Dataset Information

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

ABSTRACT:

Background

Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.

Results

We found that deletion of an ?-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (?tubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ?tubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ?tubB strain. These results suggested that the ?tubB strain was released from CCR.

Conclusion

Trichoderma reesei ?-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting ?-tubulin gene in T. reesei. The disruption of ?-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

SUBMITTER: Shibata N

PROVIDER: S-EPMC7869464 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Shibata Nozomu N Kakeshita Hiroshi H Igarashi Kazuaki K Takimura Yasushi Y Shida Yosuke Y Ogasawara Wataru W Koda Tohru T Hasunuma Tomohisa T Kondo Akihiko A

Biotechnology for biofuels 20210208 1

<h4>Background</h4>Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.<h4>Results</h4>We f ...[more]

PMID: 33557925

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Project description:BackgroundCellulolytic enzyme production in filamentous fungi requires a release from carbon catabolite repression (CCR). The protein CRE1/CreA (CRE = catabolite responsive element) is a key transcription factor (TF) that is involved in CCR and represses cellulolytic gene expression. CRE1/CreA represents the functional equivalent of Mig1p, an important Saccharomyces cerevisiae TF in CCR that exerts its repressive effect by recruiting a corepressor complex Tup1p-Cyc8p. Although it is known from S. cerevisiae that CRE1/CreA might repress gene expression via interacting with the corepressor complex Tup1-Cyc8, this mechanism is unconfirmed in other filamentous fungi, since the physical interaction has not yet been verified in these organisms. The precise mechanism on how CRE1/CreA achieves transcriptional repression after DNA binding remains unknown.ResultsThe results from tandem affinity purification and bimolecular fluorescence complementation revealed a direct physical interaction between the TF CRE1/CreA and the complex Tup1-Cyc8 in the nucleus of cellulolytic fungus Trichoderma reesei and Penicillium oxalicum. Both fungi have the ability to secrete a complex arsenal of enzymes to synergistically degrade lignocellulosic materials. In P. oxalicum, the protein PoCyc8, a subunit of complex Tup1-Cyc8, interacts directly with TF PoCreA and histone H3 lysine 36 (H3K36) methyltransferase PoSet2 in the nucleus. The di-methylation level of H3K36 in the promoter of prominent cellulolytic genes (cellobiohydrolase-encoding gene Pocbh1/cel7A and endoglucanase-encoding gene Poegl1/cel7B) is positively correlated with the expression levels of TF PoCreA. Since the methylation of H3K36 was also demonstrated to be a repression marker of cellulolytic gene expression, it appears feasible that the cellulolytic genes are repressed via PoCreA-Tup1-Cyc8-Set2-mediated transcriptional repression.ConclusionThis study verifies the long-standing conjecture that the TF CRE1/CreA represses gene expression by interacting with the corepressor complex Tup1-Cyc8 in filamentous fungi. A reasonable explanation is proposed that PoCreA represses gene expression by recruiting complex PoTup1-Cyc8. Histone methyltransferase Set2, which methylates H3K36, is also involved in the regulatory network by interacting with PoCyc8. The findings contribute to the understanding of CCR mechanism in filamentous fungi and could aid in biotechnologically relevant enzyme production.

Project description:BackgroundMicrobial production of bioactive secondary metabolites is challenging as most of the encoding genes are silent; and even if they are activated, the biosynthetic pathways are usually complex. Sorbicillinoids with multifunctional bioactivities are examples of these problems, which if solved can result in a more sustainable, simple supply of these important compounds to the pharmaceutical industry. As an excellent producer of cellulosic enzymes, Trichoderma reesei can secrete various sorbicillinoids.ResultsHere, we obtained a T. reesei mutant strain JNTR5 from the random mutation during overexpression of gene Tr69957 in T. reesei RUT-C30. JNTR5 exhibited a significant constitutive increase in sorbicillinoids production without affecting the cellulosic enzyme production. Confocal laser scanning microscope (CLSM) results indicated that sorbicillinoids were distributed in both mycelium and spores of JNTR5 with blue and green fluorescence. Compared with RUT-C30, JNTR5 displayed different cell morphology, reduced growth rate, and increased sporulation, but a similar biomass accumulation. Furthermore, transcriptome analysis revealed that all genes belonging to the sorbicillinoid gene cluster were upregulated, while most cellulase-encoding genes were downregulated. The cell wall integrity of JNTR5 was damaged, which might benefit the cellulase secretion and contribute to the almost unchanged cellulase and hemicellulase activity given that the damaged cell wall can enhance the secretion of the enzymes.ConclusionsFor the first time, we constructed a sorbicillinoids hyperproduction T. reesei platform with comparable cellulosic enzymes production. This outperformance of JNTR5, which is strain-specific, is proposed to be attributed to the overexpression of gene Tr69957, causing the chromosome remodeling and subsequently changing the cell morphology, structure, and the global gene expression as shown by phenotype and the transcriptome analysis of JNTR5. Overall, JNTR5 shows great potential for industrial microbial production of sorbicillinoids from cellulose and serves as an excellent model for investigating the distribution and secretion of yellow pigments in T. reesei.

Dataset Information

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

Background

Results

Conclusion

Publications

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

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