Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Free

Abstract

T23C (::Tn) is an isogenic gene replacement mutant of the wild-type strain T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh) strain was investigated in aerobic glucose-limited chemostat cultures, grown at a dilution rate of 0.10 h, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass of the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the ::Tn mutation on the growth efficiency was anticipated. Indeed, the biomass yield of the Pdh strain [0.44 g biomass (g glucose)] was significantly lower than that of wild-type [0.52 g biomass (g glucose)]. The effect of the mutation on biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the ::Tn mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucose-limited growth of at low growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in this yeast. Respiratory-deficient cells did not contribute to glucose metabolism in the chemostat cultures and were probably formed upon plating.

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1994-03-01
2024-03-29
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References

  1. Dixon G. H., Kornberg H. L. 1959; Assay methods for key enzymes of the glyoxylate cycle. Biochem J 72:3 p
    [Google Scholar]
  2. Hohmann S., Cederberg H. 1990; Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5 . Eur J Biochem 188:615–621
    [Google Scholar]
  3. Holzer H., Goedde W. H. 1957; Zwei Wege von Pyruvat zu Acetyl-Coenzym A in Hefe. Biochem Z 329:175–191
    [Google Scholar]
  4. Kresze G. B., Ronft H. 1981; Pyruvate dehydrogenase from baker’s yeast. I. Purification and some kinetic and regulatory properties. Eur J Biochem 119:573–579
    [Google Scholar]
  5. Lichko L., Okorokov L. A. 1991; Purification and some properties of membrane-bound and soluble pyrophosphatases of yeast vacuoles. Yeast 7:805–812
    [Google Scholar]
  6. Lundin M., Deopugari S. W., Lichko L., , Pereira da Silva L., Baltscheffski H. 1992; Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae . Biochim Biophys Acta 1098:217–223
    [Google Scholar]
  7. Oura E. 1972 The effect of aeration on the growth energetics and biochemical composition of baker's yeast. PhD thesis, University of Helsinki; Finland:
    [Google Scholar]
  8. Parelukar S. J., Semones G. B., Rolf M. J., Lievense J. C., Lim H. C. 1986; Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae . Biotechnol Bioeng 28:700–710
    [Google Scholar]
  9. Petrik M., Käppeli O., Fiechter A. 1983; An expanded concept for the glucose effect in the yeast Saccharomyces uvarum: involvement of short- and long-term regulation. J Gen Microbiol 129:43–49
    [Google Scholar]
  10. Postma E., Verduyn C., Scheffers W. A., van Dijken J. P. 1989; Enzymic analysis of the Crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae . Appl Environ Microbiol 53:468–477
    [Google Scholar]
  11. Pringle J. R., Preston R. A., Adams A. E. M., , Stearns T., , Drubin D. G., Haarer B. K., Jones E. W. 1989; Fluorescence microscopy for yeast. Methods Cell Biol 31:358–429
    [Google Scholar]
  12. Skowronek P., Krummeck G., Haferkamp O., Rödel G. 1990; Flow cytometry as a tool to discriminate respiratory-competent and respiratory-deficient yeast cells. Curr Genet 18:265–267
    [Google Scholar]
  13. Sonnleitner B. 1991; Dynamics of yeast metabolism and regulation. Bioprocess Eng 6:187–193
    [Google Scholar]
  14. Srere P. A. 1969; Citrate synthase. Methods Enzymol 13:3–11
    [Google Scholar]
  15. Steensma H. Y., Holterman L., Dekker I., van Sluis C. A., Wenzel T. J. 1990; Molecular cloning of the gene for the El a subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae . Eur J Biochem 191:769–774
    [Google Scholar]
  16. van Urk H., Mak P. R., Scheffers W. A., van Dijken J. P. 1988; Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess. Yeast 4:283–291
    [Google Scholar]
  17. van Urk H., Schipper D., Breedveld G. J., Mak P. R., Scheffers W. A., van Dijken J. P. 1989; Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim Biophys Acta 992:78–86
    [Google Scholar]
  18. Verduyn C., Postma E., Scheffers W. A., van Dijken J. P. 1990; Physiology of Saccharomyces cerevisiae in anaerobic glucose- limited chemostat cultures. J Gen Microbiol 136:395–403
    [Google Scholar]
  19. Verduyn C., Stouthamer A. H., Scheffers W. A., van Dijken J. P. 1991; A theoretical evaluation of growth yields of yeasts. Antonie Eeeuwenhoek 59:49–63
    [Google Scholar]
  20. Verduyn C., Postma E., Scheffers W. A., van Dijken J. P. 1992; Effect of benzoic acid on metabolic fluxes in yeast: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517
    [Google Scholar]
  21. Visser W., Scheffers W. A., Batenburg-van der Vegte W. H., van Dijken J. P. 1990; Oxygen requirements of yeasts. Appl Environ Microbiol 56:3785–3792
    [Google Scholar]
  22. Wenzel T. J., van den Berg M. A., Visser W., van den Berg J. A., Steensma H. Y. 1992; Characterization of mutants lacking the El a subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae . Eur J Biochem 209:697–705
    [Google Scholar]
  23. Wetlaufer D. B. 1962; Ultraviolet spectra of proteins and amino acids. Adv Protein Chem 17:303–390
    [Google Scholar]
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