1887

Abstract

Lactose (1,4----galactopyranosyl--glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by (anamorph ). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on -galactose alone does not induce cellulases. Consequently, the hypothesis was tested that -galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of were therefore performed at different dilution rates with -galactose, lactose, galactitol and -glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the (encoding cellobiohydrolase 2, Cel6A) promoter region and the glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that -galactose indeed induces gene transcription and leads to Cel7A and Cel6A accumulation at a low (=0·015 h) but not at higher dilution rates. At the same dilution rate, growth on -glucose did not lead to promoter activation or Cel6A formation but a basal level, lower than that observed on -galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0·015 h than -galactose and induced cellulases even at growth rates up to 0·042 h. Results of chemostats with an equimolar mixture of -galactose and -glucose essentially mimicked the behaviour on -galactose alone, whereas an equimolar mixture of -galactose and galactitol, the first intermediate of a recently described second pathway of -galactose catabolism, led to cellulase induction at =0·030 h. It is concluded that -galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28719-0
2006-05-01
2024-11-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/5/1507.html?itemId=/content/journal/micro/10.1099/mic.0.28719-0&mimeType=html&fmt=ahah

References

  1. Andreotti R. E., Medeiros J. E., Roche C., Mandels M. 1980; Effects of strain and substrate on production of cellulases by Trichoderma reesei mutants. In Bioconversion and Bioengineering Symposium 2 pp 353–371 Edited by Ghose T. K. New Delhi: BERC, IIT;
    [Google Scholar]
  2. Aro N., Pakula T., Penttilä M. 2005; Transcriptional regulation of plant cell wall degradation by filamentous fungi. FEMS Microbiol Rev 29:719–739 [CrossRef]
    [Google Scholar]
  3. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 2005 Current Protocols in Molecular Biology New York: Wiley Interscience;
    [Google Scholar]
  4. Bhat P. J., Murthy T. V. 2001; Transcriptional control of the GAL / MEL regulon of yeast Saccharomyces cerevisiae : mechanism of galactose-mediated signal transduction. Mol Microbiol 40:1059–1066 [CrossRef]
    [Google Scholar]
  5. Fekete E., Karaffa L., Sandor E., Banyai I., Seiboth B., Gyemant G., Sepsi A., Szentirmai A., Kubicek C. P. 2004; The alternative d-galactose degrading pathway of aspergillus nidulans proceeds via l-sorbose. Arch Microbiol 181:35–44 [CrossRef]
    [Google Scholar]
  6. Frey P. A. 1996; The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J 10:461–470
    [Google Scholar]
  7. Geysens S., Pakula T., Uusitalo J., Dewerte I., Penttilä M., Contreras R. 2005; Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei : a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30. Appl Environ Microbiol 71:2910–2924 [CrossRef]
    [Google Scholar]
  8. Gruber F., Visser J., Kubicek C. P., de Graaf L. H. 1990a; Cloning of the Trichoderma reesei pyrG -gene and its use as a homologous marker for a high-frequency transformation system. Curr Genet 18:447–451 [CrossRef]
    [Google Scholar]
  9. Gruber F., Visser J., Kubicek C. P., de Graaff L. H. 1990b; The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG -negative mutant strain. Curr Genet 18:71–76 [CrossRef]
    [Google Scholar]
  10. Holden H. M., Rayment I., Thoden J. B. 2003; Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 278:43885–43888 [CrossRef]
    [Google Scholar]
  11. Ilmen M., Thrane C., Penttila M. 1996; The glucose repressor gene cre1 of Trichoderma : isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 251:451–460
    [Google Scholar]
  12. Ilyés H., Fekete É., Karaffa L., Sándor E., Szentirmai A., Kubicek C. P. 2004; CreA-mediated carbon catabolite repression of β -galactosidase formation in Aspergillus nidulans is growth rate dependent. FEMS Microbiol Lett 235:147–151
    [Google Scholar]
  13. Mach R. L., Peterbauer C. K., Payer K., Jaksits S., Woo S. L., Zeilinger S., Kullnig C. M., Lorito M., Kubicek C. P. 1999; Expression of two major chitinase genes of Trichoderma atroviride ( T. harzianum P1) is triggered by different regulatory signals. Appl Environ Microbiol 65:1858–1863
    [Google Scholar]
  14. Meyer J., Walker-Jonah A., Hollenberg C. P. 1991; Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae . Mol Cell Biol 11:5454–5461
    [Google Scholar]
  15. Mischak H., Hofer F., Messner R. 10 other authors 1989; Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei . Biochim Biophys Acta 990:1–7 [CrossRef]
    [Google Scholar]
  16. Pail M., Peterbauer T., Seiboth B., Hametner C., Druzhinina I., Kubicek C. P. 2004; The metabolic role and evolution of l-arabinitol 4-dehydrogenase of Hypocrea jecorina . Eur J Biochem 271:1864–1872 [CrossRef]
    [Google Scholar]
  17. Pakula T. M., Salonen K., Uusitalo J., Penttilä M. 2005; The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei . Microbiology 151:135–143 [CrossRef]
    [Google Scholar]
  18. Penttilä M. E. 1998; Heterologous protein production in Trichoderma . In Trichoderma and Gliocladium pp 356–383 Edited by Harman G. E., Kubicek C. P. London, UK: Taylor & Francis;
    [Google Scholar]
  19. Peterson G. L. 1983; Determination of total protein. Methods Enzymol 91:95–105
    [Google Scholar]
  20. Pettersson H., Pettersson G. 2001; Kinetics of the coupled reaction catalysed by a fusion protein of β -galactosidase and galactose dehydrogenase. Biochim Biophys Acta 1549155–160 [CrossRef]
    [Google Scholar]
  21. Pirt S. J., Callow D. S. 1960; Studies on the growth of Penicillium chrysogenum in continuous flow culture with reference to penicillin production. J Appl Bacteriol 23:87–98 [CrossRef]
    [Google Scholar]
  22. Schmoll M., Franchi L., Kubicek C. P. 2005; Envoy, a PAS/LOV domain protein of Hypocrea jecorina (anamorph Trichoderma reesei ), modulates cellulase gene transcription in response to light. Eukaryot Cell 4:1998–2007 [CrossRef]
    [Google Scholar]
  23. Seiboth B., Hofmann G., Kubicek C. P. 2002a; Lactose metabolism and cellulase production in Hypocrea jecorina : the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction. Mol Genet Genomics 267:124–132 [CrossRef]
    [Google Scholar]
  24. Seiboth B., Karaffa L., Sandor E., Kubicek C. 2002b; The Hypocrea jecorina gal10 (uridine 5′-diphosphate-glucose 4-epimerase- encoding) gene differs from yeast homologues in structure, genomic organization and expression. Gene 295:143–149 [CrossRef]
    [Google Scholar]
  25. Seiboth B., Hartl L., Pail M., Fekete E., Karaffa L., Kubicek C. P. 2004; The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose. Mol Microbiol 51:1015–1025 [CrossRef]
    [Google Scholar]
  26. Seiboth B., Hartl L., Salovuori N., Lanthaler K., Robson G. D., Vehmaanperä J., Penttilä M. E., Kubicek C. P. 2005; Role of the bga1 -encoded extracellular β -galactosidase of Hypocrea jecorina in cellulase induction by lactose. Appl Environ Microbiol 71:851–857 [CrossRef]
    [Google Scholar]
  27. Sternberg D., Mandels G. R. 1979; Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 139:761–769
    [Google Scholar]
  28. Thoden J. B., Holden H. M. 2005; The molecular architecture of galactose mutarotase/UDP-galactose 4-epimerase from Saccharomyces cerevisiae . J Biol Chem 280:21900–21907 [CrossRef]
    [Google Scholar]
  29. Vaheri M., Leisola M., Kaupinnen V. 1979; Transglycosylation products of the cellulase system of Trichoderma reesei . Biotechnol Lett 1:41–46 [CrossRef]
    [Google Scholar]
  30. Zeilinger S., Schmoll M., Pail M., Mach R. L., Kubicek C. P. 2003; Nucleosome transactions on the Hypocrea jecorina ( Trichoderma reesei ) cellulase promoter cbh2 associated with cellulase induction. Mol Genet Genomics 270:46–55 [CrossRef]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.28719-0
Loading
/content/journal/micro/10.1099/mic.0.28719-0
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