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.

Keywords: 2A peptide; Cellobiohydrolase; Endoglucanase; Pichia pastoris; Protein fusion.

Fig. 1

Alignment and predicted translation of the native and optimized nucleotide sequences of the 1D-2A region of FMDV. Base positions with an asterisk correspond to modifications that were made for codon optimization

Fig. 2

Schematic diagram showing the strategy for construction of the expression vector pPIC9-cbhII2AeglII. Vector construction is described in “Materials and methods”. SP native signal peptide, 2A self-processing linker sequence, 5′AOX 5′ promoter region of AOX1, 3′AOX1 3′ sequences of AOX1, TT transcription termination sequence of AOX1

Fig. 3

Plate assay for detection of cellulase activity in transformed P. pastoris cells. CMC agar plates were stained with Congo Red and enzyme activity is detected by the presence of a halo around the colony. a P. pastoris colonies grown for 72 h under methanol induction. b Supernatants (2 μL) from induced cultures collected at different times spotted directly onto CMC plate. (1) Recombinant yeast transformed with pPIC9 (negative control); (2) clone FUSAOX

Fig. 4

SDS-PAGE analysis of cell-free supernatants of recombinant yeasts induced with 0.5 % methanol for 72 h. (1) pPIC9 (negative control); (2) clone EGLAOX; (3) clone CBHAOX; (4) clone FUSAOX; M: Unstained protein molecular weight marker (Thermo Scientific). The position of the bands corresponding to CBHII and EGII is indicated by arrows

Fig. 5

Purification of individual enzymes after protein fusion cleavage. The supernatant from clone FUSAOX was applied on a Q-Sepharose and proteins were eluted on a NaCl gradient. Sample fractions were analyzed on 12 % SDS-PAGE. CBHII was eluted on fractions 10–13 and EGII on fractions 15 and 16. The rectangles indicate individual enzymes. Fractions 12 and 16 were tested for cellulase activity. Only relevant bands of the unstained protein molecular weight marker (Thermo Scientific) are indicated by arrows at the right side of the panel

Fig. 6

Deglycosylation test. Culture supernatants from the negative control (pPIC9) and clones EGLAOX, CBHAOX and FUSAOX were treated (+) or not (−) with PNGase F and analyzed on 12 % SDS-PAGE. M: Unstained protein molecular weight marker (Thermo Scientific)

Fig. 7

HPLC profiles of end products released by enzymatic hydrolysis of paper filter after incubation for 16 h at 50 °C. The supernatants from clones EGLAOX (a), CBHAOX (b), FUSAOX (c) or a combination of EGLAOX + CBHAOX (d) were incubated with filter paper and incubated for 16 h at 50 °C. Hydrolysis products were analyzed by HPLC. The concentration of cellobiose after the reaction is shown in each graph. G1 glucose, G2 cellobiose, G3 cellotriose