SpringerLink
Log in

Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5′-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Meyers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Aro N, Saloheimo A, Ilmén M, Penttilä M (2001) ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. J Biol Chem 276:24309–24314

    Article  CAS  Google Scholar 

  • Aro N, Ilmén M, Saloheimo A, Penttilä M (2003) ACEI is a repressor of cellulase and xylanase genes in Trichoderma reesei. Appl Environ Microbiol 69:56–65

    Article  CAS  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Chavez R, Almarza C, Schachter K, Peirano A, Bull P, Eyzaguirre J (2001) Structure analysis of the endoxylanase A gene from penicillum purpurogenum. Biol Res 34:217–226

    Article  CAS  Google Scholar 

  • Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6:23–28

    Article  CAS  Google Scholar 

  • Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJM, Yao J, Ward M (2003) Transcription regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278:31988–31997

    Article  Google Scholar 

  • Henrique-Silva F, El-Gogary S, Carle-Urioste JC, Matheucci Jr. E, Crivellaro O, El-Dorry H (1996) Two regulatory regions controlling basal and cellulose-induced expression of the gene encoding cellobiohydrolase I of Trichoderma reesei are adjacent to its TATA box. Biochem Biophys Res Commun 302:849–854

    Google Scholar 

  • Hynes MJ, Corrick CM, King JA (1983) 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 3:1430–14391

    Article  CAS  Google Scholar 

  • Ilmén M, Thrane C, Penttilä 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

    PubMed  Google Scholar 

  • Ilmén M, Saloheimo A, Onnela ML, Penttilä M (1997) Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63:1298–1306

    Article  Google Scholar 

  • Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherchia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451

    Article  CAS  Google Scholar 

  • Kawamori M, Morikawa Y, Takasawa S (1986) Induction and production of cellulases by l-sorbose in Trichoderma reesei. Appl Microbiol Biotechnol 24:449–453

    CAS  Google Scholar 

  • Kimura T, Suzuki H, Furuhashi H, Aburatani T, Morimoto K, Sakka K, Ohmiya K (2002) Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae. Biosci Biotechnol Biochem 66:285–292

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998) Molecular characterization and heterologus expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64:555–563

    Article  CAS  Google Scholar 

  • Okada H, Wakamatsu M, Takano Y, Nogawa M, Morikawa Y (1999) Expression of two Trichoderma reesei xylanases in the fission yeast Schizosaccharomyces pombe. J Biosci Bioeng 88:563–566

    Article  CAS  Google Scholar 

  • Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderama reesei. Gene 61:155–164

    Article  Google Scholar 

  • Ruiz-Roldan MC, Di Pietro A, Huertas-Gonzalez MD, Roncero MI (1999) Two xylanase genes of the vascular with pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants. Mol Gen Genet 261:530–536

    Article  CAS  Google Scholar 

  • Saarelainen R, Paloheimo M, Fagerström R, Suominen PL, Nevalainen HKM (1993) Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2. Mol Gen Genet 241:497–503

    Article  CAS  Google Scholar 

  • Saloheimo A, Aro N, Ilmén M, Penttilä M (2000) Isolation of the ace1 gene encoding a Cys2-His2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei. J Biol Chem 275:5817–5825

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning, third edn. Cold Spring Harbor Laboratory, Cold Spring Harbor New York

    Google Scholar 

  • Srinivasa BR, Swaminathan KR, Ganapathy C, Roy RP, Murthy SK, Vithayathil PJ (1991) The primary structure of xylanase from Thermoascus aurantiacus. Protein Seq Data Anal 4:15–20

    PubMed  CAS  Google Scholar 

  • Strauss J, Mach RL, Zeilinger S, Hartler G, Stöffler G, Wolschek M, Kubicek CP (1995) Cre1, the carbon catabolite repressor protein from Trichoderma reesei. FEBS Lett 376:103–107

    Article  CAS  Google Scholar 

  • Tenkanen M, Puls J, Poutanen (1992) Two major xylanases of Trichoderma reesei. Enzyme Microb Technol 14:566–574

    Article  CAS  Google Scholar 

  • Thomson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  Google Scholar 

  • Törrönen A, Rouvinen J (1995) Structual comparison of two major endo-1,4-β-xylanases from Trichoderma reesei. Biochemistry 34:847–856

    Article  Google Scholar 

  • Törrönen A, Mach RL, Messner R, Gonzalez R, Kalkkinen N, Harkki A, Kubicek CP (1992) The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Bio/Technology 10:1461–1467

    PubMed  Google Scholar 

  • Törrönen A, Harkki A, Rouvinen J (1994) Three-dimensional structure of endo-1,4-β-xylanase II from Trichoderma reesei: two conformational states in the active site. EMBO J 13:2493–2501

    Article  Google Scholar 

  • Wurleitner E, Pera L, Wacenovsky C, Cziferszky A, Zeilinger S, Kubicek CP, Mach RL (2003) Transcriptional regulation of xyn2 in Hypocrea jecorina. Eukaryot Cell 2:150–158

    Article  CAS  Google Scholar 

  • Xu J, Takakuwa N, Nogawa M, Okada H, Morikawa Y (1998) A third xylanase from Trichoderama reesei PC-3-7. Appl Microbiol Biotechnol 49:718–724

    Article  CAS  Google Scholar 

  • Xu J, Nogawa M, Okada H, Morikawa Y (2000) Regulation of xyn3 gene expression in Trichoderama reesei PC-3-7. Appl Microbiol Biotechnol 54:370–375

    Article  CAS  Google Scholar 

  • Zeilinger S, Mach RL, Schindler M, Herzog P, Kubicek CP (1996) Different inducibility of expression of the the two xylanase genes xyn1 and xyn2 in Trichoderma reesei. J Biol Chem 271:25624–25629

    Article  CAS  Google Scholar 

  • Zeilinger S, Ebner A, Mach RL, Marosits T, Kubicek CP (2001) The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element. Mol Genet Genomics 266:56–63

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. M. Nogawa

    Present address: Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, 386-5867, Japan

Authors and Affiliations

  1. Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata, 940-2188, Japan

    W. Ogasawara, Y. Shida, T. Furukawa, R. Shimada, S. Nakagawa, M. Kawamura, T. Yagyu, A. Kosuge, J. Xu, M. Nogawa, H. Okada & Y. Morikawa

Authors
  1. W. Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y. Shida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. Furukawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. Shimada
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Nakagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. Kawamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. T. Yagyu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. Kosuge
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J. Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. Nogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. H. Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Y. Morikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y. Morikawa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ogasawara, W., Shida, Y., Furukawa, T. et al. Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei . Appl Microbiol Biotechnol 72, 995–1003 (2006). https://doi.org/10.1007/s00253-006-0365-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0365-y

Keywords

Search

Navigation