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Molecular Biotechnology

, Volume 57, Issue 11–12, pp 1010–1017 | Cite as

Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing

  • Magali Tanghe
  • Barbara Danneels
  • Andrea Camattari
  • Anton Glieder
  • Isabel Vandenberghe
  • Bart Devreese
  • Ingeborg Stals
  • Tom Desmet
Article

Abstract

The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

Keywords

Lytic polysaccharide monooxygenase Pichia pastoris Trichoderma reesei Cel61A (TrCel61A) N-terminal processing Cellulose hydrolysis Auxiliary activity family 9 

Notes

Acknowledgments

The authors wish to thank the Agency for Innovation by Science and Technology (IWT) Flanders for financial support (Ph.D.-grant to M.T.). The study was carried out in the frame of the MRP Project “Ghent Bio-economy” granted by Ghent University.

Supplementary material

12033_2015_9887_MOESM1_ESM.docx (63 kb)
Supplementary material 1 (DOCX 62 kb)
12033_2015_9887_MOESM2_ESM.docx (14 kb)
Supplementary material 2 (DOCX 14 kb)
12033_2015_9887_MOESM3_ESM.docx (23 kb)
Supplementary material 3 (DOCX 23 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Magali Tanghe
    • 1
  • Barbara Danneels
    • 1
  • Andrea Camattari
    • 2
    • 3
  • Anton Glieder
    • 3
  • Isabel Vandenberghe
    • 4
  • Bart Devreese
    • 4
  • Ingeborg Stals
    • 5
  • Tom Desmet
    • 1
  1. 1.Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial TechnologyGhent UniversityGhentBelgium
  2. 2.Bioprocessing Technology InstituteA*STAR (Agency for Science, Technology and Research)CentrosSingapore
  3. 3.Institute of Molecular Biotechnology, NAWI GrazGraz University of TechnologyGrazAustria
  4. 4.Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and MicrobiologyGhent UniversityGhentBelgium
  5. 5.BioTec Laboratory, Department of Applied BiosciencesGhent UniversityGhentBelgium

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