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Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A.

Jouhten P, Rintala E, Huuskonen A, Tamminen A, Toivari M, Wiebe M, Ruohonen L, Penttilä M, Maaheimo H - BMC Syst Biol (2008)

Bottom Line: 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.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. 13C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: VTT Technical Research Centre of Finland, Espoo, Finland. paula.jouhten@vtt.fi

ABSTRACT

Background: The yeast Saccharomyces cerevisiae 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 S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce.

Results: Metabolic flux distributions of S. cerevisiae CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O2 in the inlet gas were quantified by 13C-MFA. Metabolic flux ratios from fractional [U-13C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of S. cerevisiae.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 CO2. 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.

Conclusion: 13C-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.

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Average yields in S. cerevisiae CEN.PK113-1A glucose-limited chemostat (D = 0.1 h-1) cultures. Average yields of biomass (41.6 C-mmol g biomass-1 [19]), CO2, ethanol and glycerol on glucose (C-mol/C-mol) in the [U-13C]glucose labelled replicate cultivations of S. cerevisiae CEN.PK113-1A in glucose-limited chemostat (D = 0.1 h-1) in different oxygenation conditions: 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas.
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Figure 1: Average yields in S. cerevisiae CEN.PK113-1A glucose-limited chemostat (D = 0.1 h-1) cultures. Average yields of biomass (41.6 C-mmol g biomass-1 [19]), CO2, ethanol and glycerol on glucose (C-mol/C-mol) in the [U-13C]glucose labelled replicate cultivations of S. cerevisiae CEN.PK113-1A in glucose-limited chemostat (D = 0.1 h-1) in different oxygenation conditions: 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas.

Mentions: S. cerevisiae CEN.PK113-1A was grown in glucose-limited chemostats at a dilution rate of 0.1 h-1 in five different oxygenation conditions (20.9%, 2.8%, 1.0%, 0.5% and 0.0% O2 in the inlet gas). The corresponding average specific oxygen uptake rates (OUR) at these oxygen concentrations were 2.7, 2.5, 1.7, 1.2, and 0.0 mmol O2 g biomass-1 h-1 as derived from a number of replicate chemostat cultivations [32]. The specific uptake rate of glucose, excretion rates of acetate, ethanol and glycerol and the biomass concentration in the different oxygenation conditions in the 13C labelled replicate cultivations are given in Table 1. Net ethanol production was not observed in the aerobic cultures provided with 20.9% oxygen, indicating a fully respiratory metabolism. In 2.8% oxygen, slight ethanol excretion was observed indicating a shift to respiro-fermentative metabolism. In lower oxygen conditions, ethanol excretion rates increased further and the highest ethanol excretion rate was observed in anaerobic conditions, in which the metabolism was completely fermentative. As expected [42], the concentration of biomass was five times lower in anaerobic than in fully aerobic cultivations. Net production of glycerol was observed only in anaerobic cultivations. When only 0.5% oxygen was provided, ethanol and CO2 yields exceeded the yield of biomass (Figure 1). In anaerobic conditions the biomass yield was only one fourth of the yield of the main product ethanol. The carbon balances closed between 96–113% in all the cultures (41.6 C-mmol/g DW [19]).


Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A.

Jouhten P, Rintala E, Huuskonen A, Tamminen A, Toivari M, Wiebe M, Ruohonen L, Penttilä M, Maaheimo H - BMC Syst Biol (2008)

Average yields in S. cerevisiae CEN.PK113-1A glucose-limited chemostat (D = 0.1 h-1) cultures. Average yields of biomass (41.6 C-mmol g biomass-1 [19]), CO2, ethanol and glycerol on glucose (C-mol/C-mol) in the [U-13C]glucose labelled replicate cultivations of S. cerevisiae CEN.PK113-1A in glucose-limited chemostat (D = 0.1 h-1) in different oxygenation conditions: 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2507709&req=5

Figure 1: Average yields in S. cerevisiae CEN.PK113-1A glucose-limited chemostat (D = 0.1 h-1) cultures. Average yields of biomass (41.6 C-mmol g biomass-1 [19]), CO2, ethanol and glycerol on glucose (C-mol/C-mol) in the [U-13C]glucose labelled replicate cultivations of S. cerevisiae CEN.PK113-1A in glucose-limited chemostat (D = 0.1 h-1) in different oxygenation conditions: 20.9%, 2.8%, 1.0%, 0.5% and 0.0% oxygen of the chemostat inlet gas.
Mentions: S. cerevisiae CEN.PK113-1A was grown in glucose-limited chemostats at a dilution rate of 0.1 h-1 in five different oxygenation conditions (20.9%, 2.8%, 1.0%, 0.5% and 0.0% O2 in the inlet gas). The corresponding average specific oxygen uptake rates (OUR) at these oxygen concentrations were 2.7, 2.5, 1.7, 1.2, and 0.0 mmol O2 g biomass-1 h-1 as derived from a number of replicate chemostat cultivations [32]. The specific uptake rate of glucose, excretion rates of acetate, ethanol and glycerol and the biomass concentration in the different oxygenation conditions in the 13C labelled replicate cultivations are given in Table 1. Net ethanol production was not observed in the aerobic cultures provided with 20.9% oxygen, indicating a fully respiratory metabolism. In 2.8% oxygen, slight ethanol excretion was observed indicating a shift to respiro-fermentative metabolism. In lower oxygen conditions, ethanol excretion rates increased further and the highest ethanol excretion rate was observed in anaerobic conditions, in which the metabolism was completely fermentative. As expected [42], the concentration of biomass was five times lower in anaerobic than in fully aerobic cultivations. Net production of glycerol was observed only in anaerobic cultivations. When only 0.5% oxygen was provided, ethanol and CO2 yields exceeded the yield of biomass (Figure 1). In anaerobic conditions the biomass yield was only one fourth of the yield of the main product ethanol. The carbon balances closed between 96–113% in all the cultures (41.6 C-mmol/g DW [19]).

Bottom Line: 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.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. 13C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: VTT Technical Research Centre of Finland, Espoo, Finland. paula.jouhten@vtt.fi

ABSTRACT

Background: The yeast Saccharomyces cerevisiae 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 S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce.

Results: Metabolic flux distributions of S. cerevisiae CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O2 in the inlet gas were quantified by 13C-MFA. Metabolic flux ratios from fractional [U-13C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of S. cerevisiae.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 CO2. 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.

Conclusion: 13C-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.

Show MeSH
Related in: MedlinePlus