title: Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.
abstract: Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.
authors.affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Haon
authors.affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.
authors.forename: Sacha
authors.initials: S
authors.lastname: Grisel
authors.affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.
authors.forename: David
authors.initials: D
authors.lastname: Navarro
authors.affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York NY, USA.
authors.forename: Antoine
authors.initials: A
authors.lastname: Gruet
authors.affiliation: INRA, UMR 1163 Biodiversité et Biotechnologie Fongiques Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques Marseille, France.
authors.forename: Jean-Guy
authors.initials: JG
authors.lastname: Berrin
authors.affiliation: Architecture et Fonction des Macromolècules Biologiques, CNRS-Aix-Marseille University UMR 7257 Marseille, France ; INRA, USC 1408 AFMB Marseille, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
keywords: CAZymes; Pichia pastoris; automation; expression platform; factorial approach; glycoside hydrolase; recombinant proteins