Skip to main content
SpringerLink
Log in

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

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

Abstract

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l−1 for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l−1.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Balance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236

    Article  PubMed  Google Scholar 

  • Breathnach R, Benoist C, O'Hara K, Chambon P (1978) Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon–intron boundaries. Proc Natl Acad Sci U S A 75:4853–4857

    Article  PubMed  CAS  Google Scholar 

  • Della Cioppa GR, Garger SJ, Holtz RB, McCulloch MJ, Sverlow GG (1996) Method for making stable, extracellular tyrosinase and synthesis of polyphenolic polymers therefrom. US Patent 5,486,351

  • Duran N, Esposito E (2000) Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment: a review. Appl Catal, B Environ 28:83–99

    Article  CAS  Google Scholar 

  • Ebbelar CEM, Wichers HJ, van den Bosch T, Oyevaar JI, Recourt K (1995) URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Espin JC, Van Leeuwen J, Wichers HJ (1999) Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinases by serine proteases. J Agric Food Chem 47:3509–3517

    Article  PubMed  CAS  Google Scholar 

  • Fujita Y, Uraga Y, Ichisima E (1995) Molecular cloning and nucleotide sequence of the prototyrosinase gene, melO, from Aspergillus oryzae and expression of the gene in yeast cells. Biochim Biophys Acta 1261:151–154

    PubMed  Google Scholar 

  • Garcia-Borron JC, Solano F (2002) Molecular anatomy of tyrosinase and its related proteins: beyond the histine-bound metal catalytic center. Pigment Cell Res 15:162–173

    Article  PubMed  CAS  Google Scholar 

  • Gerdemann C, Christoph E, Krebs B (2002) The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc Chem Res 35:183–191

    Article  PubMed  CAS  Google Scholar 

  • Gordon CL, Khalaj V, Ram AFJ, Archer DB, Brookman JL, Trinci APJ, Jeenes DJ, Doonan JH, Wells B, Punt PJ, van den Hondel CAMJJ, Robson GD (2000) Glucoamylase: fluorescent protein fusions to monitor protein secretion in Aspergillus niger. Microbiology 146:415–426

    PubMed  CAS  Google Scholar 

  • Gouka RJ, Punt PJ, van den Hondel CAMJJ (1997) Efficient production of secreted proteins by Aspergillus niger: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11

    Article  PubMed  CAS  Google Scholar 

  • Halaouli S, Asther M, Kruus K, Guo L, Hamdi M, Sigoillot J-C, Asther M, Lomascolo A (2005) Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications. J Appl Microbiol 98:332–343

    Article  PubMed  CAS  Google Scholar 

  • Haq I, Ali S, Qadeer MA (2002) Biosynthesis of L-DOPA by Aspergillus oryzae. Bioresour Technol 85:25–29

    Article  PubMed  Google Scholar 

  • Herpoël I, Moukha S, Lesage-Meessen L, Sigoillot J-C, Asther M (2000) Selection of Pycnoporus cinnabarinus strains for laccase production. FEMS Microbiol Lett 183:301–306

    Article  PubMed  Google Scholar 

  • Jolivet S, Arpin N, Wichers HJ, Pellon G (1998) Agaricus bisporus browning: a review. Mycol Res 102:1459–1483

    Article  CAS  Google Scholar 

  • Kanda K, Sato T, Ishii S, Enei H, Ejiri SI (1996) Purification and properties of tyrosinase isozymes from the gill of Lentinus edodes fruiting body. Biosci Biotechnol Biochem 60:1273–1278

    Article  PubMed  CAS  Google Scholar 

  • Kanda S, Aimi T, Masumoto S, Nakano K, Kitamoto Y, Morigana T (2004) NCBI Database. URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Kupper U, Niedermann DM, Travaglini G, Lerch K (1989) Isolation and characterization of the tyrosinase gene from Neurospora crassa. J Biol Chem 264:17250–17258

    PubMed  CAS  Google Scholar 

  • Kupper U, Linden ML, Cao K, Lerch K (1990) Expression of tyrosinase in vegetative cultures of Neurospora crassa transformed with a metallothionein promoter/protyrosinase fusion gene. Curr Genet 18:331–335

    Article  PubMed  CAS  Google Scholar 

  • Kurahashi Y, Pontzen R (1998) Carpropamid: a new melanin biosynthesis inhibitor. Pflanzenschutz-Nachr Bayer 51:245–256

    Google Scholar 

  • Langfelder K, Glaser P, Brakhage AA (2003) NCBI Database. URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Lantto R, Niku-Paavola ML, Schonberg C, Buchert J (2002) A tyrosinase enzyme. International Patent WO 02/14484A1

  • Lomascolo A, Cayol J-L, Roche M, Guo L, Robert J-L, Record E, Lesage-Meessen L, Ollivier B, Sigoillot J-C, Asther M (2002) Molecular clustering of Pycnoporus strains from various geographic origins and isolation of monokaryotic strains for laccase hyperproduction. Mycol Res 106:1193–1203

    Article  CAS  Google Scholar 

  • Lomascolo A, Record E, Herpoël-Gimbert I, Delattre M, Robert J-L, Georis J, Dauvrin T, Sigoillot J-C, Asther M (2003) Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. J Appl Microbiol 94:618–624

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  • Marbach K, Stahl U (1996) NCBI Database. URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Nakamura M, Nakajima T, Ohba Y, Yamauchi S, Lee BR, Ichishima E (2000) Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis. Biochem J 350:537–545

    Article  PubMed  CAS  Google Scholar 

  • Otterbein L, Record E, Longhi S, Asther M, Moukha S (2000) Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur J Biochem 267:1619–1625

    Article  PubMed  CAS  Google Scholar 

  • Punt PJ, van den Hondel CA (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol 216:447–457

    Article  PubMed  CAS  Google Scholar 

  • Record E, Moukha S, Asther M, Asther M (2001) Cloning and expression in phospholipid containing cultures of the gene encoding the specific phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence that the pg/pi-tp is tandemly arranged with the putative 3-ketoacyl-CoA thiolase gene. Gene 10:61–72

    Article  Google Scholar 

  • Record E, Punt PJ, Chamkha M, Labat M, van den Hondel CAMJJ, Asther M (2002) Expression of the Pycnporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem 269:602–609

    Article  PubMed  CAS  Google Scholar 

  • Reese MG (2001) Application of a time-dealy neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26:51–56

    Article  PubMed  CAS  Google Scholar 

  • Riley PA (1997) Molecules in focus: melanin. Int J Biochem Cell Biol 29:1235–1239

    Article  PubMed  CAS  Google Scholar 

  • Robb DA, Gutteridge S (1981) Polypeptide composition of two fungal tyrosinases. Phytochemistry 20:1481–1485

    Article  CAS  Google Scholar 

  • Sato K (2002) URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Schulte U, Align V, Hoheisel J, Brandt P, Fartmann B, Holland R, Nyakatura G, Mewes HW, Mannhaupt G (2003) NCBI Database. URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi

  • Seo SY, Sharma VK, Sharma N (2003) Mushroom tyrosinase: recent prospects. J Agric Food Chem 51:2837–2853

    Article  PubMed  CAS  Google Scholar 

  • Soler-Rivas C, Jolivet S, Arpin N, Olivier JM, Wichers HJ (1999) Biochemical and physiological aspects of brown blotch disease of Agaricus bisporus. FEMS Microbiol Rev 23:591–614

    Article  PubMed  CAS  Google Scholar 

  • Thalmann CR, Lötzbeyer T (2002) Enzymatic cross-linking of proteins with tyrosinase. Eur Food Res Technol 214:276–281

    Article  CAS  Google Scholar 

  • Van Gelder CWG, Flurkey WH, Wichers HJ (1997) Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 45:1309–1323

    Article  PubMed  Google Scholar 

  • van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CA (1987) Development of homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75

    Article  PubMed  Google Scholar 

  • Wessels JGH, Mulder GH, Springer J (1987) Expression of dikaryon specific and non-specific mRNAs of Schizophyllum commune in relation to environmental conditions and fruiting. J Gen Microbiol 133:2557–2561

    CAS  Google Scholar 

  • Wichers HJ, Gerritsen YA, Chapelon CGJ (1996) Tyrosinase isoforms from the fruitbodies of Agaricus bisporus. Phytochemistry 43:333–337

    Article  CAS  Google Scholar 

  • Wichers HJ, Recourt K, Hendriks M, Ebbelaar CE, Biancone G, Hoeberichts FA, Mooibroek H, Soler-Rivas C (2003) Cloning, expression, and characterisation of two tyrosinase cDNAs from Agaricus bisporus. Appl Microbiol Biotechnol 61:336–341

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We warmly thank K. Kruus (VTT, Finland) for the internal tyrosinase peptides determination, P. Mansuelle (UMR 6560, IFR Jean Roche, Marseille, France) for the N-terminal amino sequence analysis, D. Navarro for technical assistance, S. Pagès (CNRS, Marseille, France) and J.-L. Cayol (IRD, Marseille, France) for helpful comments on molecular biology engineering. This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and management of Living Resources”, proposal number QLK1-2002-02208, “Novel cross-linking enzymes and their consumer acceptance for structure engineering of foods”, acronym CROSSENZ. It does not reflect its views and in no way anticipates the Commission's future policy in this area.

Author information

Authors and Affiliations

  1. UMR 1163 INRA-Université de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 de Biotechnologie Agro-Industrielle de Marseille, 163 Avenue de Luminy, Case 925, 13288, Marseille Cedex 09, France

    Sonia Halaouli, Eric Record, Jean-Claude Sigoillot, Marcel Asther & Anne Lomascolo

  2. UR 180 de Microbiologie et Biotechnologie des Environnements Chauds, IFR 86 de Biotechnologie Agro-Industrielle de Marseille, 163 Avenue de Luminy, Case 925, 13288, Marseille Cedex 09, France

    Laurence Casalot

  3. Laboratoire de Microbiologie Alimentaire et des Procédés de l'Environnement, Institut National des Sciences Appliquées et de Technologie, BP 676, 1076, Tunis, Tunisia

    Moktar Hamdi

Authors
  1. Sonia Halaouli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Record
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laurence Casalot
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Moktar Hamdi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jean-Claude Sigoillot
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marcel Asther
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anne Lomascolo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anne Lomascolo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Halaouli, S., Record, E., Casalot, L. et al. Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger . Appl Microbiol Biotechnol 70, 580–589 (2006). https://doi.org/10.1007/s00253-005-0109-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-005-0109-4

Keywords

Search

Navigation