Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution

Abstract

Background β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. Results We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. Conclusions D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris

“…Considering that the expression of UPR-related genes containing calnexin ( CNE1 ), Pdi oxidase ( ERO1 ), UPR activated transcription factor ( HAC1 ), ER chaperone ( KAR2 ) and protein disulfide isomerase ( PDI1 ) could reflect the ER stress [ 8 , 35 – 38 ]. qRT-PCR analysis was used to determine the relative expression levels of these genes between WT and mutants (Fig.…”

“…[ 7 ]. In addition, the fusion of new modules or the introduction of foreign gene mutations can also improve the expression level of target proteins [ 8 , 9 ].…”

Characterization of a GH5 endoxylanase from Penicillium funiculosum and its synergism with GH16 endo-1,3(4)-glucanase in saccharification of sugarcane bagasse

“…In addition, its capability for appropriate folding and transportation of proteins renders it an excellent tool for recombinant protein production 20 . P. pastoris also secretes fewer native proteins thereby enabling easier purification and biochemical characterization of recombinant proteins in supernatant 21 , 22 . To express this protein, the alpha secretion signal peptide of the p PICZαA vector was used to direct the secretion of the protein under the AOX1 promoter.…”

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases

“…Chen and coworkers identified two mutant α-galactosidases from Penicillium janczewskii zalesk with a higher galactosidase activity, potentially useful in feed manufacturing [ 47 ]. Kao and coworkers used a 96-deep-well plate screening to identify a β-glucosidase with an enhanced specific activity [ 48 ]. However, since this system afforded just limited results for the site-saturation mutagenesis of Tf Hex (only four prospective amino acid exchanges were identified), we resorted to the site-directed mutagenesis approach, as detailed further.…”