Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.     

The ability to use nitrate confers advantage to <Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> over <Emphasis Type="Italic">S. cerevisiae</Emphasis> and can explain its adaptation to industrial fermentation processes

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.     

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.     

Effects of single and combined cell treatments based on low pH and high concentrations of ethanol on the growth and fermentation of Dekkera bruxellensis and Saccharomyces cerevisiae

The alcoholic fermentation in Brazil displays some peculiarities because the yeast used is recycled in a non-aseptic process. After centrifugation, the cells are treated with acid to control the bacterial growth. However, it is difficult to manage the indigenous yeasts without affecting the main culture of Saccharomyces cerevisiae. This work evaluated how the cell treatment could be modified to combat contaminant yeasts based on the differential sensitivities to low pH and high concentrations of ethanol displayed by an industrial strain of S. cerevisiae and three strains of Dekkera bruxellensis, which are common contaminant yeasts in Brazilian fermentation processes. The tests were initially performed in rich medium with a low pH or a high concentration of ethanol to analyse the yeast growth profile. Then, the single and combined effects of low pH and ethanol concentration on the yeast cell viability were evaluated under non-proliferative conditions. The effects on the fermentation parameters were also verified. S. cerevisiae grew best when not subjected to the stresses, but this yeast and D. bruxellensis had similar growth kinetics when exposed to a low pH or increased ethanol concentrations. However, the combined treatments of low pH (2.0) and ethanol (11 or 13 %) resulted in a decrease of D. bruxellensis cell viability almost three times higher than of S. cerevisiae, which was only slightly affected by all cell treatments. The initial viability of the treated cells was restored within 8 h of growth in sugar cane juice, with the exception of the combined treatment for D. bruxellensis. The ethanol-based cell treatment, in despite of slowing the fermentation, could decrease and maintain D. bruxellensis population under control while S. cerevisiae was taking over the fermentation along six fermentative cycles. These results indicate that it may be possible to control the growth of D. bruxellensis without major effects on S. cerevisiae. The cells could be treated between the fermentation cycles by the parcelled addition of 13 % ethanol to the tanks in which the yeast cream is treated with sulphuric acid at pH 2.0.     

Fermentation characteristics of Dekkera bruxellensis strains

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)⁻¹ and growth rates varied from 0.037 to 0.050 h⁻¹. The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0–13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72–84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.     

The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae

The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis

Aim: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. Methods and Results: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small‐scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1 : 2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l^?1 (0·22 mol l^?1)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1 : 5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1 : 10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0·42 ± 0·01 g ethanol (g glucose)^?1 were observed for both yeasts in 1 : 10 hydrolysate. In small‐scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1 : 5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1 : 2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. Conclusions: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. Significance and Impact of the study: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.     

Engineering of the glycerol decomposition pathway and cofactor regulation in an industrial yeast improves ethanol production

Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD⁺-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.     

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl‐CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl‐CoA was engineered by over‐expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP⁺‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100 h was with a strain harboring the phosphoketolase pathway to supply acetyl‐CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl‐CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol. Biotechnol. Bioeng. 2013; 110: 2216–2224. © 2013 Wiley Periodicals, Inc.     

Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates

The yeast Dekkera bruxellensis is a major contaminant of industrial fermentations, such as those used for the production of biofuel and wine, where it outlasts and, under some conditions, outcompetes the major industrial yeast Saccharomyces cerevisiae. In order to investigate the level of inter-strain variation that is present within this economically important species, the genomes of four diverse D. bruxellensis isolates were compared. While each of the four strains was shown to contain a core diploid genome, which is clearly sufficient for survival, two of the four isolates have a third haploid complement of chromosomes. The sequences of these additional haploid genomes were both highly divergent from those comprising the diploid core and divergent between the two triploid strains. Similar to examples in the Saccharomyces spp. clade, where some allotriploids have arisen on the basis of enhanced ability to survive a range of environmental conditions, it is likely these strains are products of two independent hybridisation events that may have involved multiple species or distinct sub-species of Dekkera. Interestingly these triploid strains represent the vast majority (92%) of isolates from across the Australian wine industry, suggesting that the additional set of chromosomes may confer a selective advantage in winery environments that has resulted in these hybrid strains all-but replacing their diploid counterparts in Australian winery settings. In addition to the apparent inter-specific hybridisation events, chromosomal aberrations such as strain-specific insertions and deletions and loss-of-heterozygosity by gene conversion were also commonplace. While these events are likely to have affected many phenotypes across these strains, we have been able to link a specific deletion to the inability to utilise nitrate by some strains of D. bruxellensis, a phenotype that may have direct impacts in the ability for these strains to compete with S. cerevisiae.     

Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions

Industrial fermentation of lignocellulosic hydrolysates to ethanol requires microorganisms able to utilise a broad range of carbon sources and generate ethanol at high yield and productivity. D. bruxellensis has recently been reported to contaminate commercial ethanol processes, where it competes with Saccharomyces cerevisiae [4, 26]. In this work Brettanomyces/Dekkera yeasts were studied to explore their potential to produce ethanol from renewable sources under conditions suitable for industrial processes, such as oxygen-limited and low-pH conditions. Over 50 strains were analysed for their ability to utilise a variety of carbon sources, and some strains grew on cellobiose and pentoses. Two strains of D. bruxellensis were able to produce ethanol at high yield (0.44 g g⁻¹ glucose), comparable to those reported for S. cerevisiae. B. naardenensis was shown to be able to produce ethanol from xylose. To obtain ethanol from synthetic lignocellulosic hydrolysates we developed a two-step fermentation strategy: the first step under aerobic conditions for fast production of biomass from mixtures of hexoses and pentoses, followed by a second step under oxygen limitation to promote ethanol production. Under these conditions we obtained biomass and ethanol production on synthetic lignocellulosic hydrolysates, with ethanol yields ranging from 0.2 to 0.3 g g⁻¹ sugar. Hexoses, xylose and arabinose were consumed at the end of the process, resulting in 13 g l⁻¹ of ethanol, even in the presence of furfural. Our studies showed that Brettanomyces/Dekkera yeasts have clear potential for further development for industrial processes aimed at production of ethanol from renewable sources.     

Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl₂ (2.1 Å; R = 18.2%, R_free = 21.9%), and in complex with glutamate, MgCl₂, and ADP (2.7 Å; R = 19.0%, R_free = 24.2%). Inspection of these structures reveals an unusual binding pocket for the α-carboxylate of the glutamate substrate and an ATP-independent Mg²⁺ coordination site, clarifying the Mg²⁺ dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.     

Dekkera bruxellensis and Lactobacillus vini Form a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process

The ethanol production process of a Swedish alcohol production plant was dominated by Dekkera bruxellensis and Lactobacillus vini, with a high number of lactic acid bacteria. The product quality, process productivity, and stability were high; thus, D. bruxellensis and L. vini can be regarded as commercial ethanol production organisms.     

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.