Skip to main content
SpringerLink
Log in

Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1–1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].

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
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

ABTS:

2,2′-Azino-bis-[3-ethylthiazoline-6-sulphonate]

AX:

Arabinoxylans

2,6-DMP:

2,6-Dimethoxyphenol

GRAS:

Generally recognized as safe

riceMaL:

Recombinant Melanocarpus albomyces laccase produced in rice

rMaL:

Recombinant M. albomyces laccase produced in Trichoderma reesei

MaL:

Native M. albomyces laccase

ricePycL:

Recombinant Pycnoporus cinnabarinus laccase produced in rice

PycL:

Native Pycnoporus cinnabarinus laccase

TSP:

Total soluble proteins

References

  • Bailey MR, Wooddard SL, Callaway E, Beifuss K, Magallanes-Lundback M, Lane JR, Horn ME, Mallubhotla H, Delaney DD, Ward M, Van Gastel F, Howard JA, Hood EE (2004) Improved recovery of active recombinant laccase from maize seed. Appl Microbiol Biotechnol 63:390–397

    Article  PubMed  CAS  Google Scholar 

  • Chanliaud E, Saulnier L, Thibault J-F (1995) Alkaline extraction and characterisation of heteroxylans from maize bran. J Cereal Chem 21:195–203

    Article  CAS  Google Scholar 

  • Claparols MI, Bassie L, Miro B, Del Duca S, Rodriguez-Montesinos J, Christou P, Serafini-Fracassini D, Capell T (2004) Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase. Transgenic Res 13:195–199

    Article  PubMed  CAS  Google Scholar 

  • Conesa A, Punt PJ, van Luijk N, van den Hondel CAMJJ (2001) The secretion pathway in filamentous fungi: a biotechnological view. Fungal Genet Biol 33:155–217

    Article  PubMed  CAS  Google Scholar 

  • de Pater BS, van der Mark F, Rueb S, Katagiri F, Chua NH, Schilperoort RA, Hensgens LA (1992) The promoter of the rice gene GOS2 is active in various different monocot tissues and binds rice nuclear factor ASF-1. Plant J 2:837–844

    Article  PubMed  Google Scholar 

  • Figueroa-Espinoza MC, Rouau X (1998) Oxidative cross-linking of pentosans by a fungal laccase and horseradish peroxidase: mechanism of linkage between feruloylated arabinoxylans. Cereal Chem 75:259–265

    Article  CAS  Google Scholar 

  • Goddijn OJM, Pen J (1995) Plants as bioreactors. Trends Biotechnol 13:379–387

    Article  CAS  Google Scholar 

  • Gonzalez T, Terron MC, Yague S, Zapico E, Galletti GC, Gonzalez AE (2000) Pyrolysis/gas chromatography/mass spectrometry monitoring of fungal-biotreated distillery wastewater using Trametes sp. I62 (CECT 20197). Rapid Commun Mass Spectrom 14:1417–1424

    Article  PubMed  CAS  Google Scholar 

  • Greco G Jr, Toscanoa G, Cioffi M, Gianfreda L, Sannino F (1999) Dephenolisation of olive mill waste-waters by olive husk. Water Res 33:3046–3050

    Article  CAS  Google Scholar 

  • Hakulinen N, Kiiskinen L-L, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J (2002) Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nat Struct Mol Biol 9:601–605

    CAS  Google Scholar 

  • Herpoël I, Jeller H, Fang G, Petit-Conil M, Bourbonnais R, Robert J-L, Asther M, Sigoillot J-C (2002) Efficient enzymatic delignification of wheat straw pulp by a sequential xylanase-laccase treatment. J Pulp Paper Sci 28:67–71

    Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    Article  PubMed  CAS  Google Scholar 

  • Hood EE, Bailey MR, Beifuss K, Magallanes-Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA (2003) Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotechnol J 1:129–140

    Article  PubMed  CAS  Google Scholar 

  • James J, Simpson BK (1996) Application of enzymes in food processing. Crit Rev Food Sci Nutr 36:437–463

    Article  PubMed  CAS  Google Scholar 

  • Kiiskinen LL, Viikari L, Kruus K (2002) Purification and characterization of a novel laccase from the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 59:198–204

    Article  PubMed  CAS  Google Scholar 

  • Kiiskinen L-L, Saloheimo M (2004) Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the Ascomycete Melanocarpus albomyces. Appl Environ Microbiol 70:137–144

    Article  PubMed  CAS  Google Scholar 

  • Kiiskinen LL, Kruus K, Bailey M, Ylosmaki E, Siika-aho M, Saloheimo M (2004) Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology 150:3065–3074

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Lomascolo A, Cayol JL, Roche M, Guo L, Robert JL, Record E, Lesage-Meessen L, Ollivier B, Sigoillot JC, 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 JL, Georis J, Dauvrin T, Sigoillot JC, 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 

  • Micard V Thibault JF (1999) Oxidative gelation of sugar-beet pectins: use of laccases and hydration properties of the cross-linked pectins. Carbohydr Polym 39:265–273

    Article  Google Scholar 

  • Minussi RC, Pastore GM, Duran N (2002) Potential applications of laccase in the food industry. Trends Food Sci Technol 13:205–216

    Article  CAS  Google Scholar 

  • Niku-Paavola ML, Karhunen E, Salola P, Raunio V (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254:877–883

    PubMed  CAS  Google Scholar 

  • Ooms G, Hooykaas PJ, Van Veen RJ, Van Beelen P, Regensburg-Tuink TJ, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15–29

    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 

  • Palmieri G, Giardina P, Bianco C, Fontanella B, Sannia G (2000) Copper induction of laccase isoenzymes in the lignolytic fungus Pleurotus ostreatus. Appl Environ Microbiol 66:920–924

    Article  PubMed  CAS  Google Scholar 

  • Patel M, Johnson JS, Brettell RIS, Jacobsen J, Xue GP (2000) Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol Breed 6:113–123

    Article  CAS  Google Scholar 

  • Piacquadio P, De Stefano G, Sammartino M, Sciancalepore V (1997) Phenols removal from apple juice by laccase immobilized on Cu2+-chelate regenerable carrier. Biotechnol Tech 11:515–517

    Article  CAS  Google Scholar 

  • Pen J, Molendijk L, Quax WJ, Sijmons PC, van Ooyen AJJ, ven den Elzen PJM, Rietveld K, Hoekema A (1992) Production of active Bacillus licheniformis alpha-amylase in tobacco and its applications in starch liquefaction. Biotechnology 10:292–296

    Article  PubMed  CAS  Google Scholar 

  • Pen J, Verwoerd TC, van Paridon PA, Beudeker RF, ven den Elzen PJM, Geerse K, van der Klis JD, Versteegh HAJ, van Ooyen AJJ, Hoekema A (1993) Phytase-containing transgenic seeds as a novel feed additive for improved phosphorus utilization. Biotechnology 11:811–814

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Rose AB (2002) Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA 8:1444–1453

    Article  PubMed  CAS  Google Scholar 

  • Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226

    Article  PubMed  CAS  Google Scholar 

  • Saulnier L, Thibault J-F (1999) Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran heteroxylans. J Sci Food Agric 79:396–402

    Article  CAS  Google Scholar 

  • Sigoillot C, Record E, Belle V, Robert J-L, Levasseur A, Punt PJ, Van den hondel CAMJJ, Fournel A, Sigoillot J-C, Asther M (2004) Natural and recombinant fungal laccases for paper pulp bleaching. Appl Microbiol Biotechnol 64:346–352

    Article  PubMed  CAS  Google Scholar 

  • Sonoki T, Kajita S, Ikeda S, Uesugi M, Tatsumi K, Katayama Y, Iimura Y (2005) Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants. Appl Microbiol Biotechnol 67:138–142

    Article  PubMed  CAS  Google Scholar 

  • Takaiwa F, Kikuchi S, Oono K (1989) The complete nucleotide-sequence of new type cDNA coding for rice storage protein glutelin. Nucleic Acids Res 17:3289–3289

    Article  PubMed  CAS  Google Scholar 

  • Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26

    CAS  Google Scholar 

  • Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578

    Article  PubMed  CAS  Google Scholar 

  • Vain P, Finer KR, Engler DE, Pratt RC, Finer JJ (1996) Intron-mediated enhancement of gene expression in maize (Zea mays L) and bluegrass (Poa pratensis L). Plant Cell Rep 15:489–494

    Article  CAS  Google Scholar 

  • Ziegler MT, Thomas SR, Danna KJ (2000) Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6:37–46

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are deeply grateful to David Navarro (UMR 1163 INRA, Marseille, France), Outi Liehunen and Birgit Hillebrandt (VTT, Finland) for technical assistance, Christophe Flaudrops (AFMB, CNRS, Marseille, France) for mass spectrometry analysis and Willem Broekaert and Yves Hatzfeld (CropDesign NV) for their valuable contribution in construct design. This work has been carried out with financial support from the Commission of the European Communities, specifically the 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. CropDesign NV, A BASF Plant Science Company, Technologiepark 3, 9052, Zwijnaarde-Gent, Belgium

    Chris de Wilde & Zhongyi Zhou

  2. UMR 1163 Biotechnologie des Champignons Filamenteux INRA, Universités de Provence et de la Méditerranée, ESIL, Case 925, 163 Avenue de Luminy, 13288, Marseille Cedex 09, France

    Eva Uzan, Eric Record, Marcel Asther & Anne Lomascolo

  3. VTT Biotechnology, P.O. Box 1500, 02044, VTT, Espoo, Finland

    Kristiina Kruus, Martina Andberg & Johanna Buchert

Authors
  1. Chris de Wilde
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eva Uzan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhongyi Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kristiina Kruus
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martina Andberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Johanna Buchert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eric Record
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marcel Asther
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. 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

de Wilde, C., Uzan, E., Zhou, Z. et al. Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases. Transgenic Res 17, 515–527 (2008). https://doi.org/10.1007/s11248-007-9124-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-007-9124-9

Keywords

Search

Navigation