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Simultaneous enhancement of the beta-exo synergism and exo-exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability.

ABSTRACT: BACKGROUND:Cellulase is the one of the largest contributors to the high production costs of the lignocellulose-based biorefineries. As the most widely used cellulase producer, Trichoderma reesei has two weaknesses, deficiencies in ?-glucosidase and cellobiohydrolase II. This work aimed at solving this problem by simultaneous enhancement of the beta-exo synergism and exo-exo synergism in T. reesei cellulase to increase the cellulose degrading capability, i.e. enhanced co-expression of the ?-glucosidase gene the cellobiohydrolase II gene of T. reesei. RESULTS:Enhanced co-expression of the ?-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 was found successful in overcoming the two weaknesses. Filter paper activities of T. reesei cellulase were greatly elevated, which were 7.21?±?0.45 (E7, Aabgl1 and Trcbh2) and 7.69?±?0.42 (F6, Anbgl1 and Trcbh2) FPIU/mL. They were much higher than that of the parental strain Rut-C30, 2.45?±?0.36 FPIU/mL. Enzymatic hydrolysis yields were also improved, from 67.22?±?1.61% by Rut-C30 cellulase to 87.98?±?0.65% by E7 cellulase and 86.50?±?1.01% by F6 cellulase. The substrate loading for 1 g glucose release from SECS were decreased, from 2.9637 g SECS using Rut-C30 cellulase to 2.0291 g SECS using E7 cellulase and 2.0573 g SECS using F6 cellulase. As a result, the efficiency of the process from SECS to glucose was substantially improved. CONCLUSIONS:Enhanced co-expression of the ?-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 in T. reesei was proven triumphal in the simultaneous enhancement of the beta-exo synergism and exo-exo synergism in T. reesei cellulase. This strategy also improved the cellulase production, enzymatic hydrolysis yield and the efficiency of the process from SECS to glucose in the context of on-site cellulase production. This work is a commendable attempt in the cellulase composition optimization at the transcriptional level.

SUBMITTER: Fang H

PROVIDER: S-EPMC6337788 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Simultaneous enhancement of the beta-exo synergism and exo-exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability.

Fang Hao H Zhao Runze R Li Chaofeng C Zhao Chen C

Microbial cell factories 20190118 1

<h4>Background</h4>Cellulase is the one of the largest contributors to the high production costs of the lignocellulose-based biorefineries. As the most widely used cellulase producer, Trichoderma reesei has two weaknesses, deficiencies in β-glucosidase and cellobiohydrolase II. This work aimed at solving this problem by simultaneous enhancement of the beta-exo synergism and exo-exo synergism in T. reesei cellulase to increase the cellulose degrading capability, i.e. enhanced co-expression of the ...[more]

PMID: 30657063

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Project description:BackgroundCellulolytic enzymes produced by the filamentous fungus Trichoderma reesei are commonly used in biomass conversion. The high cost of cellulase is still a significant challenge to commercial biofuel production. Improving cellulase production in T. reesei for application in the cellulosic biorefinery setting is an urgent priority.ResultsTrichoderma reesei hyper-cellulolytic mutant SS-II derived from the T. reesei NG14 strain exhibited faster growth rate and more efficient lignocellulosic biomass degradation than those of RUT-C30, another hyper-cellulolytic strain derived from NG14. To identify any genetic changes that occurred in SS-II, we sequenced its genome using Illumina MiSeq. In total, 184 single nucleotide polymorphisms and 40 insertions and deletions were identified. SS-II sequencing revealed 107 novel mutations and a full-length wild-type carbon catabolite repressor 1 gene (cre1). To combine the mutations of RUT-C30 and SS-II, the sequence of one confirmed beneficial mutation in RUT-C30, cre196, was introduced in SS-II to replace full-length cre1, forming the mutant SS-II-cre196. The total cellulase production of SS-II-cre196 was decreased owing to the limited growth of SS-II-cre196. In contrast, 57 genes mutated only in SS-II were selected and knocked out in RUT-C30. Of these, 31 were involved in T. reesei growth or cellulase production. Cellulase activity was significantly increased in five deletion strains compared with that in two starter strains, RUT-C30 and SS-II. Cellulase production of T. reesei Δ108642 and Δ56839 was significantly increased by 83.7% and 70.1%, respectively, compared with that of RUT-C30. The amount of glucose released from pretreated corn stover hydrolyzed by the crude enzyme from Δ108642 increased by 11.9%.ConclusionsThe positive attribute confirmed in one cellulase hyper-producing strain does not always work efficiently in another cellulase hyper-producing strain, owing to the differences in genetic background. Genome re-sequencing revealed novel mutations that might affect cellulase production and other pathways indirectly related to cellulase formation. Our strategy of combining the mutations of two strains successfully identified a number of interesting phenotypes associated with cellulase production. These findings will contribute to the creation of a gene library that can be used to investigate the involvement of various genes in the regulation of cellulase production.

Project description: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 ?-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 ?-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.

Project description:BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Project description:Background:Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production. Results:In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of ?cre1 strain. Conclusions:A new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

Dataset Information

Simultaneous enhancement of the beta-exo synergism and exo-exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability.

Publications

Simultaneous enhancement of the beta-exo synergism and exo-exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability.

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