3.06 Tesis doctorado

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Browsing 3.06 Tesis doctorado by Author "Aceituno Aceituno, Felipe Eduardo"

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    Systemic characterization of the oxygen response of the wine yeast saccharomyces cerevisiae EC1118 in oenological conditions.
    (2013) Aceituno Aceituno, Felipe Eduardo; Agosin T., Eduardo; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    Discrete oxygen additions play a critical role in alcoholic fermentation. However, few studies have quantified the fate of the dissolved oxygen and its impact on wine yeast cell physiology, under oenological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the wine making process, by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. Metabolic Flux Analysis was performed with a custom-built model. This indicated that when the dissolved oxygen concentration increases from 1.2 to 2.7μM, yeast cells change from a fully fermentative to a mixed respiro-fermentative metabolism. This transition was 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 for all oxygen conditions, suggesting the limitation of mitochondrial NADH reoxidation as the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite high sugar concentrations, indicating that oxygen overrides glucose repression, which was thought to be the cause of the Crabtree effect. Genes associated with other processes such as proline uptake, mannoproteins and oxidative stress are also significantly affected by oxygen. Our data suggests that oxygen can be beneficial at low levels, improving proline uptake and reducing acetic acid. On the contrary, high oxygen levels can reduce mannoproteins, important for haze removal and wine mouthfeel, and even stress yeast to the point of reducing its metabolic capacity when in excess, highlighting the dual role of oxygen in “making or breaking wines”. Even though oxygen was promptly consumed under oenological fermentation conditions, we wondered which pathways accounted for biological oxygen consumption. By means of a genome-scale, unbiased metabolic model, we determined that most oxygen is consumed by respiration. This is concordant with our experimental analyses that showed a minor contribution of non-respiratory pathways (such as ergosterol and lipid biosynthesis) to the overall oxygen consumption, and a major contribution of the respiratory-coupled proline assimilation pathway. Altogether, these results strongly indicate that oxygen consumption, when limiting, is more critical for nitrogen assimilation and respiration than for lipid synthesis. To the best of our knowledge, this work is the first to globally assess the effects of oxygen on yeast physiology under a high-sugar, nitrogen-limited culture setting simulating oenological conditions. From this data, we determined optimal and deleterious oxygen conditions that can be chosen or avoided in wine making. Regarding yeast physiology, while several data can be extrapolated from carbon-limited physiology (such as TCA splitting in anaerobiosis), it is striking that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation, contrary to the widespread belief that respiration is under glucose repression in these conditions. Nevertheless, respiration does not surpass fermentation as the main energy-providing pathway, in a way analogous to what is seen in cancer cells that show the “Warburg effect”, this is, aerobic ethanol production. Therefore, the study of the oxygen response in nitrogen-limited yeast cells can provide relevant information on how to manage oxygen to impact positively wine quality, but is also as a model for other relevant biological systems.