Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose

Shibata, Nozomu; Kakeshita, Hiroshi; Igarashi, Kazuaki; Takimura, Yasushi; Shida, Yosuke; Ogasawara, Wataru; Koda, Tohru; Hasunuma, Tomohisa; Kondo, Akihiko

doi:10.1186/s13068-021-01887-0

Cited by 19 publications

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“…We selected the act1 promoter as a constitutive promoter unaffected by carbon source utilization, which showed similar expression levels as pyruvate decarboxylase 1 (Trire2_121534) in RNAseq results obtained under cellulose and glucose-containing culture conditions in the E1AB1 strain 54 . A cassette having XYR1 V821F under constitutively active act1 promoter was introduced at the ace1 locus, and the strain was named E1AB1-X.…”

Section: Resultsmentioning

confidence: 99%

Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the industrial fungus Trichoderma reesei

Arai

Ichinose

Shibata

et al. 2022

Sci Rep

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Trichodermareesei is a widely used host for producing cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Here, we report a genetic modification strategy for industrial T.reesei that enables enzyme production using simple glucose without inducers, such as cellulose, lactose and sophorose. Previously, the mutated XYR1V821F or XYR1A824V was known to induce xylanase and cellulase using only glucose as a carbon source, but its enzyme composition was biased toward xylanases, and its performance was insufficient to degrade lignocellulose efficiently. Therefore, we examined combinations of mutated XYR1V821F and constitutively expressed CRT1, BGLR, VIB1, ACE2, or ACE3, known as cellulase regulators and essential factors for cellulase expression to the T.reesei E1AB1 strain that has been highly mutagenized for improving enzyme productivity and expressing a ß-glucosidase for high enzyme performance. The results showed that expression of ACE3 to the mutated XYR1V821F expressing strain promoted cellulase expression. Furthermore, co-expression of these two transcription factors also resulted in increased productivity, with enzyme productivity 1.5-fold higher than with the conventional single expression of mutated XYR1V821F. Additionally, that productivity was 5.5-fold higher compared to productivity with an enhanced single expression of ACE3. Moreover, although the DNA-binding domain of ACE3 had been considered essential for inducer-free cellulase production, we found that ACE3 with a partially truncated DNA-binding domain was more effective in cellulase production when co-expressed with a mutated XYR1V821F. This study demonstrates that co-expression of the two transcription factors, the mutated XYR1V821F or XYR1A824V and ACE3, resulted in optimized enzyme composition and increased productivity.

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Section: Resultsmentioning

confidence: 99%

Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the industrial fungus Trichoderma reesei

Arai

Ichinose

Shibata

et al. 2022

Sci Rep

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“…The E1AB1 strain shows higher sacchari cation activity and higher enzyme productivity than the hyperproducing PC-3-7 strain [15], but the desirable characteristics are only available under induced conditions. Thus, we aimed to construct an inexpensive inducer-free enzyme production system by disrupting the repressors and genetically expressing various factors associated with cellulase transcription in the industrial E1AB1strain.…”

Section: Discussionmentioning

confidence: 99%

“…3b, 3c). In the E1AB1 strain used in this study, CCR is thought to be dysfunctional due to the cre1 mutation [8], tubB mutation [15], and so on. It needs to be examined whether the elimination of the repressor is essential for this system in the ancestral wild-type strain.…”

Section: Discussionmentioning

confidence: 99%

“…The enzyme produced by the E1AB1 T. reesei strain [14], which expresses AaBGL1 in the egl1 promoter, enhances the degradation of NaOH-pretreated rice straw, thereby reducing the amount of required cellulase. Interestingly, the E1AB1 strain not only exhibited improved sacchari cation activity but also enhanced protein secretion; this was reportedly caused by the disruption of the alpha-tubulin gene [15] and the insertion of aabgl1 in the tubulin gene locus. Furthermore, it was found that ΔtubB acquired a phenotype that reduced CCR even in the presence of high concentrations of glucose.…”

Section: Introductionmentioning

confidence: 99%

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Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

Arai

Ichinose

Shibata

et al. 2021

Preprint

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Background: Trichoderma reesei (Hypocrea jecorina) is a filamentous fungus that can produce extremely high levels of protein; consequently, it is utilized as a host for the production of cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Several hyper-producer strains of T. reesei have been bred for use in industrial production, but they generally require inducers to achieve high production capacities. The most commonly used inducers are soluble sugars produced by the degradation of cellulose; however, the dependence on cellulose degradation is problematic because cellulose is insoluble and has poor handling properties as a carbon source. Furthermore, once cellulose is decomposed, little cellulase is produced, making it difficult to produce the enzyme continuously and efficiently. The aim of this study was to establish a simple, inducer-free, cellulase production system using glucose as the sole carbon source.Results: Here, we focused on transcription factors that regulate both cellulase and hemicellulase genes. First, we verified that the previously reported Xylanase regulator 1 (Xyr1) mutation had a glucose-blind phenotype in T. reesei, and confirmed that constitutive expression of the V821F mutation in Xyr1 produced high levels of proteins, especially hemicellulase and cellulase, even in inducer-free conditions. However, the majority of proteins were hemicellulases. To reproduce cellulase/hemicellulase production similar to those observed under induced conditions, an activator of cellulase expression 3 (Ace3) was expressed in Xyr1V821F expressed strain additionally. As a result, the T. reesei strain constitutively expressing Xyr1V821F and Ace3 exhibited a 1.5-fold increase than Xyr1V821F expressed only in protein productivity under inducer-free conditions. Notably, the enzyme composition significantly improved for cellulases ratio and similar to that induced by cellulose. Furthermore, the enzymes exhibited a high saccharification efficiency when compared to that of produced by the strain expressing only the mutated Xyr1.Conclusions: This work shows that the constitutive expression of mutated Xyr1 and Ace3 can increase cellulase and hemicellulase production in T. reesei without inducers. This inducer-free enzyme production method could provide an effective system to reduce costs and simplify production processes, and is expected to be applied in the production of various proteins.

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“…Thus, inhibiting the phosphorylation of the C-terminal region of Cre1 could be another promising strategy to increase cellulase production without affecting growth. Another approach for relieving catabolite repression involves the deletion of the alpha-tubulin tubB gene in T. reesei , which led to the upregulation of several cellulase and hemicellulase genes, as well as genes encoding transporters of cellobiose and other sugars, when grown in a medium containing either glucose or cellobiose, suggesting that tubB could be involved in sugar sensing as well, and therefore analogous to Cre1 [ 98 ].…”

Section: Transcriptional Regulators Involved In the Regulation Of Cel...mentioning

confidence: 99%

Factors regulating cellulolytic gene expression in filamentous fungi: an overview

2022

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The growing demand for biofuels such as bioethanol has led to the need for identifying alternative feedstock instead of conventional substrates like molasses, etc. Lignocellulosic biomass is a relatively inexpensive feedstock that is available in abundance, however, its conversion to bioethanol involves a multistep process with different unit operations such as size reduction, pretreatment, saccharification, fermentation, distillation, etc. The saccharification or enzymatic hydrolysis of cellulose to glucose involves a complex family of enzymes called cellulases that are usually fungal in origin. Cellulose hydrolysis requires the synergistic action of several classes of enzymes, and achieving the optimum secretion of these simultaneously remains a challenge. The expression of fungal cellulases is controlled by an intricate network of transcription factors and sugar transporters. Several genetic engineering efforts have been undertaken to modulate the expression of cellulolytic genes, as well as their regulators. This review, therefore, focuses on the molecular mechanism of action of these transcription factors and their effect on the expression of cellulases and hemicellulases.

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Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose

Cited by 19 publications

References 50 publications

Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the industrial fungus Trichoderma reesei

Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the industrial fungus Trichoderma reesei

Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

Factors regulating cellulolytic gene expression in filamentous fungi: an overview

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