Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Jun;62(6):2145–2151. doi: 10.1128/aem.62.6.2145-2151.1996

Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei.

E Margolles-Clark 1, C K Hayes 1, G E Harman 1, M Penttilä 1
PMCID: PMC167992  PMID: 8787411

Abstract

The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.

Full Text

The Full Text of this article is available as a PDF (662.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aho S., Olkkonen V., Jalava T., Paloheimo M., Bühler R., Niku-Paavola M. L., Bamford D. H., Korhola M. Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. Eur J Biochem. 1991 Sep 15;200(3):643–649. doi: 10.1111/j.1432-1033.1991.tb16227.x. [DOI] [PubMed] [Google Scholar]
  2. Carsolio C., Gutiérrez A., Jiménez B., Van Montagu M., Herrera-Estrella A. Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10903–10907. doi: 10.1073/pnas.91.23.10903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Harkki A., Mäntylä A., Penttilä M., Muttilainen S., Bühler R., Suominen P., Knowles J., Nevalainen H. Genetic engineering of Trichoderma to produce strains with novel cellulase profiles. Enzyme Microb Technol. 1991 Mar;13(3):227–233. doi: 10.1016/0141-0229(91)90133-u. [DOI] [PubMed] [Google Scholar]
  5. Hayes C. K., Klemsdal S., Lorito M., Di Pietro A., Peterbauer C., Nakas J. P., Tronsmo A., Harman G. E. Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum. Gene. 1994 Jan 28;138(1-2):143–148. doi: 10.1016/0378-1119(94)90797-8. [DOI] [PubMed] [Google Scholar]
  6. Hynes M. J., Corrick C. M., King J. A. Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations. Mol Cell Biol. 1983 Aug;3(8):1430–1439. doi: 10.1128/mcb.3.8.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jarai G., Buxton F. Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger. Curr Genet. 1994 Sep;26(3):238–244. doi: 10.1007/BF00309554. [DOI] [PubMed] [Google Scholar]
  8. Karhunen T., Mäntylä A., Nevalainen K. M., Suominen P. L. High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol Gen Genet. 1993 Dec;241(5-6):515–522. doi: 10.1007/BF00279893. [DOI] [PubMed] [Google Scholar]
  9. Leplatois P., Le Douarin B., Loison G. High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae. Gene. 1992 Dec 1;122(1):139–145. doi: 10.1016/0378-1119(92)90041-m. [DOI] [PubMed] [Google Scholar]
  10. Mischak H., Hofer F., Messner R., Weissinger E., Hayn M., Tomme P., Esterbauer H., Küchler E., Claeyssens M., Kubicek C. P. Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei. Biochim Biophys Acta. 1989 Jan 27;990(1):1–7. doi: 10.1016/s0304-4165(89)80003-0. [DOI] [PubMed] [Google Scholar]
  11. Nehlin J. O., Ronne H. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. EMBO J. 1990 Sep;9(9):2891–2898. doi: 10.1002/j.1460-2075.1990.tb07479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nyyssönen E., Penttilä M., Harkki A., Saloheimo A., Knowles J. K., Keränen S. Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei. Biotechnology (N Y) 1993 May;11(5):591–595. doi: 10.1038/nbt0593-591. [DOI] [PubMed] [Google Scholar]
  13. Penttilä M. E., André L., Lehtovaara P., Bailey M., Teeri T. T., Knowles J. K. Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene. 1988;63(1):103–112. doi: 10.1016/0378-1119(88)90549-5. [DOI] [PubMed] [Google Scholar]
  14. Penttilä M., Nevalainen H., Rättö M., Salminen E., Knowles J. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene. 1987;61(2):155–164. doi: 10.1016/0378-1119(87)90110-7. [DOI] [PubMed] [Google Scholar]
  15. Schultz L. D., Hofmann K. J., Mylin L. M., Montgomery D. L., Ellis R. W., Hopper J. E. Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast. Gene. 1987;61(2):123–133. doi: 10.1016/0378-1119(87)90107-7. [DOI] [PubMed] [Google Scholar]
  16. Teeri T. T., Kumar V., Lehtovaara P., Knowles J. Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi. Anal Biochem. 1987 Jul;164(1):60–67. doi: 10.1016/0003-2697(87)90367-8. [DOI] [PubMed] [Google Scholar]
  17. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Usui T., Matsui H., Isobe K. Enzymic synthesis of useful chito-oligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium sulfate. Carbohydr Res. 1990 Aug 1;203(1):65–77. doi: 10.1016/0008-6215(90)80046-6. [DOI] [PubMed] [Google Scholar]
  19. Verdoes J. C., van Diepeningen A. D., Punt P. J., Debets A. J., Stouthamer A. H., van den Hondel C. A. Evaluation of molecular and genetic approaches to generate glucoamylase overproducing strains of Aspergillus niger. J Biotechnol. 1994 Aug 15;36(2):165–175. doi: 10.1016/0168-1656(94)90052-3. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES