Correlation between TCA cycle flux and glucose uptake rate during respiro-fermentative growth of Saccharomyces cerevisiae.

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using (13)C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO(2) allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g(-1) h(-1)), while the highest acetate production rate of 2.1 mmol g(-1) h(-1) was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l(-1) , pH 5.0, 30 degrees C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g(-1) h(-1) to about 1.7 mmol g(-1) h(-1) was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO(2) to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.

[1]  J. Gancedo,et al.  The early steps of glucose signalling in yeast. , 2008, FEMS microbiology reviews.

[2]  Carl Johan Franzén,et al.  Characterization of glucose transport mutants of Saccharomyces cerevisiae during a nutritional upshift reveals a correlation between metabolite levels and glycolytic flux. , 2008, FEMS yeast research.

[3]  Lars M Steinmetz,et al.  Systematic screens for human disease genes, from yeast to human and back. , 2008, Molecular bioSystems.

[4]  A. Kornberg,et al.  Di- and triphosphopyridine nucleotide isocitric dehydrogenases in yeast. , 1951, The Journal of biological chemistry.

[5]  Uwe Sauer,et al.  Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts. , 2005, FEMS yeast research.

[6]  Ronald W. Davis,et al.  Systematic screen for human disease genes in yeast , 2002, Nature Genetics.

[7]  G. Stephanopoulos,et al.  Metabolic Engineering: Principles And Methodologies , 1998 .

[8]  Jacky L. Snoep,et al.  Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae , 2004, Applied and Environmental Microbiology.

[9]  J. Pronk,et al.  Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae , 2004, Journal of Biological Chemistry.

[10]  H. Y. Steensma,et al.  The Two Acetyl-coenzyme A Synthetases of Saccharomyces cerevisiae Differ with Respect to Kinetic Properties and Transcriptional Regulation* , 1996, The Journal of Biological Chemistry.

[11]  U. Sauer,et al.  Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast , 2005, Genome Biology.

[12]  Thomas Szyperski,et al.  Metabolic-Flux Profiling of the Yeasts Saccharomyces cerevisiae and Pichia stipitis , 2003, Eukaryotic Cell.

[13]  L. Olsson,et al.  Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae , 2007, Proceedings of the National Academy of Sciences.

[14]  Zhikang Yin,et al.  Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs , 2003, Molecular microbiology.

[15]  U. Sauer,et al.  Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. , 2001, European journal of biochemistry.

[16]  Saeed Tavazoie,et al.  Ras and Gpa2 Mediate One Branch of a Redundant Glucose Signaling Pathway in Yeast , 2004, PLoS biology.

[17]  J. Heijnen,et al.  Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid , 2008, The FEBS journal.

[18]  L. Gustafsson,et al.  Energy balance calculations as a tool to determine maintenance energy requirements under stress conditions , 1993 .

[19]  J. Heijnen,et al.  Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites. , 2005, FEMS yeast research.

[20]  Concetta Compagno,et al.  Fermentative lifestyle in yeasts belonging to the Saccharomyces complex , 2007, The FEBS journal.

[21]  Johannes Boonstra,et al.  HXT5 expression is determined by growth rates in Saccharomyces cerevisiae , 2002, Yeast.

[22]  Christoph Wittmann,et al.  Metabolic flux screening of Saccharomyces cerevisiae single knockout strains on glucose and galactose supports elucidation of gene function. , 2007, Journal of biotechnology.

[23]  R. H. De Deken,et al.  The Crabtree Effect: A Regulatory System in Yeast , 1966 .

[24]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[25]  T Szyperski,et al.  13C-NMR, MS and metabolic flux balancing in biotechnology research , 1998, Quarterly Reviews of Biophysics.

[26]  Lisbeth Olsson,et al.  A systems biology approach to study glucose repression in the yeast Saccharomyces cerevisiae , 2007, Biotechnology and bioengineering.

[27]  J. Nielsen,et al.  Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae. , 1997, Microbiology.

[28]  Nicola Zamboni,et al.  FiatFlux – a software for metabolic flux analysis from 13C-glucose experiments , 2005, BMC Bioinformatics.

[29]  J. Nielsen,et al.  Network Identification and Flux Quantification in the Central Metabolism of Saccharomyces cerevisiae under Different Conditions of Glucose Repression , 2001, Journal of bacteriology.

[30]  Duboc,et al.  An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. , 2000, Enzyme and microbial technology.

[31]  E. Lander,et al.  Remodeling of yeast genome expression in response to environmental changes. , 2001, Molecular biology of the cell.

[32]  W. A. Scheffers,et al.  Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation , 1992, Yeast.

[33]  G. Stephanopoulos CHAPTER 1 – The Essence of Metabolic Engineering , 1998 .

[34]  U. Sauer,et al.  High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. , 2004, Analytical biochemistry.

[35]  Christoph Wittmann,et al.  Fluxome analysis using GC-MS , 2007, Microbial cell factories.

[36]  Jens Nielsen,et al.  Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C‐labelled glucose , 2004, Yeast.

[37]  S. Hohmann Osmotic Stress Signaling and Osmoadaptation in Yeasts , 2002, Microbiology and Molecular Biology Reviews.

[38]  J. Thevelein,et al.  Osmotic Stress-Induced Gene Expression in Saccharomyces cerevisiae Requires Msn1p and the Novel Nuclear Factor Hot1p , 1999, Molecular and Cellular Biology.

[39]  L. Guarente,et al.  Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae , 1985, Molecular and cellular biology.

[40]  Thomas Lengauer,et al.  Metabolic screening of Saccharomyces cerevisiae single knockout strains reveals unexpected mobilization of metabolic potential , 2006 .

[41]  Bernhard Ø Palsson,et al.  Integrated analysis of metabolic phenotypes in Saccharomyces cerevisiae , 2004, BMC Genomics.

[42]  Uwe Sauer,et al.  TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates. , 2004, Microbiology.

[43]  L. Bisson,et al.  On the trail of an elusive flux sensor. , 2003, Research in microbiology.