4 research outputs found
Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A
<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h<sup>-1 </sup>with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O<sub>2 </sub>in the inlet gas were quantified by <sup>13</sup>C-MFA. Metabolic flux ratios from fractional [U-<sup>13</sup>C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of <it>S. cerevisiae</it>.</p> <p>While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO<sub>2</sub>. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.</p> <p>Conclusion</p> <p><sup>13</sup>C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.</p
Anhydrobiosis in yeast: is it possible to reach anhydrobiosis for yeast grown in conditions with severe oxygen limitation?
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Decoupling nutrient signaling from growth rate causes aerobic glycolysis and deregulation of cell size and gene expression
To survive and proliferate, cells need to coordinate their metabolism, gene expression, and cell division. To understand this coordination and the consequences of its failure, we uncoupled biomass synthesis from nutrient signaling by growing, in chemostats, yeast auxotrophs for histidine, lysine, or uracil in excess of natural nutrients (i.e., sources of carbon, nitrogen, sulfur, and phosphorus), such that their growth rates (GRs) were regulated by the availability of their auxotrophic requirements. The physiological and transcriptional responses to GR changes of these cultures differed markedly from the respective responses of prototrophs whose growth-rate is regulated by the availability of natural nutrients. The data for all auxotrophs at all GRs recapitulated the features of aerobic glycolysis, fermentation despite high oxygen levels in the growth media. In addition, we discovered wide bimodal distributions of cell sizes, indicating a decoupling between the cell division cycle (CDC) and biomass production. The aerobic glycolysis was reflected in a general signature of anaerobic growth, including substantial reduction in the expression levels of mitochondrial and tricarboxylic acid genes. We also found that the magnitude of the transcriptional growth-rate response (GRR) in the auxotrophs is only 40–50% of the magnitude in prototrophs. Furthermore, the auxotrophic cultures express autophagy genes at substantially lower levels, which likely contributes to their lower viability. Our observations suggest that a GR signal, which is a function of the abundance of essential natural nutrients, regulates fermentation/respiration, the GRR, and the CDC.National Institutes of Health (U.S.) (GM046406)National Institute of General Medical Sciences (U.S.) (Center for Quantitative Biology (GM071508