Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.
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.
Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium. Magali.Tanghe@ugent.be.
Magali
M
Tanghe
Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
Barbara
B
Danneels
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.
Andrea
A
Camattari
Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria.
Anton
A
Glieder
Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium.
Isabel
I
Vandenberghe
Laboratory of Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium.
Bart
B
Devreese
BioTec Laboratory, Department of Applied Biosciences, Ghent University, V. Vaerwijckweg 1, 9000, Ghent, Belgium.
Ingeborg
I
Stals
Laboratory of Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
Tom
T
Desmet
D000595:Amino Acid Sequence; D018533:Biomass; D002482:Cellulose / Q000737:chemistry / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D006868:Hydrolysis; D000072235:Mating Factor; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D010455:Peptides; D011134:Polysaccharides; D000072235:Mating Factor; D002482:Cellulose; D006899:Mixed Function Oxygenases
Auxiliary activity family 9; Cellulose hydrolysis; Lytic polysaccharide monooxygenase; N-terminal processing; Pichia pastoris; Trichoderma reesei Cel61A (TrCel61A)