. The accumulation was always preceded by an increase in the rate of O₂ utilization by the cultures. The latter coincided approximately with an increase in specific activity of NADH oxidase, pyruvate oxidase and NADH peroxidase. H₂O₂ was not a product of NADH oxidase in vitro but was formed in substantial quantities from O₂ during oxidation of pyruvate. The three enzymes were induced by O₂ and H₂O₂; the induction of NADH oxidase responded to lower levels of O₂ (but not of H₂O₂) than the pyruvate oxidase or the NADH peroxidase.Abbreviations MRSG Mann, Rogosa and Sharpe medium (1960) with glucose as fermentation source - TPP thiamin pyrophosphate     

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H₂O₂ oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O₂ have been studied with ten freshwater and marine strains. Generally, O₂ was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O₂ concentrations of below 15 M (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O₂ as electron acceptor (up to one doubling of protein). However, O₂ was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O₂ tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O₂ concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 M. The specific rates of O₂ uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O₂ concentrations in the gas phase.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Selection of Vaginal H2O2-Generating Lactobacillus Species for Probiotic Use

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H₂O₂. This paper describes the selection of H₂O₂-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H₂O₂ level, was chosen to study: (a) the kinetics of H₂O₂ production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H₂O₂ in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H₂O₂L⁻¹) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H₂O₂. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H₂O₂ since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract. Received: 17 November 1998 / Accepted: 17 December 1998     

An anaerobic bacterium,Bacteroides thetaiotaomicron,uses a consortium of enzymes to scavenge hydrogen peroxide

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H₂O₂ that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H₂O₂ in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H₂O₂, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H₂O₂ formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H₂O₂ was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.     

Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h⁻¹). Accumulation of H₂O₂ in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H₂O₂ accumulated only temporarily during the lag phase. H₂O₂ is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min⁻¹ mg of protein⁻¹), which reduced oxygen to water, whereby superoxide was formed as a by-product. H₂O₂ originated from the dismutation of superoxide by superoxide dismutase. Although H₂O₂ was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H₂O₂-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H₂O₂ and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H₂O₂.     

Purification and characterization of an H<Subscript>2</Subscript>O-forming NADH oxidase from <Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria

Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O₂. When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O₂-free N₂ carrier-gas) to microoxic (sparged with 3% O₂/97% N₂ mixed carrier-gas) growth conditions in the mid exponential phase (OD₆₆₀=1.0). When the strain grew under 3% O₂/97% N₂, the medium remains anoxic. Thirty minutes after beginning aeration with 3% O₂, the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H₂O, and the estimated K_m for oxygen was 61.9 M. These data demonstrate that an O₂-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.Abbreviations NRIC NODAI Research Institute-Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

The role of thiamine inYersinia kristensenii resistance to antibiotics and heavy metals

The resistance to divalent metal ions, antibiotics and H₂O₂ was investigated inYersinia kristensenii strains 13, 15, 18 by performing subcultivations with CdSO₄ (20 and 100mg/L) in nutrient agar (NA) and M9 medium with thiamine. Metal resistance of all three strains in NA was the same and decreased in the following sequence: Ni>Zn=Co>Cd. The chloramphenicol (Cmp) resistance ranged between 32 and 256 mg/L and the H₂O₂ sensitivity was very low or even zero. In the presence of thiamine the metal resistance sequence changed to Zn=Cd>Ni, Co, Ni and Co tolerance being 10–20 mg/L. Cmp resistance of all strains increased to 256 mg/L and H₂O₂ sensitivity also rose. In Cd-treated cultures, the ratio of glucose to thiamine in culture medium affected Cd resistance. At normal content of glucose and thiamine (5 g/L and 5mg/L), Cd resistance markedly decreased coincident with thiamine exhaustion in these slowly-growing cultures. The Cmp resistance decreased to 16 mg/L, Ni and Co intolerance and H₂O₂ hypersensitivity appeared. At lowered glucose or thiamine levels (5 g/L and 2.5 mg/L or 2.5 g/L and 5 mg/L) a marginal decrease of Cd resistance took place in response to limited glucose uptake. Low thiamine or low-glucose cultures were resistant to H₂O₂, and exhibited a small decrease in Cmp resistance and a low Ni, Co tolerance. The adaptation of strain 15 to Cd induced only a small decrease of Cd resistance. Lowered glucose-to-thiamine ratio in culture medium probably induced in Cd-treated cultures a response triggering Cd resistance.     

Selection of symbiotically energy efficient strains of Rhizobium japonicum by their ability to induce a H2-uptake hydrogenase in the free-living state

The capacity of inducing a H₂-uptake hydrogenase in free-living cultures was examined in 21 strains of Rhizobium japonicum. Four strains were found to take up H₂ at rapid rates after 3 days of growth on agar slants inside sealed vials provided with an atmosphere of 5% H₂ in air. Soybean nodules from these strains lost little or no H₂ in air and their bacteroids oxidized H₂ at rates that were similar to those observed in free-living cultures. In contrast, three randomly chosen strains of R. japonicum that showed no H₂-uptake capacity in free-living state produced nodules which lost large amounts of H₂ and the corresponding bacteroids had no hydrogenase activity. A screening procedure is described for the selection of Rhizobium strains producing high energy-efficient nodules based on a test of their ability to induce a H₂-uptake hydrogenase in asymbiotic conditions.     

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain ofAzotobacter vinelandii

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N,N-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O₂-limited chemostat cultures. The results showed that the mutant strain grew and used O₂ less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochromeo,a ₁, oxidase branch of the respiratory chain may be important in the physiology ofA. vinelandii under O₂-limiting growth conditions.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Growth and adaptive hydrogen production of Rhodospirillum rubrum (F1) in anaerobic dark cultures

Rhodospirillum rubrum (F₁) maintained electron balance mainly by producing propionate, formate and H₂ during fermentation metabolism. H₂ formation was inversely correlated with the production of propionate.In diluted, growing cultures high amounts of H₂ and only traces or no propionate were produced from pyruvate. In dense cultures or in resting cultures without (NH₄)₂SO₄, however, propionate was formed from pyruvate in relatively high amounts Cultures always produced much more propionate than H₂ from fructose in contrast to cells with pyruvate. Kinetic studies of growth and excretion of fermentation products indicated that the enzyme system for H₂ formation is adaptive. Chloramphenicol (3 μg/ml) completely inhibited the formation of H₂ if the cells were not adapted to fermentation metabolism. The production of propionate, on the other hand, was not prevented by chloramphenicol after shifting the cells from aerobic dark culture with malate to fermentation conditions with pyruvate.H₂ formation was not influenced by sodium ascorbate but it was significantly decreased by K₃[Fe(CN)₆].Poly(β-hydroxybutyric acid) was also synthesized by the cells during anaerobic dark metabolism especially in dense cultures, probably favoured by the rapid acidification of the medium. Formate can also accumulate in the fermentation metabolism, especially in young growing cultures.These results give an explanation for the differing reports in the literature on the fermentation metabolism of R. rubrum.     

MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Peroxidase mediated hydrogen peroxide production in barley roots grown under stress conditions

All applied metals (Co, Al, Cu, Cd) and NaCl inhibited barley root growth. No root growth inhibition was caused by drought exposure, in contrast to cold treatment. 0.01 mM H₂O₂ stimulated root growth and GA application did not affect root growth at all. Other activators and inhibitors of H₂O₂ production (SHAM, DTT, 10 mM H₂O₂, 2,4-D) inhibited root growth. Loss of cell viability was most significant after Al treatment, followed by Cd and Cu, but no cell death was induced by Co. Drought led to slight increase in Evans blue uptake, whereas neither NaCl nor cold influenced this parameter. DTT treatment caused slight increase in Evans blue uptake and significant increases were detected after 2,4-D and 10 mM H₂O₂ treatment, but were not induced by others stressors. Metal exposure increased guaiacol-POD activity, which was correlated with oxidation of NADH and production of H₂O₂. Exposure to drought caused a minor change in NADH oxidation, but neither H₂O₂ production nor guaiacol-POD activity was increased. Cold and NaCl application decreased all monitored activities. Increase in NADH oxidation and guaiacol-POD activity was caused by 10 mM H₂O₂ and 0.01 mM 2,4-D treatment, which also caused enhancement of H₂O₂ production. Slight inhibition of all activities was caused by 0.01 mM H₂O₂, GA, DTT; more pronounced inhibition was detected after SHAM treatment. The role of H₂O₂ production mediated by POD activity in relation to root growth and cell viability under exposure to some abiotic stress factors is discussed.     

Components of antioxidant systems in the cells of aerotolerant sulfate-reducing bacteria of the genus <Emphasis Type="Italic">Desulfovibrio</Emphasis> (strains A2 and TomC) isolated from metal mining waste

Two strains of sulfate-reducing bacteria of the genus Desulfovibrio (A2 and TomC) isolated from metal mining waste were able to grow on agar Postgate C nutrient medium under microaerobic conditions. Since their growth in liquid nutrient medium was just slightly affected by 1% O₂ (initial concentration in the gas phase) and 0.05–0.1 mM H₂O₂, these strains were relatively oxygen-tolerant. Only the presence of oxidants in high concentrations (5–10% О₂ or 0.3–1.0 mM H₂O₂) resulted in practically complete inhibition of their growth. Strain A2 was more resistant to oxidative stresses than strain TomC. Activities of the key enzymes of antioxidant defense—superoxide dismutase (SOD), catalase, and peroxidase—were revealed in the cell-free extracts of strain A2 grown under strict anaerobic conditions. While strain TomC was found to possess no peroxidase activity, its catalase activity was much higher than that of strain A2 (36 and 2 U/mg protein, respectively). SOD activity of both strains was almost the same (5 U/mg protein). Sublethal H₂O₂ doses (concentration of 0.05–0.15 mM and exposure for 45–240 min) resulted in a drastic increase of catalase activity, especially in strain A2. Sublethal О₂ doses (1–2% in the gas phase) had no significant effect on activities of the antioxidant enzymes of both strains. The cytochrome composition determined from the absolute absorption spectra of the whole cells of strains TomC and A2 revealed the presence of the c heme (438 and 831 pmol/mg protein) and the d heme (336 and 303 pmol/mg protein, respectively). The presence of the d heme indicated the presence of the bd heme–heme quinol oxidase, which together with the c heme may provide for the functioning of the electron transport segment of the antioxidant defensive system, which is responsible for aerotolerance of sulfate-reducing bacteria.     

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H₂O₂ and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H₂O₂. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H₂O₂. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H₂O₂, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.     

Engineering a Saccharomyces cerevisiae Wine Yeast That Exhibits Reduced Ethanol Production during Fermentation under Controlled Microoxygenation Conditions

We recently showed that expressing an H₂O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD⁺ reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.