1887

Abstract

Prolonged cultivation of in aerobic, glucose-limited chemostat cultures (dilution rate, 0·10 h) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg l after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an ‘evolved’ strain revealed a decreased , while was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose l, the specific growth rate of the evolved strain was lower than that of the parental strain (0·28 and 0·37 h, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type are exposed to excess glucose, the evolved strain exhibited a delay of ∼90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary ‘driving force’ for several of the observed changes remains to be elucidated.

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2005-05-01
2019-10-22
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References

  1. Adams, J., Paquin, C., Oeller, P. W. & Lee, L. W. ( 1985; ). Physiological characterization of adaptive clones in evolving populations of the yeast, Saccharomyces cerevisiae. Genetics 110, 173–185.
    [Google Scholar]
  2. Bergmeyer, H. U. ( 1974; ). Methods of Enzymatic Analysis, 2nd edn. New York & London: Academic Press.
  3. Bisson, L. F. ( 1988; ). High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control. J Bacteriol 170, 4838–4845.
    [Google Scholar]
  4. Bisson, L. F. & Fraenkel, D. G. ( 1983; ). Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 80, 1730–1734.[CrossRef]
    [Google Scholar]
  5. Boer, V. M., de Winde, J. H., Pronk, J. T. & Piper, M. D. ( 2003; ). The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J Biol Chem 278, 3265–3274.[CrossRef]
    [Google Scholar]
  6. Boles, E. & Hollenberg, C. P. ( 1997; ). The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21, 85–111.[CrossRef]
    [Google Scholar]
  7. Brown, C. M. & Hough, J. S. ( 1965; ). Elongation of yeast cells in continuous culture. Nature 206, 676–678.[CrossRef]
    [Google Scholar]
  8. Brown, C. J., Todd, K. M. & Rosenzweig, R. F. ( 1998; ). Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment. Mol Biol Evol 15, 931–942.[CrossRef]
    [Google Scholar]
  9. Chambers, A., Packham, E. A. & Graham, I. R. ( 1995; ). Control of glycolytic gene expression in the budding yeast (Saccharomyces cerevisiae). Curr Genet 29, 1–9.[CrossRef]
    [Google Scholar]
  10. de Jong-Gubbels, P., Vanrolleghem, P., van Dijken, J. P. & Pronk, J. T. ( 1995; ). Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol. Yeast 11, 407–418.[CrossRef]
    [Google Scholar]
  11. Daran-Lapujade, P., Jansen, M. L., Daran, J. M., Van Gulik, W., de Winde, J. H. & Pronk, J. T. ( 2004; ). Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae: a chemostat culture study. J Biol Chem 279, 9125–9138.[CrossRef]
    [Google Scholar]
  12. Diderich, J. A., Schuurmans, J. M., van Gaalen, M. C., Kruckeberg, A. L. & van Dam, K. ( 2001; ). Functional analysis of the hexose transporter homologue HXT5 in Saccharomyces cerevisiae. Yeast 18, 1515–1524.[CrossRef]
    [Google Scholar]
  13. Dunham, M. J., Badrane, H., Ferea, T. L., Adams, J., Brown, P. O. & Rosenzweig, R. F. ( 2002; ). Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 99, 16144–16149.[CrossRef]
    [Google Scholar]
  14. Ferea, T. L., Botstein, D., Brown, P. O. & Rosenzweig, R. F. ( 1999; ). Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc Natl Acad Sci U S A 96, 9721–9726.[CrossRef]
    [Google Scholar]
  15. Flikweert, M. T., Kuyper, M., van Maris, A. J. A., Kotter, P., van Dijken, J. P. & Pronk, J. T. ( 1999; ). Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity. Biotechnol Bioeng 66, 42–50.[CrossRef]
    [Google Scholar]
  16. Flores, N., Xiao, J., Berry, A., Bolivar, F. & Valle, F. ( 1996; ). Pathway engineering for the production of aromatic compounds in Escherichia coli. Nat Biotechnol 14, 620–623.[CrossRef]
    [Google Scholar]
  17. Forrest, W. W. & Walker, D. J. ( 1971; ). The generation and utilization of energy during growth. Adv Microb Physiol 5, 213–274.
    [Google Scholar]
  18. Francis, J. C. & Hansche, P. E. ( 1972; ). Directed evolution of metabolic pathways in microbial populations. I. Modification of the acid phosphatase pH optimum in S. cerevisiae. Genetics 70, 59–73.
    [Google Scholar]
  19. Hall, B. G. & Hauer, B. ( 1993; ). Acquisition of new metabolic activities by microbial populations. Methods Enzymol 224, 603–613.
    [Google Scholar]
  20. Iyer, V. R., Horak, C. E., Scafe, C. S., Botstein, D., Snyder, M. & Brown, P. O. ( 2001; ). Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409, 533–538.[CrossRef]
    [Google Scholar]
  21. Jansen, M. L. A., de Winde, J. H. & Pronk, J. T. ( 2002; ). Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiae to excess maltose. Appl Environ Microbiol 68, 4259–4265.[CrossRef]
    [Google Scholar]
  22. Jansen, M. L., Daran-Lapujade, P., de Winde, J. H., Piper, M. D. & Pronk, J. T. ( 2004; ). Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl Environ Microbiol 70, 1956–1963.[CrossRef]
    [Google Scholar]
  23. Kovarova-Kovar, K. & Egli, T. ( 1998; ). Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62, 646–666.
    [Google Scholar]
  24. Kubitschek, H. E. ( 1970; ). Introduction to Research with Continuous Cultures. Englewood Cliffs, NJ: Prentice Hall.
  25. Kuyper, M., Winkler, A. A., van Dijken, J. P. & Pronk, J. T. ( 2004; ). Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 4, 655–664.[CrossRef]
    [Google Scholar]
  26. Lange, H. C. & Heijnen, J. J. ( 2001; ). Statistical reconciliation of the elemental and polymeric biomass composition of Saccharomyces cerevisiae. Biotechnol Bioeng 75, 334–344.[CrossRef]
    [Google Scholar]
  27. Mäenpää, P. H., Raivio, K. O. & Kekomäki, M. P. ( 1968; ). Liver adenine nucleotides: fructose-induced depletion and its effect on protein synthesis. Science 161, 1253–1254.[CrossRef]
    [Google Scholar]
  28. Martinez-Pastor, M. T., Marchler, G., Schuller, C., Marchler-Bauer, A., Ruis, H. & Estruch, F. ( 1996; ). The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15, 2227–2235.
    [Google Scholar]
  29. Mashego, M. R., van Gulik, W. M., Vinke, J. L. & Heijnen, J. J. ( 2003; ). Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae. Biotechnol Bioeng 83, 395–399.[CrossRef]
    [Google Scholar]
  30. Mashego, M. R., Jansen, M. L., Vinke, J. L., van Gulik, W. M. & Heijnen, J. J. ( 2005; ). Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats. FEMS Yeast Res 5, 419–430.[CrossRef]
    [Google Scholar]
  31. Monod, J. ( 1942; ). Recherche sur la Croissance des Cultures Bactériennes. Paris: Hermann et Cie.
  32. Nishi, K., Park, C. S., Pepper, A. E., Eichinger, G., Innis, M. A. & Holland, M. J. ( 1995; ). The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein. Mol Cell Biol 15, 2646–2653.
    [Google Scholar]
  33. Noorman, H. J., Baksteen, J., Heijnen, J. J., Luyben, K. & Ch, A. M. ( 1991; ). The bioreactor overflow device: an undesired selective separator in continuous cultures? J Gen Microbiol 13, 2171–2177.
    [Google Scholar]
  34. Novick, A. & Szilard, L. ( 1950a; ). Experiments with the chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci U S A 36, 708–719.[CrossRef]
    [Google Scholar]
  35. Novick, A. & Szilard, L. ( 1950b; ). Description of the chemostat. Science 112, 715–716.[CrossRef]
    [Google Scholar]
  36. Oura, E. ( 1972; ). Reactions leading to the formation of yeast cell material from glucose and ethanol. PhD thesis, University of Helsinki.
  37. Özcan, S. & Johnston, M. ( 1999; ). Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63, 554–569.
    [Google Scholar]
  38. Petit, T., Diderich, J. A., Kruckeberg, A. L., Gancedo, C. & van Dam, K. ( 2000; ). Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae. J Bacteriol 182, 6815–6818.[CrossRef]
    [Google Scholar]
  39. Piper, M. D., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S., Nielsen, J. & Pronk, J. T. ( 2002; ). Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277, 37001–37008.[CrossRef]
    [Google Scholar]
  40. Postma, E., Scheffers, W. A. & van Dijken, J. P. ( 1989; ). Kinetics of growth and glucose transport in glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066. Yeast 5, 159–165.[CrossRef]
    [Google Scholar]
  41. Ramos, J., Szkutnicka, K. & Cirillo, V. P. ( 1988; ). Relationship between low- and high-affinity glucose transport systems of Saccharomyces cerevisiae. J Bacteriol 170, 5375–5377.
    [Google Scholar]
  42. Reifenberger, E., Freidel, K. & Ciriacy, M. ( 1995; ). Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on glycolytic flux. Mol Microbiol 16, 157–167.[CrossRef]
    [Google Scholar]
  43. Richard, P., Teusink, B., Hemker, M. B., van Dam, K. & Westerhoff, H. V. ( 1996; ). Sustained oscillations in free-energy state and hexose phosphates in yeast. Yeast 12, 731–740.[CrossRef]
    [Google Scholar]
  44. Rieger, M., Käppeli, O. & Fiechter, A. ( 1983; ). The role of limited respiration in the complete oxidation of glucose by Saccharomyces cerevisiae. J Gen Microbiol 129, 653–661.
    [Google Scholar]
  45. Rosenzweig, R. F., Sharp, R. R., Treves, D. S. & Adams, J. ( 1994; ). Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli. Genetics 137, 903–917.
    [Google Scholar]
  46. Rutherford, J. C., Jaron, S. & Winge, D. R. ( 2003; ). Aft1p and Aft2p mediate iron-responsive gene expression in yeast through related promoter elements. J Biol Chem 278, 27636–27643.[CrossRef]
    [Google Scholar]
  47. Sauer, U. ( 2001; ). Evolutionary engineering of industrially important microbial phenotypes. Adv Biochem Eng Biotechnol 73, 129–169.
    [Google Scholar]
  48. Schmitt, M. E., Brown, T. A. & Trumpower, T. L. ( 1990; ). A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18, 3091–3092.[CrossRef]
    [Google Scholar]
  49. Senn, H., Lendenmann, U., Snozzi, M., Hamer, G. & Egli, T. ( 1994; ). The growth of Escherichia coli in glucose-limited chemostat cultures: a re-examination of the kinetics. Biochim Biophys Acta 1201, 424–436.[CrossRef]
    [Google Scholar]
  50. Stouthamer, A. H. ( 1973; ). A theoretical study on the amount of ATP required for synthesis of microbial cell material. Antonie van Leeuwenhoek 39, 545–565.[CrossRef]
    [Google Scholar]
  51. Teusink, B., Diderich, J. A., Westerhoff, H. V., van Dam, K. & Walsh, M. C. ( 1998a; ). Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50 %. J Bacteriol 180, 556–562.
    [Google Scholar]
  52. Teusink, B., Walsh, M. C., van Dam, K. & Westerhoff, H. V. ( 1998b; ). The danger of metabolic pathways with turbo design. Trends Biochem Sci 23, 162–169.[CrossRef]
    [Google Scholar]
  53. Tusher, V. G., Tibshirani, R. & Chu, G. ( 2001; ). Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98, 5116–5121.[CrossRef]
    [Google Scholar]
  54. van Dijken, J. P., Harder, W., Beardsmore, A. J. & Quayle, J. R. ( 1978; ). Dihydroxyacetone: an intermediate in the assimilation of methanol by yeasts? FEMS Microbiol Lett 4, 97–102.[CrossRef]
    [Google Scholar]
  55. van Helden, J., Andre, B. & Collado-Vides, J. ( 2000; ). A web site for the computational analysis of yeast regulatory sequences. Yeast 16, 177–187.[CrossRef]
    [Google Scholar]
  56. van Hoek, P. ( 2000; ). Fermentative capacity in aerobic cultures of bakers' yeast. PhD thesis, Technical University of Delft.
  57. 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 Microbiol 64, 4226–4233.
    [Google Scholar]
  58. van Maris, A. J. A., 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 Res 1, 139–149.[CrossRef]
    [Google Scholar]
  59. van Schie, B. J., Rouwenhorst, R. J., van Dijken, J. P. & Kuenen, J. G. ( 1989; ). Selection of glucose-assimilating variants of Acinetobacter calcoaceticus LMD 79.41 in chemostat culture. Antonie van Leeuwenhoek 55, 39–52.[CrossRef]
    [Google Scholar]
  60. 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.[CrossRef]
    [Google Scholar]
  61. van Urk, H., Voll, W. S. L., Scheffers, W. A. & van Dijken, J. P. ( 1990; ). Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl Environ Microbiol 56, 282–286.
    [Google Scholar]
  62. Verduyn, C., van Dijken, J. P. & Scheffers, W. A. ( 1984; ). Colorimetric alcohol assays with alcohol oxidase. J Microbiol Methods 2, 15–25.[CrossRef]
    [Google Scholar]
  63. Verduyn, C., Postma, E., Scheffers, W. A. & van Dijken, J. P. ( 1992; ). Effect of benzoic acid on metabolic fluxes in yeasts: a continuous study on regulation of respiration and alcoholic fermentation. Yeast 8, 501–517.[CrossRef]
    [Google Scholar]
  64. Walsh, M. C., Smits, H. P., Scholte, M. & van Dam, K. ( 1994; ). Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol 176, 953–958.
    [Google Scholar]
  65. Weikert, C., Sauer, U. & Bailey, J. E. ( 1997; ). Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties. Microbiology 143, 1567–1574.[CrossRef]
    [Google Scholar]
  66. Weusthuis, R. A., Luttik, M. A. H., Scheffers, W. A., van Dijken, J. P. & Pronk, J. T. ( 1994; ). Is the Kluyver effect in yeast caused by product inhibition? Microbiology 140, 1723–1729.[CrossRef]
    [Google Scholar]
  67. Wick, L. M., Weilenmann, H. & Egli, T. ( 2002; ). The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics. Microbiology 148, 2889–2902.
    [Google Scholar]
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Expression profiles of genes whose expression is signficantly changed in the evolved strain compared to its parental strain in glucose-limited chemostat cultivation ( PDF file, 84 kb) Potentially duplicated regions of chromosomes III, V and X after 200 generations of selected evolution as suggested by transcriptome analysis ( PDF file, 27 kb)

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Expression profiles of genes whose expression is signficantly changed in the evolved strain compared to its parental strain in glucose-limited chemostat cultivation ( PDF file, 84 kb) Potentially duplicated regions of chromosomes III, V and X after 200 generations of selected evolution as suggested by transcriptome analysis ( PDF file, 27 kb)

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