MOSES

Investigation of dynamics of the central metabolism (glycolysis, PPP, anaplerotic reactions, purines) of yeast Saccharomyces cerevisiae in anaerobic conditions

Steady state metabolic fluxes and metabolite concentrations of yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1

This investigation examines the effect of benzoic acid, a food preservative, on the ATP levels of yeast cells.

This study aims to establish the optimum conditions and assay methods for measuring ATP levels

To see changes in ATP levels in cells with induced ABC transporters. Cells with Pdr12 pump by 10 mM benzoic acid are used.

ATP levels of cells stressed with higher concentrations of benzoic acid (30 mM and 50 mM).

Effect of benzoate treatment (high concentrations) on ATP levels and Pdr12 expression after pretreatment of cells with low concentrations of benzoic acid.

Cell survival was determined under different benzoic acid concentrations

The steady state anaerobic culture (D = 0.1 h-1) was pertrubed by sudden increase of the extracellular glucose up to 1 g/L and both extra- and intracellular transient metabolite concentrations were measured

Steady state fluxes in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1

Steady state concentrations of metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1

Biomass weight during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Dynamics of extracellular metabolites (glc, pyr, suc, lac, gly, ac, etoh, fum, mal, cit, including loss of akg, g3p, 2pg, 3pg, r5p, f6p, g6p, 6pg) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Dynamics of intracellular metabolites (pyr, suc, fum, mal, akg, pep, g3p, 2pg, 3pg, cit, r5p, f6p, g6p, 6pg, ATP, ADP, AMP, UTP, GTP, inosine, NAD+, IMP, UDP, NADP+, CTP, AdenyloSuccinate, NADPH, trehalose) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines,
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Dynamics of macromolecules (total RNA) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

experimentally measured extracellular fluxes in yeast Saccharomyces cerevisiae in anaerobic glucose limited chemostat (D=0.1 h-1) on minimal medium

Steady state concentrations of extracellular metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D = 0.1 h-1 on minimal medium

We will compare two different procedures to extract ATP from yeast cells: Standard kit procedure (hot Tris/EDTA) and Serrano procedure (cold perchloric acid). In addition we have tested different condition as it turned out that some are important.

Monitoring of partial pressure of CO2 in off-gas. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Dynamics of intracellular metabolites (pyr, suc, fum, mal, akg, pep, g3p, 2pg, 3pg, cit, r5p, f6p, g6p, 6pg, ATP, ADP, AMP, UTP, GTP, inosine, NAD+, IMP, UDP, NADP+, CTP, AdenyloSuccinate, NADPH, trehalose) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines,
...

Dynamics of extracellular metabolites (glc, pyr, suc, lac, gly, ac, etoh, fum, mal, cit, including loss of akg, g3p, 2pg, 3pg, r5p, f6p, g6p, 6pg) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Cellular size and granularity (measured by FACS) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Dynamics of macromolecules (total RNA) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Biomass weight during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, etc) were measured.

Steady state concentrations of intracellular metabolites in yeast Saccharomyces cerevisiae anaerobic chemostat at D = 0.1 h-1 on minimal medium. All metabolite concentrations are in mmol/L(CV).

Steady state concentrations of extracellular metabolites in yeast Saccharomyces cerevisiae anaerobic chemostat at D = 0.1 h-1 on minimal medium. All metabolite concentrations are in mmol/L(R) except CO2, which is in parts of the partial pressure.

Steady state metabolic fluxes measured in glucose-limited chemostat of Saccharomyces cerevisiae at D = 0.1 h-1 growing on minimal medium. Fluxes are: glucose, ethanol, glycerol, acetate, succinate, pyruvate, lactate, citrate, malate, a-ketoglutarate, fumarate

The principles of Stealthy Engineering (Adamczyk et al.: Biotechnology Journal 2012; 7(7):877-83) are illustrated in this model by emulating a cross engineering intervention between L. lactis and S. cerevisiae.

The case study consists of replacing the native glucose uptake system of L. lactis with that native to the yeast S. cerevisiae. A modified version of Hoefnagel et al.’s model of L. lacrtis’ central metabolism was used as starting point. The total functional replacement of the PTS with the
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The model includes glycolysis, pentosephosphate pathway, purine salvage reactions, purine de novo synthesis, redox balance and biomass growth. The network balances adenylate pool as opened moiety.

Assay methodologies for individual glycolytic isoenzymes from the Mendes Group, University of Manchester, UK

This is the general protocol for the glycolytic enzyme measurements. Detailed Information for each Enzyme can be found in the SOP: Assays for measuring the activities of the individual glycolytic isoenzymes of Saccharomyces cerevisiae

SOP for growing yeast in anaerobic conditions

Biomass sampling - SOP for sampling of biomass

Metabolic perturbations - SOP for metabolic perturbations (i.e. glucose pulse)

Yeast strains used in the project

General protocol for measuring the kinetic parameters of the purified glycolytic enzymes from Saccharomyces cerevisiae - SOP for measuring the kinetic parameters of the purified glycolytic isoenzymes

Sample preparation - SOP for sampling, preparation of cell-free extracts, and determination of total extracted protein

Sample preparation - SOP for sampling, preparation of cell extracts, and general assay set-up

The kit is used for the quantitative detection of ATP in yeast cells by luciferase driven
bioluminescence. ATP extraction is done by boiling cells in TE buffer.

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Talk given by Maksim Zakhartsev (Hohenheim University, Stuttgart, Germany, member of MOSES, ZucAt and ExtremoPharm projects)