Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient, Nitrogen-Limited Enological Condition

Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago, Chile.
Applied and Environmental Microbiology (Impact Factor: 3.67). 12/2012; 78(23). DOI: 10.1128/AEM.02305-12


Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations

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    • "All these results are in contrast to those by Aceituno et al. (2012) who described acetic acid production to take place only under fully anaerobic conditions. Probably, the use of nitrogen-limited chemostat by Aceituno et al. (2012) is related to the differences they found in the pattern of acetic acid production, as compared to other authors. "
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    ABSTRACT: We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process.
    Applied Microbiology and Biotechnology 01/2015; 99(9). DOI:10.1007/s00253-014-6321-3 · 3.34 Impact Factor
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    • "Gene expression Two additional parameters were included to account for transcriptomic (gene expression) information. Eighteen normalized microarray expression experiments (6 for aerobic and 12 for anaerobic cultivations) of different yeast strains growing on glucose were obtained (a) from a published work (Lai et al., 2006) using the query-driven search tool SPELL (Serial Pattern of Expression Levels Locator) (Hibbs et al., 2007) from the Saccharomyces Genome Database (SGD) (Cherry et al., 2012), and (b) from two works from our group (Aceituno et al., 2012; Orellana et al., 2014). All data was preprocessed, disregarding genes that were not included in Yeast 5 or that were essential for biomass growth, using single-knockout gene analysis (Edwards and Palsson, 2000; Zomorrodi et al., 2012). "
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    ABSTRACT: Dynamic flux balance analysis (dFBA) has been widely employed in metabolic engineering to predict the effect of genetic modifications and environmental conditions in the cell's metabolism during dynamic cultures. However, the importance of the model parameters used in these methodologies has not been properly addressed. Here, we present a novel and simple procedure to identify dFBA parameters that are relevant for model calibration. The procedure uses metaheuristic optimization and pre/post regression diagnostics, fixing iteratively the model parameters that do not have a significant role. We evaluated this protocol in a Saccharomyces cerevisiae dFBA framework calibrated for aerobic fed-batch and anaerobic batch cultivations. The model structures achieved have only significant, sensible and uncorrelated parameters and are able to calibrate different experimental data. We show that consumption, suboptimal growth and production rates are more useful for calibrating dynamic S. cerevisiae metabolic models than Boolean gene expression rules, biomass requirements and ATP maintenance.
    Metabolic Engineering 09/2014; 25:159 - 173. DOI:10.1016/j.ymben.2014.07.004 · 6.77 Impact Factor
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    • "In the past recent years, systems biology tools have been extensively used to study the physiology of the model yeast S. cerevisiae. Nevertheless, the application of such tools to the understanding of yeast physiology under winemaking conditions is still limited [9-14]. "
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    ABSTRACT: Background Saccharomyces cerevisiae is the most relevant yeast species conducting the alcoholic fermentation that takes place during winemaking. Although the physiology of this model organism has been extensively studied, systematic quantitative physiology studies of this yeast under winemaking conditions are still scarce, thus limiting the understanding of fermentative metabolism of wine yeast strains and the systematic description, modelling and prediction of fermentation processes. In this study, we implemented and validated the use of chemostat cultures as a tool to simulate different stages of a standard wine fermentation, thereby allowing to implement metabolic flux analyses describing the sequence of metabolic states of S. cerevisae along the wine fermentation. Results Chemostat cultures mimicking the different stages of standard wine fermentations of S. cerevisiae EC1118 were performed using a synthetic must and strict anaerobic conditions. The simulated stages corresponded to the onset of the exponential growth phase, late exponential growth phase and cells just entering stationary phase, at dilution rates of 0.27, 0.04, 0.007 h−1, respectively. Notably, measured substrate uptake and product formation rates at each steady state condition were generally within the range of corresponding conversion rates estimated during the different batch fermentation stages. Moreover, chemostat data were further used for metabolic flux analysis, where biomass composition data for each condition was considered in the stoichiometric model. Metabolic flux distributions were coherent with previous analyses based on batch cultivations data and the pseudo-steady state assumption. Conclusions Steady state conditions obtained in chemostat cultures reflect the environmental conditions and physiological states of S. cerevisiae corresponding to the different growth stages of a typical batch wine fermentation, thereby showing the potential of this experimental approach to systematically study the effect of environmental relevant factors such as temperature, sugar concentration, C/N ratio or (micro) oxygenation on the fermentative metabolism of wine yeast strains.
    Microbial Cell Factories 06/2014; 13(1):85. DOI:10.1186/1475-2859-13-85 · 4.22 Impact Factor
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