title: Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.
abstract: The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coproduce two different cellulases. For that purpose the eglII (endoglucanase II) and cbhII (cellobiohydrolase II) genes from Trichoderma reesei were fused in-frame separated by the self-processing 2A peptide sequence from the foot-and-mouth disease virus. The protein fusion construct was placed under the control of the strong inducible AOX1 promoter. Analysis of culture supernatants from methanol-induced yeast transformants showed that the protein fusion was effectively processed. Enzymatic assay showed that the processed enzymes were fully functional with the same catalytic properties of the individual enzymes produced separately. Furthermore, when combined both enzymes acted synergistically on filter paper to produce cellobiose as the main end-product. Based on these results we propose that P. pastoris should be considered as an alternative platform for the production of cellulases at competitive costs.
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. juliaamorim@yahoo.com.br.
authors.forename: Juliana
authors.initials: J
authors.lastname: de Amorim Araújo
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. tulcesar@gmail.com.
authors.forename: Túlio César
authors.initials: TC
authors.lastname: Ferreira
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. mrrubini@gmail.com.
authors.forename: Marciano Régis
authors.initials: MR
authors.lastname: Rubini
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. ana.gomez@ciens.ucv.ve.
authors.forename: Ana Gilhema Gomez
authors.initials: AG
authors.lastname: Duran
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. janicedemarco@unb.br.
authors.forename: Janice Lisboa
authors.initials: JL
authors.lastname: De Marco
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. lmoraes@unb.br.
authors.forename: Lidia Maria Pepe
authors.initials: LM
authors.lastname: de Moraes
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil. ftorres@unb.br.
authors.forename: Fernando Araripe Gonçalves
authors.initials: FA
authors.lastname: Torres
keywords: 2A peptide; Cellobiohydrolase; Endoglucanase; Pichia pastoris; Protein fusion