Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei

Abstract

Background: The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei.

Results: In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high β-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize β-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose.

Conclusions: In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.

Keywords: Cellulase; Transglycosylation; Trichoderma reesei; cre1; β-Disaccharides; β-Glucosidase.

Fig. 1

Deletion of the cre1 gene in T. reesei SP4. a Deletion of cre1 and excision of the pyrG marker in the Δcre1 strains. b Growth of T. reesei SP4 and Δcre1 strain SDC11 on the MM plate containing Avicel (0.5%) as sole carbon source. c Growth of T. reesei SP4 and SDC11 on the MM plate containing both glucose (1.0%) and Avicel (0.5%) as carbon sources

Fig. 2

Cellulase production by the Δcre1 strain SDC11. FPA activity (a), EG activity (b), CBH activity (c), BGL activity (d), extracellular protein (e) and intracellular protein (f) were measured at 3d, 5d, and 7d of cellulase-inducing cultivation. Data are the means of three independent experiments; error bars show standard deviations

Fig. 3

Saccharification of differently pretreated concob residues by T. reesei SDC11. a Glucose released from saccharification of acid-pretreated corncob residue every 24 h. b Glucose released from saccharification of delignified corncob residue every 24 h. Data are the means of three independent experiments; error bars show standard deviations

Fig. 4

Overexpression of the A. niger BGLA-encoding gene bglA in T. reesei SDC11. a Detection of β-glucosidase activity on the CMC-esculin plate. b SDS-PAGE analysis of the fermentation broth during cellulase-inducing cultivation. c, d The BGL and FPA activities produced during cellulase-inducing cultivation, respectively. e, f The extracellular protein and intracellular protein that were measured at 3d, 5d, and 7d of cellulase-inducing cultivation, respectively. Data are the means of three independent experiments; error bars show standard deviations

Fig. 5

Saccharification of different pretreated concob residues by the BGLA-overexpressing strain SCB18. a Glucose released from saccharification of acid-pretreated corncob residue every 24 h. b Glucose released from saccharification of delignified corncob residue every 24 h. Data are the means of three independent experiments; error bars show standard deviations

Fig. 6

Transglycosylation capability of the fermentation broth produced by T. reesei SCB18 under different glucose concentrations. a Glucose conversion rate (%) by determining glucose residual content at 1d, 2d, and 3d of transglycosylation reaction. b Analysis of the transglycosylation products containing glucose and β-disaccharides by thin-layer chromatography (TLC) at 2d. Data are the means of three independent experiments; error bars show standard deviations

Fig. 7

Cellulase production by T. reesei SDC11 using the transglycosylation product as inducer. Comparison of FPA activities using different carbon sources at the same concentration (2%) after 3d of cultivation. M, G, L, and C represent the transglycosylation product, glucose, lactose, and cellulose, respectively. Data are the means of three independent experiments; error bars show standard deviations

Fig. 8

Schematic model illustrating the principle for constructing the BGLA-overexpressing cellulase system with pleiotropic functions. The cellulase expression in T. reesei (SP4) is repressed through the carbon catabolite repressor Cre1 when glucose is present. Deletion of the cre1 gene results in glucose derepression and enhanced cellulase expression. Further overexpression of the A. niger bglA gene produces the T. reesei (SCB18) cellulase with high efficiency for saccharification of different cellulosic substrates. Meanwhile, the cellulase in T. reesei could also be induced by β-disaccharides (cellobiose and sophorose). The BGLA-overexpressing cellulase system produced by T. reesei (SCB18) can conversely synthesize these β-disaccharides with its transglycosylation activity from glucose as carbon source, which could be further used as cellulase inducers