1887

Abstract

Metabolic responses of to different physical and chemical environmental conditions were investigated in glucose batch culture by GC-MS-detected mass isotopomer distributions in proteinogenic amino acids from C-labelling experiments. For this purpose, GC-MS-based metabolic flux ratio analysis was extended from bacteria to the compartmentalized metabolism of . Generally, was shown to have low catabolic fluxes through the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. Notably, respiratory TCA cycle fluxes exhibited a strong correlation with the maximum specific growth rate that was attained under different environmental conditions, including a wide range of pH, osmolarity, decoupler and salt concentrations, but not temperature. At pH values of 4·0 to 6·0 with near-maximum growth rates, the TCA cycle operated as a bifurcated pathway to fulfil exclusively biosynthetic functions. Increasing or decreasing the pH beyond this physiologically optimal range, however, reduced growth and glucose uptake rates but increased the ‘cyclic’ respiratory mode of TCA cycle operation for catabolism. Thus, the results indicate that glucose repression of the TCA cycle is regulated by the rates of growth or glucose uptake, or signals derived from these. While sensing of extracellular glucose concentrations has a general influence on the TCA cycle activity, the growth-rate-dependent increase in respiratory TCA cycle activity was independent of glucose sensing.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26845-0
2004-04-01
2020-04-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/4/mic1501085.html?itemId=/content/journal/micro/10.1099/mic.0.26845-0&mimeType=html&fmt=ahah

References

  1. Alexander M. A., Jeffries T. W.. 1990; Respiratory efficiency and metabolite partitioning as regulatory phenomena in yeasts. Enzyme Microb Technol12:2–19[CrossRef]
    [Google Scholar]
  2. Bailey J. E.. 1999; Lessons from metabolic engineering for functional genomics and drug discovery. Nat Biotechnol17:616–618[CrossRef]
    [Google Scholar]
  3. Boles E., de Jong-Gubbels P., Pronk J. T.. 1998; Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme. J Bacteriol180:2875–2882
    [Google Scholar]
  4. Carlson M.. 1999; Glucose repression in yeast. Curr Opin Microbiol2:202–207[CrossRef]
    [Google Scholar]
  5. Causton H. C., Ren B., Koh S. S.. & 7 other authors. 2001; Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell12:323–337[CrossRef]
    [Google Scholar]
  6. Christensen B., Gombert A. K., Nielsen J.. 2002; Analysis of flux estimates based on 13C-labelling experiments. Eur J Biochem269:2795–2800[CrossRef]
    [Google Scholar]
  7. Dauner M., Sauer U.. 2000; GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog16:642–669[CrossRef]
    [Google Scholar]
  8. Dauner M., Bailey J. E., Sauer U.. 2001; Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng76:144–156[CrossRef]
    [Google Scholar]
  9. DeRisi J. L., Iyer V. R., Brown P. O.. 1997; Exploring the metabolic and genetic control of gene expression on a genomic scale. Science278:680–686[CrossRef]
    [Google Scholar]
  10. Diderich J. A., Schepper M., van Hoek P..8 other authors 1999; Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae. J Biol Chem274:15350–15359[CrossRef]
    [Google Scholar]
  11. Diderich J. A., Raamsdonk L. M., Kruckeberg A. L., Berden J. A., Van Dam K.. 2001; Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted. Appl Environ Microbiol67:1587–1593[CrossRef]
    [Google Scholar]
  12. Dos Santos M. M., Gombert A. K., Christensen B., Olsson L., Nielsen J.. 2003; Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates. Eukaryot Cell2:599–608[CrossRef]
    [Google Scholar]
  13. Emmerling M., Dauner M., Ponti A., Fiaux J., Hochuli M., Szyperski T., Wüthrich K., Bailey J. E., Sauer U.. 2002; Metabolic flux responses to pyruvate kinase knockout in Escherichia coli. J Bacteriol184:152–164[CrossRef]
    [Google Scholar]
  14. Fiaux J., Cakar Z. P., Sonderegger M., Wüthrich K., Szyperski T., Sauer U.. 2003; Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis. Eukaryot Cell2:170–180[CrossRef]
    [Google Scholar]
  15. Fischer E., Sauer U.. 2003a; Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem270:880–891[CrossRef]
    [Google Scholar]
  16. Fischer E., Sauer U.. 2003b; A novel metabolic cycle catalyzes glucose oxidation and anaplerosis in hungry Escherichia coli. J Biol Chem278:46446–46451[CrossRef]
    [Google Scholar]
  17. Fischer E., Zamboni N., Sauer U.. 2004; High-throughput metabolic flux analysis based on gas-chromatography-mass spectrometry derived 13C constraints. Anal Biochem (in press)
    [Google Scholar]
  18. Gancedo J. M.. 1998; Yeast carbon catabolite repression. Microbiol Mol Biol Rev62:334–361
    [Google Scholar]
  19. Gasch A. P., Spellman P. T., Kao C. M., Carmel-Harel O., Eisen M. B., Storz G., Botstein D., Brown P. O.. 2000; Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell11:4241–4257[CrossRef]
    [Google Scholar]
  20. Gombert A. K., Moreira dos Santos M., Christensen B., Nielsen J.. 2001; Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression. J Bacteriol183:1441–1451[CrossRef]
    [Google Scholar]
  21. Hellerstein M. K.. 2003; In vivo measurement of fluxes through metabolic pathways: the missing link in functional genomics and pharmaceutical research. Annu Rev Nutr23:379–402[CrossRef]
    [Google Scholar]
  22. Herwig C., Doerries C., Marison I., von Stockar U.. 2001; Quantitative analysis of the regulation scheme of invertase expression in Saccharomyces cerevisiae. Biotechnol Bioeng76:247–258[CrossRef]
    [Google Scholar]
  23. Hohmann S.. 2002; Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev66:300–372[CrossRef]
    [Google Scholar]
  24. Jiao Z., Baba T., Mori H., Shimizu K.. 2003; Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement. FEMS Microbiol Lett220:295–301[CrossRef]
    [Google Scholar]
  25. Klapa M. I., Aon J. C., Stephanopoulos G.. 2003; Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry. Eur J Biochem270:3525–3542[CrossRef]
    [Google Scholar]
  26. Maaheimo H., Fiaux J., Cakar Z. P., Bailey J. E., Sauer U., Szyperski T.. 2001; Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids. Eur J Biochem268:2464–2479[CrossRef]
    [Google Scholar]
  27. Meijer M. M., Boonstra J., Verkleij A. J., Verrips C. T.. 1998; Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux. J Biol Chem273:24102–24107[CrossRef]
    [Google Scholar]
  28. Möller K., Christensen B., Förster J., Piskur J., Nielsen J., Olsson L.. 2002; Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes. Biotechnol Bioeng77:186–193[CrossRef]
    [Google Scholar]
  29. Ozcan S., Dover J., Rosenwald A. G., Wolfl S., Johnston M.. 1996; Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc Natl Acad Sci U S A93:12428–12432[CrossRef]
    [Google Scholar]
  30. Palmieri L., Vozza A., Agrimi G., De Marco V., Runswick M. J., Palmieri F., Walker J. E.. 1999; Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate. J Biol Chem274:22184–22190[CrossRef]
    [Google Scholar]
  31. Petersen S., de Graaf A. A., Eggeling L., Möllney M., Wiechert W., Sahm H.. 2000; In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum. J Biol Chem275:35932–35941[CrossRef]
    [Google Scholar]
  32. Rolland F., Winderickx J., Thevelein J. M.. 2002; Glucose-sensing and -signalling mechanisms in yeast. FEMS Yeast Res2:183–201[CrossRef]
    [Google Scholar]
  33. Sauer U.. 2004; High-throughput phenomics: experimental methods for mapping fluxomes. Curr Opin Biotechnol15: (in press
    [Google Scholar]
  34. Sauer U., Lasko D. R., Fiaux J., Hochuli M., Glaser R., Szyperski T., Wüthrich K., Bailey J. E.. 1999; Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol181:6679–6688
    [Google Scholar]
  35. Sauer U., Canonaco F., Heri S., Perrenoud A., Fischer E.. 2004; The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem (in press). DOI [CrossRef] PMID 14660605
    [Google Scholar]
  36. Smits P. H., Hauf J., Muller S., Hobley T. J., Zimmermann F. K., Hahn-Hagerdal B., Nielsen J., Olsson L.. 2000; Simultaneous overexpression of enzymes of the lower part of glycolysis can enhance the fermentative capacity of Saccharomyces cerevisiae. Yeast16:1325–1334[CrossRef]
    [Google Scholar]
  37. van Dijken J. P., Bauer J., Brambilla L.. & 20 other authors. 2000; An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol26:706–714[CrossRef]
    [Google Scholar]
  38. Van Hoek P., Van Dijken J. P., Pronk J. T.. 1998; Effect of specific growth rate on fermentative capacity of baker's yeast. Appl Environ Microbiol64:4226–4233
    [Google Scholar]
  39. van Maris A. J., Bakker B. M., Brandt M., Boorsma A., Teixeira de Mattos M. J., Grivell L. A., Pronk J. T., Blom J.. 2001; Modulating the distribution of fluxes among respiration and fermentation by overexpression of HAP4 in Saccharomyces cerevisiae. FEMS Yeast Res1:139–149[CrossRef]
    [Google Scholar]
  40. Van Winden W.. 2002; Verifying assumed biosynthetic pathways, metabolic precursors and estimated measurement errors of amino acids, trehalose and levulinic acid using redundant 2D [13C, 1H] COSY NMR data. In Department of Bioprocess Technology pp.229–245 Delft: Delft University of Technology;
    [Google Scholar]
  41. Van Winden W. A., Van Gulik W. M., Schipper D., Verheijen P. J., Krabben P., Vinke J. L., Heijnen J. J.. 2003; Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [13C,1H] COSY NMR measurements and cumulative bondomer simulation. Biotechnol Bioeng83:75–92[CrossRef]
    [Google Scholar]
  42. Varela C., Agosin E., Baez M., Klapa M., Stephanopoulos G.. 2003; Metabolic flux redistribution in Corynebacterium glutamicum in response to osmotic stress. Appl Microbiol Biotechnol60:547–555[CrossRef]
    [Google Scholar]
  43. Verduyn C., Postma E., Scheffers W. A., Van Dijken J. P.. 1992; Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast8:501–517[CrossRef]
    [Google Scholar]
  44. Wiechert W.. 2001; 13C metabolic flux analysis. Metab Eng3:195–206[CrossRef]
    [Google Scholar]
  45. Winzeler E. A., Shoemaker D. D., Astromoff A.. & 22 other authors. 1999; Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science285:901–906[CrossRef]
    [Google Scholar]
  46. Wittmann C., Heinzle E.. 2001; Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis. Eur J Biochem268:2441–2455[CrossRef]
    [Google Scholar]
  47. Wittmann C., Heinzle E.. 2002; Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria. Appl Environ Microbiol68:5843–5859[CrossRef]
    [Google Scholar]
  48. Wittmann C., Hans M., Heinzle E.. 2002a; In vivo analysis of intracellular amino acid labelings by GC/MS. Anal Biochem307:379–382[CrossRef]
    [Google Scholar]
  49. Wittmann C., Hans M., Bluemke W.. 2002b; Metabolic physiology of aroma-producing Kluyveromyces marxianus. Yeast19:1351–1363[CrossRef]
    [Google Scholar]
  50. Ye L., Kruckeberg A. L., Berden J. A., van Dam K.. 1999; Growth and glucose repression are controlled by glucose transport in Saccharomyces cerevisiae cells containing only one glucose transporter. J Bacteriol181:4673–4675
    [Google Scholar]
  51. Yin Z., Wilson S., Hauser N. C., Tournu H., Hoheisel J. D., Brown A. J.. 2003; Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs. Mol Microbiol48:713–724[CrossRef]
    [Google Scholar]
  52. Zamboni N., Sauer U.. 2003; Knockout of the high-coupling cytochrome aa3 oxidase reduces TCA cycle fluxes in Bacillus subtilis. FEMS Microbiol Lett226:121–126[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26845-0
Loading
/content/journal/micro/10.1099/mic.0.26845-0
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error