Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

Juliana De Amorim Araújo,Janice Lisboa De Marco,Fernando Araripe Gonçalves Torres,Marciano Régis Rubini,Ana Gilhema Gomez Duran,Lidia Maria Pepe De Moraes,Túlio César Ferreira

doi:10.1186/s13568-015-0170-z

Juliana De Amorim Araújo, Janice Lisboa De Marco + Show 5 more

Open Access

https://doi.org/10.1186/s13568-015-0170-z

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Journal: AMB Express	Publication Date: Dec 1, 2015
Citations: 32	License type: CC BY 4.0

Affiliation: Universidade de Brasília

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.

Highlights

Cellulases represent a complex group of enzymes that act synergistically on cellulosic material to release sugars (Lynd et al 2002)
The promoter of alcohol oxidase 1 gene (AOX1) is the most commonly used in commercial expression vectors for directing expression of heterologous genes in P. pastoris since it is efficient and highly regulated by methanol (Yu et al 2013; Lünsdorf et al 2011; Sigoillot et al 2012)
A gene fusion consisting of cbhII and eglII genes from T. reesei separated by the 2A sequence was cloned into the P. pastoris expression vector pPIC9 (Fig. 2)

Summary

Introduction

Cellulases represent a complex group of enzymes that act synergistically on cellulosic material to release sugars (Lynd et al 2002). The keys of its success have been widely reported in the literature and the advantages of using this yeast as expression platform include: GRAS (Generally Recognized as Safe) status, easy molecular genetic manipulation, high level production of secreted proteins, ability to promote posttranslational modifications of higher eukaryotic and a preference for a respiratory rather than a fermentative metabolism to grow (Macauley-Patrick et al 2005; Cregg et al 2000, 2002; Hohenblum et al 2004; Ahmad et al 2014) These characteristics allow the production de Amorim Araújo et al AMB Expr (2015) 5:84 of large amounts of heterologous protein with relative technical facility and at costs lower than those of most other eukaryotic systems such as mammalian cell culture (Gellissen 2000, Higgins and Cregg 1998). The promoter of alcohol oxidase 1 gene (AOX1) is the most commonly used in commercial expression vectors for directing expression of heterologous genes in P. pastoris since it is efficient and highly regulated by methanol (Yu et al 2013; Lünsdorf et al 2011; Sigoillot et al 2012)

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