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

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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.

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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.

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Authors and Affiliations

  1. Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium

    Magali Tanghe, Barbara Danneels & Tom Desmet

  2. Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Centros, 138668, Singapore

    Andrea Camattari

  3. Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria

    Andrea Camattari & Anton Glieder

  4. Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium

    Isabel Vandenberghe & Bart Devreese

  5. BioTec Laboratory, Department of Applied Biosciences, Ghent University, V. Vaerwijckweg 1, 9000, Ghent, Belgium

    Ingeborg Stals

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  1. Magali Tanghe
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Correspondence to Magali Tanghe.

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Tanghe, M., Danneels, B., Camattari, A. et al. Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing. Mol Biotechnol 57, 1010–1017 (2015). https://doi.org/10.1007/s12033-015-9887-9

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  • DOI: https://doi.org/10.1007/s12033-015-9887-9

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