1887

Abstract

NMR isotopic filiation of C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in during anaerobic glucose fermentation. The assimilation of [3-C]aspartate led to the formation of [2,3-C]malate and [2,3-C]succinate, with equal levels of C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [3-C]glutamate. The non-random distribution of C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed C distribution suggests that the succinate dehydrogenase (SDH) complex is not active during glucose fermentation. This hypothesis was tested by deleting the gene encoding the flavoprotein subunit of the SDH complex. The growth, fermentation rate and metabolite profile of the mutant were similar to those of the parental strain, demonstrating that SDH was indeed not active. Filiation experiments indicated the reductive branch of the TCA pathway was the main pathway for succinate production if aspartate was used as the nitrogen source, and that a surplus of succinate was produced by oxidative decarboxylation of 2-oxoglutarate if glutamate was the sole nitrogen source. Consistent with this finding, a mutant displayed lower levels of succinate production on glutamate than on other nitrogen sources, and higher levels of oxoglutarate dehydrogenase activity were observed on glutamate. Thus, the reductive branch generating succinate via fumarate reductase operates independently of the nitrogen source. This pathway is the main source of succinate during fermentation, unless glutamate is the sole nitrogen source, in which case the oxidative decarboxylation of 2-oxoglutarate generates additional succinate.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26007-0
2003-09-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/9/mic1492669.html?itemId=/content/journal/micro/10.1099/mic.0.26007-0&mimeType=html&fmt=ahah

References

  1. Albers E., Larsson C., Lidén G., Niklasson C., Gustafsson L. 1996; Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl Environ Microbiol 62:3187–3195
    [Google Scholar]
  2. Albers E., Gustafsson L., Niklasson C., Lidén G. 1998; Distribution of 14C-labelled carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae . Microbiology 144:1683–1690
    [Google Scholar]
  3. Arikawa Y., Enomoto K., Muratsubakin H., Okazaki M. 1998; Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth. FEMS Microbiol Lett 165:111–116
    [Google Scholar]
  4. Atzpodien W., Gancedo J. M., Duntze W., Holzer H. 1968; Isoenzymes of malate dehydrogenase in Saccharomyces cerevisiae . Eur J Biochem 7:58–62
    [Google Scholar]
  5. Bely M., Sablayrolles J. M., Barre P. 1990; Automatic detection of assimilable nitrogen deficiencies during alcoholic fermentation in enological conditions. J Ferment Bioeng 70:246–252
    [Google Scholar]
  6. Chapman C., Bartley W. 1968; The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria. Biochem J 107:455–465
    [Google Scholar]
  7. Coleman S. T., Fang T. K., Rovinsky S. A., Turano F. J., Moye-Rowley W. S. 2001; Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae . J Biol Chem 276:244–250
    [Google Scholar]
  8. de Winde J. H., Grivell L. A. 1993; Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae . Prog Nucleic Acid Res Mol Biol 46:51–91
    [Google Scholar]
  9. 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 Bacteriol 183:1441–1451
    [Google Scholar]
  10. Gonzalez B., Francois J., Renaud M. 1997; A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast 13:1347–1355
    [Google Scholar]
  11. Gray C. T., Wimpenny J. W., Mossman M. R. 1966; Regulation of metabolism in facultative bacteria. II. Effects of aerobiosis, anaerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli . Biochim Biophys Acta 117:33–41
    [Google Scholar]
  12. Heerde E., Radler F. 1978; Metabolism of the anaerobic formation of succinic acid by Saccharomyces cerevisiae . Arch Microbiol 117:269–276
    [Google Scholar]
  13. Huet C., Menendez J., Gancedo C., François J. M. 2000; Regulation of PYC1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae . Eur J Biochem 267:6817–6823
    [Google Scholar]
  14. Iuchi S., Lin E. C. C. 1993; Adaptation of Escherichia coli to redox environments by gene expression. Mol Microbiol 9:9–15
    [Google Scholar]
  15. Keruchenko J., Keruchenko I., Gladilin K., Zuitsev V., Chirgadze Y. 1992; Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae . Biochim Biophys Acta 1122:85–92
    [Google Scholar]
  16. Klein C. J. L., Olsson L., Nielsen J. 1998; Glucose control in Saccharomyces cerevisiae : the role of MIG1 in metabolic functions. Microbiology 144:13–24
    [Google Scholar]
  17. Kroger A. 1978; Fumarate reductase as terminal acceptor of phosphorylative electron transport. Biochim Biophys Acta 505:129–145
    [Google Scholar]
  18. Kwast K. E., Burke P. V., Poyton R. O. 1998; Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast. J Exp Biol 201:1177–1195
    [Google Scholar]
  19. Lewis M. J., Rainbow C. 1963; Transamination and the liberation of 2-oxoglutarate by yeast. J Inst Brew 69:39–45
    [Google Scholar]
  20. Liu Z., Butow R. 1999; A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response to a reduction or loss of respiratory function. Mol Cell Biol 19:6720–6728
    [Google Scholar]
  21. Lupiañez J., Machado A., Nuñez de Castro I., Mayor F. 1974; Succinic acid production by yeasts grown under different hypoxic conditions. Mol Cell Biochem 3:113–116
    [Google Scholar]
  22. Maaheimo H., Fiaux J., Cakar Z., Bailey J., Sauer U., Szyperski T. 2001; Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional 13C labeling of common amino acids. Eur J Biochem 268:2464–2479
    [Google Scholar]
  23. Machado A., Nunez de Castro I., Mayor F. 1975; Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions. Mol Cell Biochem 6:93–100
    [Google Scholar]
  24. Muratsubaki H. 1987; Regulation of reductive production of succinate under anaerobic conditions in baker's yeast. J Biochem 102:705–714
    [Google Scholar]
  25. Nordström K. 1968; Yeast growth and glycerol formation. II. Carbon and redox balances. J Inst Brew 74:429–432
    [Google Scholar]
  26. Nunez de Castro I., Ugarte M., Cano A., Mayor F. 1970; Effect of glucose, galactose, and different nitrogen-sources on the activity of yeast glutamate dehydrogenase (NAD and NADP-linked) from normal strain and impaired respiration mutant. Eur J Biochem 16:567–570
    [Google Scholar]
  27. Oura E. 1977; Reaction products of yeast fermentations. Process Biochem 35:19–21
    [Google Scholar]
  28. Pines O., Even-Ram S., Elnathan S., Battat E., Aharonov O., Gibson D., Goldberg J. 1996; The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae : the role of fumarase. Appl Microbiol Biotechnol 46:393–399
    [Google Scholar]
  29. Pitson S. M., Mendz G. L., Srinivasan S., Hazell S. L. 1999; The tricarboxylic acid cycle of Helicobacter pylori . Eur J Biochem 260:258–267
    [Google Scholar]
  30. Polakis E., Bartley W. 1965; Changes in the enzyme activities of Saccharomyces cerevisiae during aerobic growth on different carbon sources. Biochem J 97:284–297
    [Google Scholar]
  31. Reed L. J., Oliver R. M. 1982; Structure–function relationships in pyruvate and alpha-ketoglutarate dehydrogenase complexes. Adv Exp Med Biol 148:231–241
    [Google Scholar]
  32. Rosenkrantz M., Kell C., Pennel E., Devenish L. 1994; The HAP2 , 3 , 4 transcriptional activator is required for derepression of the yeast citrate synthase gene CIT1 . Mol Microbiol 13:119–131
    [Google Scholar]
  33. Sablayrolles J. M., Barre P., Grenier P. 1987; Design of laboratory automatic system for studying alcoholic fermentations in anisothermal enological conditions. Biotechnol Tech 1:180–184
    [Google Scholar]
  34. Sauer U., Lasko D. R., Fiaux J., Hochuli M., Glaser R., Szyperski T., Wuthrich K., Bailey J. E. 1999; Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181:6679–6688
    [Google Scholar]
  35. Schiesti R. H., Gietz R. D. 1989; High efficiency transformation of intact cells using simple stranded nucleic acid as carrier. Curr Genet 16:339–346
    [Google Scholar]
  36. Sols A., Gancedo C., de la Fuente G. 1971; Energy-yielding metabolism in yeasts. In The Yeasts vol. 2 pp 271–307 Edited by Rose A. H., Harrison J. S. London: Academic Press;
    [Google Scholar]
  37. Spiro S., Guest J. 1991; Adaptive response to oxygen limitation in Escherichia coli . Trends Biochem Sci 16:310–314
    [Google Scholar]
  38. Wach A., Brachat A., Pöhlmann R., Philippsen P. 1994; New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae . Yeast 10:1793–1808
    [Google Scholar]
  39. Wakaï Y., Shimazaki T., Hara S. 1980; Formation of succinate during fermentation of sake mash and grape must. Hakkokogaku 58:363–368
    [Google Scholar]
  40. Wales D. S., Cartledge T. G., Lloyd D. 1980; Effects of glucose repression and anaerobiosis on the activities and subcellular distribution of tricarboxylic acid cycle and associated enzymes in Saccharomyces cerevisiae . J Gen Microbiol 116:93–98
    [Google Scholar]
  41. Whiting G. 1976; Organic acid metabolism in yeast during fermentation of alcoholic beverages. J Inst Brew 82:84–92
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26007-0
Loading
/content/journal/micro/10.1099/mic.0.26007-0
Loading

Data & Media loading...

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