Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues

Abstract

Background: Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass.

Results: In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2-BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification.

Conclusions: These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.

Keywords: BGL1; CBH2; EG2; Genetic engineering; Optimization; Saccharification; Trichderma reesei.

Fig. 1

Construction of the T. reesei cbh2 overexpression strains. a Cassettes used for cbh2 overexpression in the uracil auxotrophic strain QP4. b The cellulose agar plate used to screen for the cbh2-overexpression transformants. c PCR confirmation of the cbh2-overexpression transformant QPC67, which shows a 600-bp DNA fragment product using the primers Y-cbh2-F1 and Y-PyrG-R1

Fig. 2

RT-qPCR and SDS-PAGE analysis for the CBH2 overexpression strain QPC67 and the parental strain QP4. a qPCR analysis of the transcription levels of the cbh1, cbh2, egl1 and egl2 genes in QPC67 and QP4. b SDS-PAGE analysis of the supernatants from QPC67 and QP4

Fig. 3

Cellulase activities and the total secreted proteins of T. reesei QPC67 and QP4. a FPase activity (FPA). b Cellobiohydrolase activity (CBH). c Endoglucanase activity (EG). d The total extracellular protein. e The total intracellular protein, which was used to determine the fungal growth. Data are means of results from three independent measurements. Error bars indicate the standard deviation

Fig. 4

Saccharification of differently pretreated corncob residues by T. reesei QPC67 and QP4. a Saccharification of ACR with equal FPA activity. b Saccharification of DCR with equal protein concentration. Data are represented as the mean of three independent experiments. Error bars express the standard deviations

Fig. 5

Construction of the T. reesei egl2 overexpression strains. a Cassettes used for egl2 overexpression in the uracil auxotrophic strain QP4. b The CMC agar plate used to screen for the egl2 overexpression transformants. c PCR confirmation of the egl2- overexpression transformant QPE36, which shows an about 600-bp DNA fragment product using the primers Y-egl2-F1 and Y-PyrG-R1

Fig. 6

RT-qPCR and SDS-PAGE analysis for the EG2 overexpression strain QPE36 and the parental strain QP4. a qPCR analysis of the transcription levels of the cbh1, cbh2, egl1 and egl2 genes in QPE36 and QP4. b SDS-PAGE analysis of the supernatants from QPE36 and QP4

Fig. 7

Cellulase activities and the total secreted proteins of the EG2 overexpression strain QPE36 and the parental strain QP4. a FPase activity (FPA). b Endoglucanase activity (EG). c Cellobiohydrolase activity (CBH). d The total extracellular protein. e The total intracellular protein, which was used to determine the fungal growth. Data are means of results from three independent measurements. Error bars indicate the standard deviation

Fig. 8

Saccharification of differently pretreated corncob residues by T. reesei QPE36 and QP4. Saccharification of ACR (a) and DCR (b) with same protein concentration. Data are represented as the mean of three independent experiments. Error bars express the standard deviations

Fig. 9

Construction of the pyrG-excised strains. a Schematic representation of the forced recombination between the DR repeat regions under 5-FOA positive selection to reobtain uracil auxotrophy. b Colony morphology of the 5-FOA-resistant strains grown on the MM medium without uracil. c Colony morphology of 5-FOA-resistant strains grown on the MM medium with uracil. d PCR confirmation of the absence of the pyrG marker in the genome of the pyrG-excised T. reesei strains. e The CMC plate analysis of the pyrG-excised T. reesei strains

Fig. 10

Overexpression of bgl1 in the EG2 overexpression strain T. reesei QE51. a PCR confirmation of the BGL1 overexpression strain QEB4, which shows a 1.0-kb DNA fragment using the primers Y1 and Y2. b BGL, FPA, EG and CBH activities, which were measured after 5-day fermentation. Data are represented as the mean of three independent experiments and error bars express the standard deviations

Fig. 11

Comparison of the saccharification efficiencies towards differently pretreated corncob residues between the engineered T. reesei strains and the parental strain QP4. Saccharification of ACR (a) and DCR (b) by T. reesei QP4, QPC67, QPE36 and QEB4 with equal protein concentration. Displayed data represents averages of three independent experiments