1887

Abstract

The physiological phenotype of was investigated for different genetic and environmental conditions of glucose repression through the quantification of fluxes in the central carbon metabolism using C-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of . Batch cultivations were performed with a reference strain and a deletion mutant strain (Δ4) using [1-C]glucose as carbon source. The mutant strain was also grown on a mixture of [1-C]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the metabolic fluxes. The -mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0·11 and 0·25 h, respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0·11 h). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of upon deletion of the gene was a 20 % decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40 % in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden–Meyerhof–Parnas pathway and the tricarboxylic acid cycle (in the range of 20–30 %). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27787-0
2005-07-01
2019-11-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/7/mic1512209.html?itemId=/content/journal/micro/10.1099/mic.0.27787-0&mimeType=html&fmt=ahah

References

  1. Adams, I. P., Dack, S., Dickinson, F. M., Midgley, M. & Ratledge, L. ( 1997; ). ATP:citrate lyase from Aspergillus nidulans. Biochem Soc Trans 25, S670.
    [Google Scholar]
  2. Agger, T., Petersen, J. B., O'Connor, S. M., Murphy, R. L., Kelly, J. M. & Nielsen, J. ( 2002; ). Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase. J Biotechnol 92, 279–285.[CrossRef]
    [Google Scholar]
  3. Arst, H. N., Jr & Bailey, C. R. ( 1977; ). The regulation of carbon metabolism in Aspergillus nidulans. In Genetics and Physiology of Aspergillus nidulans, pp. 131–146. Edited by J. E. Smith & J. A. Pateman. London: Academic Press.
  4. Bradford, M. M. ( 1976; ). A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  5. Carlsen, M. & Nielsen, J. ( 2001; ). Influence of carbon source on alpha-amylase production by Aspergillus oryzae. Appl Microbiol Biotechnol 57, 346–349.[CrossRef]
    [Google Scholar]
  6. Christensen, B. & Nielsen, J. ( 2000a; ). Metabolic network analysis. A powerful tool in metabolic engineering. Adv Biochem Eng Biotechnol 66, 209–231.
    [Google Scholar]
  7. Christensen, B. & Nielsen, J. ( 2000b; ). Metabolic network analysis of Penicillium chrysogenum using 13C-labeled glucose. Biotechnol Bioeng 68, 652–659.[CrossRef]
    [Google Scholar]
  8. David, H., Åkesson, M. & Nielsen, J. ( 2003; ). Reconstruction of the central carbon metabolism of Aspergillus niger. Eur J Biochem 270, 4243–4253.[CrossRef]
    [Google Scholar]
  9. Dowzer, C. E. & Kelly, J. M. ( 1991; ). Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol 11, 5701–5709.
    [Google Scholar]
  10. Felenbok, B., Flipphi, M. & Nikolaev, I. ( 2001; ). Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. Prog Nucleic Acid Res Mol Biol 69, 149–204.
    [Google Scholar]
  11. Fischer, E. & Sauer, U. ( 2003; ). Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 270, 880–891.[CrossRef]
    [Google Scholar]
  12. Gombert, A. K., Moreira dos, S. 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.[CrossRef]
    [Google Scholar]
  13. Hondmann, D. H. & Visser, J. ( 1994; ). Carbon metabolism. In Aspergillus: 50 Years On, pp. 61–139. Edited by S. D. Martinelli & J. R. Kinghorn. Amsterdam: Elsevier.
  14. Ilyés, H., Fekete, E., Karaffa, L., Fekete, E., Sandor, E., Szentirmai, A. & Kubicek, C. P. ( 2004; ). CreA-mediated carbon catabolite repression of beta-galactosidase formation in Aspergillus nidulans is growth rate dependent. FEMS Microbiol Lett 235, 147–151.
    [Google Scholar]
  15. Kelly, J. M. ( 1994; ). Carbon catabolite repression. In Aspergillus: 50 Years On, pp. 355–367. Edited by S. D. Martinelli & J. R. Kinghorn. Amsterdam: Elsevier.
  16. Kelly, J. M. & Hynes, M. J. ( 1981; ). The regulation of phosphoenolpyruvate carboxykinase and the NADP-linked malic enzyme in Aspergillus nidulans. J Gen Microbiol 123, 371–375.
    [Google Scholar]
  17. Kelly, J. M. & Hynes, M. J. ( 1982; ). The regulation of NADP-linked isocitrate dehydrogenase in Aspergillus nidulans. J Gen Microbiol 128, 23–28.
    [Google Scholar]
  18. Kubicek, C. & Röhr, M. ( 1986; ). Citric acid fermentation. Crit Rev Biotechnol 3, 331–373.
    [Google Scholar]
  19. Manzoni, M. & Rollini, M. ( 2002; ). Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs. Appl Microbiol Biotechnol 58, 555–564.[CrossRef]
    [Google Scholar]
  20. Nielsen, J. ( 2001; ). Metabolic engineering. Appl Microbiol Biotechnol 55, 263–283.[CrossRef]
    [Google Scholar]
  21. Osmani, S. A. & Scrutton, M. C. ( 1983; ). The sub-cellular localisation of pyruvate carboxylase and of some other enzymes in Aspergillus nidulans. Eur J Biochem 133, 551–560.[CrossRef]
    [Google Scholar]
  22. Pedersen, H., Carlsen, M. & Nielsen, J. ( 1999; ). Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain. Appl Environ Microbiol 65, 11–19.
    [Google Scholar]
  23. Prathumpai, W., McIntyre, M. & Nielsen, J. ( 2004; ). The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans. Appl Microbiol Biotechnol 63, 748–753.[CrossRef]
    [Google Scholar]
  24. Raghevendran, V., Gombert, A. K., Christensen, B., Kotter, P. & Nielsen, J. ( 2004; ). Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C-labelled glucose. Yeast 21, 769–779.[CrossRef]
    [Google Scholar]
  25. Ruijter, G. & Visser, J. ( 1997; ). Carbon repression in aspergilli. FEMS Microbiol Lett 151, 103–114.[CrossRef]
    [Google Scholar]
  26. Scazzocchio, C., Gavrias, V., Cubero, B., Panozzo, C., Mathieu, M. & Felenbok, B. ( 1995; ). Carbon catabolite repression in Aspergillus nidulans: a review. Can J Bot 73, S160–S166.[CrossRef]
    [Google Scholar]
  27. Shroff, R. A., O'Connor, S. M., Hynes, M. J., Lockington, R. A. & Kelly, J. M. ( 1997; ). Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans. Fungal Genet Biol 22, 28–38.[CrossRef]
    [Google Scholar]
  28. Sims, A. H., Robson, G. D., Hoyle, D. C., Oliver, S. G., Turener, G., Prade, R. A., Russell, H. H., Dunn-Coleman, N. & Gent, M. E. ( 2004; ). Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans. Fungal Genet Biol 41, 199–212.[CrossRef]
    [Google Scholar]
  29. Singh, M., Scrutton, N. S. & Scrutton, M. C. ( 1988; ). NADPH generation in Aspergillus nidulans: is the mannitol cycle involved? J Gen Microbiol 134, 643–654.
    [Google Scholar]
  30. Szyperski, T. ( 1998; ). 13C-NMR, MS and metabolic flux balancing in biotechnology research. Q Rev Biophys 31, 41–106.[CrossRef]
    [Google Scholar]
  31. van der Veen, P., Ruijter, G. J. & Visser, J. ( 1995; ). An extreme creA mutation in Aspergillus nidulans has severe effects on d-glucose utilization. Microbiology 141, 2301–2306.[CrossRef]
    [Google Scholar]
  32. Wiechert, W. ( 2001; ). 13C metabolic flux analysis. Metab Eng 3, 195–206.[CrossRef]
    [Google Scholar]
  33. Wiechert, W. & de Graaf, A. A. ( 1996; ). In vivo stationary flux analysis by 13C labeling experiments. Adv Biochem Eng Biotechnol 54, 109–154.
    [Google Scholar]
  34. Wiechert, W. & de Graaf, A. A. ( 1997; ). Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. Biotechnol Bioeng 55, 101–117.[CrossRef]
    [Google Scholar]
  35. Wiechert, W., Mollney, M., Petersen, S. & de Graaf, A. A. ( 2001; ). A universal framework for 13C metabolic flux analysis. Metab Eng 3, 265–283.[CrossRef]
    [Google Scholar]
  36. Zamboni, N. & Sauer, U. ( 2003; ). Knockout of the high-coupling cytochrome aa3 oxidase reduces TCA cycle fluxes in Bacillus subtilis. FEMS Microbiol Lett 226, 121–126.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27787-0
Loading
/content/journal/micro/10.1099/mic.0.27787-0
Loading

Data & Media loading...

Supplements

vol. , part 7, pp. 2209 - 2221

Metabolic model [PDF](78 KB)



PDF
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