| Title: | Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing |
| Author(s): | Tanghe M; Danneels B; Camattari A; Glieder A; Vandenberghe I; Devreese B; Stals I; Desmet T; |
| Address: | "Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium. Magali.Tanghe@ugent.be. Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium. Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01, Centros, 138668, Singapore. Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria. Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium. BioTec Laboratory, Department of Applied Biosciences, Ghent University, V. Vaerwijckweg 1, 9000, Ghent, Belgium" |
| DOI: | 10.1007/s12033-015-9887-9 |
| ISSN/ISBN: | 1559-0305 (Electronic) 1073-6085 (Linking) |
| 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: | "Amino Acid Sequence Biomass Cellulose/chemistry/genetics DNA, Fungal/genetics Fermentation Fungal Proteins/*biosynthesis/genetics Hydrolysis Mating Factor Mixed Function Oxygenases/*biosynthesis/genetics Molecular Sequence Data Peptides/genetics/metabolis;" |
| Notes: | "MedlineTanghe, Magali Danneels, Barbara Camattari, Andrea Glieder, Anton Vandenberghe, Isabel Devreese, Bart Stals, Ingeborg Desmet, Tom eng Research Support, Non-U.S. Gov't Switzerland 2015/08/20 Mol Biotechnol. 2015 Dec; 57(11-12):1010-7. doi: 10.1007/s12033-015-9887-9" |
