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

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)1010-7
Number of pages8
JournalMolecular biotechnology
Volume57
Issue number11-12
DOIs
Publication statusPublished - Dec 2015

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Trichoderma
Pichia
Enzymes
Mating Factor
Polysaccharides
Processing
Mixed Function Oxygenases
Cellulose
Degradation
Proteins
Ports and harbors
Histidine
Yeast
Biomass
Fermentation
Crystalline materials
Substrates

Keywords

  • Amino Acid Sequence
  • Biomass
  • Cellulose
  • DNA, Fungal
  • Fermentation
  • Fungal Proteins
  • Hydrolysis
  • Mixed Function Oxygenases
  • Molecular Sequence Data
  • Peptides
  • Pichia
  • Polysaccharides
  • Saccharomyces cerevisiae
  • Sequence Analysis, DNA
  • Trichoderma
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris : Optimizing Yield and N-terminal Processing. / Tanghe, Magali; Danneels, Barbara; Camattari, Andrea; Glieder, Anton; Vandenberghe, Isabel; Devreese, Bart; Stals, Ingeborg; Desmet, Tom.

In: Molecular biotechnology, Vol. 57, No. 11-12, 12.2015, p. 1010-7.

Research output: Contribution to journalArticleResearchpeer-review

Tanghe, M, Danneels, B, Camattari, A, Glieder, A, Vandenberghe, I, Devreese, B, Stals, I & Desmet, T 2015, 'Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing' Molecular biotechnology, vol. 57, no. 11-12, pp. 1010-7. https://doi.org/10.1007/s12033-015-9887-9
Tanghe, Magali ; Danneels, Barbara ; Camattari, Andrea ; Glieder, Anton ; Vandenberghe, Isabel ; Devreese, Bart ; Stals, Ingeborg ; Desmet, Tom. / Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris : Optimizing Yield and N-terminal Processing. In: Molecular biotechnology. 2015 ; Vol. 57, No. 11-12. pp. 1010-7.
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