Transformation of Trichoderma reesei with the cellobiohydrolase II gene as a means for obtaining strains with increased cellulase production and specific activity

doi:10.1016/0168-1656(91)90037-V

Journal of Biotechnology

Volume 20, Issue 1, August 1991, Pages 83-94

https://doi.org/10.1016/0168-1656(91)90037-V Get rights and content

Abstract

Trichoderma reesei QM 9414 was shown to contain a single copy of both the cbh1 and cbh2 gene, coding for cellobiohydrolase I (CBH 1) and II (CBH II) synthesis. In the attempt to increase CBH II formation, multiple copies of the cbh2 gene were introduced by cotransformation with the Neurospora crassa pyr4 gene using a pyrG auxotrophic mutant of T. reesei QM 9414 (T. reesei TU-6). Transformants with mitotically stable pyrG prototrophy, were shown to display both homologous as well as ectopic integration of the cbh2 gene. The cbh2 copy number varied between 10- and 20-fold. Secretion of CBH I and II by the transformants during growth on lactose as a carbon source leading to cellulase formation was quantified by means of monoclonal antibodies. Three out of 14 transformants exhibited a 2- to 4-fold increased CBH II titer. Hence, no correlation between the cbh2 copy number and the amount of secreted CBH II was observed. The same three transformants exhibited a roughly 1.5-fold increase in specific cellulase activity (FPU). The crude cellulase mixture of transformant 3, which displayed a 3- to 4-fold elevated CBH 11 formation, exhibited an increased specific activity against crystalline cellulose in vitro. It is therefore concluded that transformation with individual cellulase genes can he a useful and simple tool to alter the quantitative pattern of cellulolytic enzymes produced by T. reesei.

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Constitutive overexpression of cellobiohydrolase 2 in Trichoderma reesei reveals its ability to initiate cellulose degradation
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Cellulose degradation results from the synergistic effect of different enzymes, but which enzyme is involved in the initial stage of cellulose degradation is still not well understood. Cellobiohydrolase 2 (CBH2) attached to the conidial surface is possibly associated with the initial stage. However, its specific mechanism is still incompletely known. This study explored the potential role of CBH2 in initiating cellulose degradation using a constitutive overexpression strategy. First, the CBH2-overexpression Trichoderma reesei strains Qgc2–5 and Qrc2–40 were constructed using the constitutive promoters Pgpd1 and PrpS30, respectively. It was found that cbh2 was expressed at a high level under the glucose conditions and was significantly higher than that of the parental strain QM9414 at the early stage of 29 h when cellulose was used as the carbon source. Particularly, the constitutive overexpression of cbh2 caused the strong expression of major cellulase-encoding genes (cbh1, eg1, and eg2) and the rapid decomposition of cellulosic material. Meanwhile, the scanning electron microscope showed that the groove-like structure of the cellulose surface was eroded seriously owing to CBH2 overexpression, which caused the cellulose surface to be smooth. These results showed that the overexpression of CHB2 caused the major cellulase enzymes to be expressed and contributed to cellulose degradation, showing the potential role of CBH2 in the initial stage of the cellulose hydrolytic process.
Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses
2020, Process Biochemistry
Citation Excerpt :
The major issue with T. reesei cellobiohydrolase I (TrCBH1) is feedback inhibition by cellobiose and other reducing sugars produced during cellulose hydrolysis [6]. Additionally, low levels of β-glucosidase and other accessory enzymes require supplementation of the T. reesei cocktail with β-glucosidase and other important enzymes from other sources, such as Aspergillus and Talaromyces species [7–9]. Recent bioprospecting in our lab identified Penicillium funiculosum NCIM1228 as having a secretome of superior saccharification competence when compared with that of T. reesei [10].
PfMig1⁸⁸, a catabolically derepressed engineered strain of the hyper-cellulolytic fungus Penicillium funiculosum NCIM1228, was investigated for the efficacy of its secretome for biomass saccharification. An inexpensive version of media containing microcrystalline cellulose, wheat bran and soya protein was optimized for producing a high-quality secretome from the PfMig1⁸⁸ strain in both shake flasks and in a 20-L bioreactor. The activities of four classes of core cellulolytic enzymes required for saccharification in the PfMig1⁸⁸ secretome, namely, cellobiohydrolase (Avicelase activity), endoglucanase (CMCase activity), β-glucosidase (PNPGase activity) and xylanase (xylanase activity), were found to be 2.29 U/mL, 28.24 U/mL, 150 U/mL and 76 U/mL, respectively. The saccharification potential of the PfMig1⁸⁸ secretome was evaluated on rice straw and sugarcane bagasse pretreated with nitric acid and/or ammonium hydroxide. Saccharification performed using a 15 % (w/v) biomass load and a 3% (w/w) enzyme load released >100 g/L sugar in the hydrolysate, irrespective of the type of biomass and pre-treatment, with >80 % hydrolysis. Furthermore, the presence of lignin in nitric acid-pretreated biomass only marginally affected saccharification. Overall, the results demonstrated that the PfMig1⁸⁸ secretome, having relatively broad substrate specificity, is a viable and efficient substitute for T. reesei-based secretomes for diverse biomass saccharification.
Mixed culture of recombinant Trichoderma reesei and Aspergillus niger for cellulase production to increase the cellulose degrading capability
2018, Biomass and Bioenergy
Citation Excerpt :
The BG of T. reesei was enhanced by genetic engineering, solving the deficiency of BG once for all [9,14,15]. For the deficiency of CBH II, the approach to solve the problem is that the promoter cbh1 was used to overexpress cbh2 gene to strengthen the exo-exo-synergism between CBH I and CBH II [3,16]. The direct addition of CBH II from the genetically engineered Pichia pastoris producing recombinant CBH II into the cellulase from T. reesei was also feasible in overcoming the deficiency of CBH II [8].
It is well known that Trichoderma reesei's cellulase is deficient in β-glucosidase and cellobiohydrolase II, hampering the synergism of cellulase components and attenuating the cellulose-degrading capability. T. reesei was engineered to enhance its cellobiohydrolase II and cellulase production. However, it was still deficient in β-glucosidase. Therefore, the mixed culture of the recombinant T. reesei and Aspergillus niger was introduced to further improve cellulase production and the performance in enzymatic hydrolysis. It was found to produce the most robust cellulase, whose filter paper activity was 12.17 ± 1.18 FPIU/mL, cellobiohydrolase activity 53.10 ± 2.95 U/mL, endoglucanase activity 716.11 ± 41.16 U/mL, and β-glucosidase activity 1.93 ± 0.28 IU/mL. It had the highest enzymatic hydrolysis yield 89.35%, the lowest cellulase input 25.18 FPIU for 1 g glucose. It just needed 1.96 g SECS (dry material) for producing 1 g glucose. This work is profound and meaningful for lignocellulose-based biorefineries.
Heterologous expression of cellobiohydrolases in filamentous fungi - An update on the current challenges, achievements and perspectives
2015, Process Biochemistry
Citation Excerpt :
Additionally, the determined gene copy number and enzyme activity were not correlated [62,76]. Kubicek-Pranz et al. [76] assumed that the increase in activity observed for 3 out of 16 isolated transformants might have resulted from a transcriptionally efficient site of gene integration rather than from the number of gene copies; regardless, the hypothesis was not tested further. In general, successful heterologous expression depends on promoter efficiency, vector integration site, origin of the expressed protein, and the type of the host strain [77].
Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well-known producers of commercial preparations of cellulolytic enzymes and efficient hosts for heterologous protein secretion. Thus, Aspergillus and Trichoderma species have been chosen as hosts for the heterologous expression of native or engineered enzymes aiming at the overproduction of single enzymes or as hosts for the secretion of multi-enzyme cocktails for on-site production in biorefineries, which is important for reducing the costs of biomass conversion. An even more interesting aspect is consolidated bioprocessing, in which a single fungus both hydrolyzes lignocellulose polymers and ferments the resulting sugars into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation engineering are obvious targets for constructing efficient enzyme producers.
Quantitative proteomic analysis of secretome of microbial consortium during saw dust utilization
2012, Journal of Proteomics
Citation Excerpt :
Although P. chrysosporium and T. reesei encode an array of cellulolytic proteins, the former is a potent lignin degrading strain and the later is an industrial cellulolytic strain. To enhance cellulolytic potential of T. reesei, a variety of mutants have been generated [20–22]. In nature, populations of microorganisms inhabiting a common environment compete for nutrients and other resources creating a profoundly important network of interactions.
Proteomics analysis of lignocellulolytic proteins by lignocellulosic biomass degrading microbes and compatible microbial consortium is a promising approach that offers a new means to enzyme discovery. The abundance of proteins in complex secretome by microbial communities would highlight key lignocellulolytic proteins for lignocellulosic biorefinery. In this study, lignocellulolytic enzymes of potent lignin degrading basidiomycota and effective cellulolytic ascomycota fungal strains, and their co-cultures were analyzed using high throughput isobaric tag for relative and absolute quantitation (iTRAQ) technique using liquid chromatography tandem mass spectrometry. Protein abundances in the iTRAQ-multiplexed samples were determined by integrating relative quantitation and exponentially modified protein abundance index (emPAI). The functional classification of the secretory proteins by individual culture and co-culture demonstrated 36.77% cellulolytic proteins, 13.06% hemicellulases, 14.09% ligninolytic proteins, 19.59% proteolytic enzymes. 7.22% hypothetical proteins and 6.87% cell morphogenesis proteins. The abundance of the proteins by individual cultures and co-cultured fungal consortium revealed that co-culturing of Phanerochaete chrysosporium with Trichoderma reesei QM6a and Trichoderma reesei Rut C30 induced the production of cellulolytic proteins and stimulated expression of hemicellulolytic enzymes. The hierarchical clustering of proteins in secretome of fungal strains and their co-cultures elucidated differential expressions of lignocellulolytic proteins by the microbial consortium.
A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation
2011, Catalysis Today
Citation Excerpt :
For a certain industrial employment of these enzymes the composition of the cocktail is crucial: some applications demand for cellulases or hemicellulases exclusively, others need exact ratios of cellulases to hemicellulases. In the past, it was demonstrated that a simple deletion or insertion of multiple copies of various hydrolytic enzyme-encoding genes from/into Hypocrea genomes did not substantially alter the composition of produced enzymes [20,21]. This insight provoked other strategies as the regulation of gene expression of (hemi)cellulases.
Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

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Journal of Biotechnology

Abstract

References (31)

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Regulation of genes involved in the utilization of carbon sources in Aspergillus nidulans

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Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant

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Enzyme Microb. Technol.

Cited by (34)

Constitutive overexpression of cellobiohydrolase 2 in Trichoderma reesei reveals its ability to initiate cellulose degradation

Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

Mixed culture of recombinant Trichoderma reesei and Aspergillus niger for cellulase production to increase the cellulose degrading capability

Heterologous expression of cellobiohydrolases in filamentous fungi - An update on the current challenges, achievements and perspectives

Quantitative proteomic analysis of secretome of microbial consortium during saw dust utilization

A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation