Current Genetics

, Volume 26, Issue 5–6, pp 422–429 | Cite as

Construction by one-step gene replacement of Trichoderma reesei strains that produce the glucoamylase P of Hormoconis resinae

  • Vesa V. Joutsjoki
Original Articles


Two one-step gene replacement vectors containing either the Hormoconis resinae glucoamylase P (gamP) genomic gene or the corresponding cDNA, each under the control of the promoter of the Trichoderma reesei cellobiohydrolase 1 gene (cbh1), were constructed and use to replace the cbh1 gene in a T. reesei strain. In both vectors the cbh1 promoter is precisely fused to the gamP protein coding region. Both the gamP cDNA and the genomic gene direct the secretion of the active glucoamylase P (GAMP) enzyme from T. reesei, which indicates that the intron sequences in the genomic gamP gene are processed in T. reesei. According to the results, a T. reesei transformant strain, in which the cbh1 gene has been replaced by a single copy of the gamP genomic gene, secretes more active GAMP than does a transformant strain having three copies of the cDNA clone in tandem orientation at the cbh1 locus.

Key words

Trichoderma reesei Homologous recombination Glucoamylase Hormoconis resinae 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aho S, Olkkonen V, Jalava T, Paloheimo M, Bühler R, Niku-Paavola M-L, Bamford DH, Korhola M (1991) Monoclonal antibodies against core and cellulase-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. Eur J Biochem 200:643–649Google Scholar
  2. Bailey MJ, Nevalainen KMH (1981) Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microbiol Technol 3:153–157Google Scholar
  3. Banerji J, Olson J, Schaffner W (1983) A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33:729–740Google Scholar
  4. Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86:153–162Google Scholar
  5. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acad Res 7:1513–1523Google Scholar
  6. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  7. Durand H, Clanet M, Tiraby G (1988) Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microbiol Technol 10:341–346Google Scholar
  8. Esser K, Mohr G (1986) Integrative transformation of filamentous fungi with resepct to biotechnological application. Process Biochem Oct:153–159Google Scholar
  9. Fagerström R (1994) Purification and specificity of recombinant Hormoconis resinae glucoamylase P and endogenous glucoamylase from Trichoderma reesei. Enzyme Microbiol Technol 16:36–42Google Scholar
  10. Fagerström R, Vainio A, Suoranta K, Pakula T, Kalkkinen N, Torkkeli H (1990) Comparison of two glucoamylases from Hormoconis resinae. J Gen Microbiol 136:913–920Google Scholar
  11. Finkelstein DB (1992) Transformation. In: Finkelstein DB, Ball C (eds) Biotechnology of filamentous fungi. Technology and products. Butterworth-Heinemann, Boston, pp 113–156Google Scholar
  12. Harkki A, Uusitalo J, Bailey M, Penttilä M, Knowles JKC (1989) A novel fungal expression system: secretion of active calf chymosin from the filamentous fungus Trichoderma reesei. Bio/Technology 7:596–603Google Scholar
  13. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933Google Scholar
  14. Joutsjoki VV, Torkkeli TK (1992) Glucoamylase P gene of Hormoconis resinae: molecular cloning, sequencing and introduction into Trichoderma reesei. FEMS Microbiol Lett 99:237–244Google Scholar
  15. Joutsjoki VV, Torkkeli TK, Nevalainen KMH (1993) Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei. Curr Genet 24:223–228Google Scholar
  16. Karhunen T, Mäntylä A, Nevalainen KMH, Suominen PL (1993) High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet 241:515–522Google Scholar
  17. Kelly JM, Hynes MJ (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479Google Scholar
  18. Konieczny SF, Emerson Jr CP (1987) Complex regulation of the muscle-specific contractile protein (troponin I) gene. Mol Cell Biol 7:3065–3075Google Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  20. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  21. Miller BL, Miller KY, Timberlake WE (1985) Direct and indirect gene replacements in Aspergillus nidulans. Mol Cell Biol 5:1714–1721Google Scholar
  22. Nevalainen H, Penttilä M, Harkki A, Teeri T, Knowles JKC (1991) The molecular biology of Trichoderma and its application to the expression of both homologous and heterologous gene. In: Leong SA, Berka R (eds) Molecular industrial mycology. Systems and applications for filamentous fungi. Marcel Dekker Inc, New York, pp 129–148Google Scholar
  23. Orr-Weaver TL, Szostack JW, Rothstein RJ (1981) Yeast transformantion: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358Google Scholar
  24. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164Google Scholar
  25. Prentice N (1982) Purification of beta-glucanase for beta-D-glucan assays. Cereal Chem 59:231–232Google Scholar
  26. Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20Google Scholar
  27. Rossi P, de Crombrugghe B (1987) Identification of a cell-specific transcriptional enhancer in the first intron of the mouse α2 (type I) collagen gene. Proc Natl Acad Sci USA 84:5590–5594Google Scholar
  28. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211Google Scholar
  29. Saloheimo M, Niku-Paavola M-L (1991) Heterologous production of a ligninolytic enzyme: expression of the Phlebia radiata laccase gene in Trichoderma reesei. Bio/Technology 9:987–990Google Scholar
  30. Scherer S, Davis RW (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci USA 76:4951–4955Google Scholar
  31. Seiboth B, Messner R, Gruber F, Kubicek CP (1992) Disruption of the Trichoderma reesei cbh2 gene coding for cellobiohydrolase II leads to a delay in the triggering of cellulase formation by cellulose. J Gen Microbiol 138: 1259–1264Google Scholar
  32. Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis M (1983) Molecular cloning of exo-cellobiohydrolase I derived from Trichoderma reesei strain L27. Bio/Technology 1:691–696Google Scholar
  33. Slater EP, Rabenau O, Karin M, Baxter JD, Beato M (1985) Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene. Mol Cell Biol 5:2984–2992Google Scholar
  34. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  35. Suominen P, Mäntylä A, Saarelainen R, Paloheimo M, Fagerström R, Parkkinen E, Nevalainen H (1992) Genetic engineering of Trichoderma reesei to produce suitable enzyme combinations for applications in the pulp and paper industry. In: Kuwahara M, Shimada M (eds) Biotechnology in pulp and paper industry. Uni Publishers Co, Ltd, Tokyo, Japan, pp 439–445Google Scholar
  36. Suominen PL, Mäntylä AL, Karhunen T, Hakola S, Nevalainen H (1993) High-frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes Mol Gen Genet 241: 523–530Google Scholar
  37. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354Google Scholar
  38. Vainio AEI, Torkkeli HT, Tuusa T, Aho SA, Fagerström BR, Korhola MP (1993) Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae. Curr Genet 24: 38–44Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Vesa V. Joutsjoki
    • 1
  1. 1.Research LaboratoriesAlko LtdHelsinkiFinland

Personalised recommendations