Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production

ABSTRACT The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.

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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

Biotechnology for Biofuels ◽

10.1186/s13068-021-01887-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Nozomu Shibata ◽

Hiroshi Kakeshita ◽

Kazuaki Igarashi ◽

Yasushi Takimura ◽

Yosuke Shida ◽

...

Keyword(s):

Trichoderma Reesei ◽

Catabolite Repression ◽

Enzyme Production ◽

Protein Production ◽

Carbon Catabolite Repression ◽

Transcriptional Regulator ◽

Secretory Protein ◽

Sugar Transporter ◽

Alpha Tubulin ◽

High Enzyme

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 α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) 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 ΔtubB 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 ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR. Conclusion Trichoderma reesei α-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 α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

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Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1521-1527.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1521-1527

Author(s):

T E Torchia ◽

R W Hamilton ◽

C L Cano ◽

J E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Negative Regulator ◽

Null Mutation ◽

Null Mutant ◽

Wild Type

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

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Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes.

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1521 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1521-1527 ◽

Cited By ~ 65

Author(s):

T E Torchia ◽

R W Hamilton ◽

C L Cano ◽

J E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Negative Regulator ◽

Null Mutation ◽

Null Mutant ◽

Wild Type

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Carbon catabolite repression in Aspergillus nidulans: a review

Canadian Journal of Botany ◽

10.1139/b95-240 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 160-166 ◽

Cited By ~ 21

Author(s):

Claudio Scazzocchio ◽

Victoria Gavrias ◽

Beatriz Cubero ◽

Cristina Panozzo ◽

Martine Mathieu ◽

...

Keyword(s):

Nitrogen Source ◽

Aspergillus Nidulans ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Experimental Methodology ◽

Structural Genes ◽

General Transcription Factor ◽

Proline Utilization ◽

Utilization Pathway

We describe the experimental methodology that led to the discovery of the creA gene in Aspergillus nidulans. This gene codes for a transcriptional repressor mediating carbon catabolite repression in many pathways in this organism. We compare both the mode and the mechanism of action in two pathways subject to CreA-mediated repression. The genes comprising the ethanol regulon are subject to carbon catabolite repression independently of the nitrogen source, while the genes involved in proline utilization are repressed by glucose only when a repressing nitrogen source is also present. In the ethanol regulon, CreA drastically represses the expression of the positive regulatory gene alcR, thus preventing the expression of the structural genes. Direct repression of the structural genes is also existant. In the proline utilization pathway, repression operates directly at the level of the structural genes. In the ethanol regulon, CreA prevents the self-induction of alcR and the induction of the structural genes by competing with the binding of the AlcR protein. In proline gene cluster, CreA does not interfere with induction mediated by PrnA but with the activity of an unknown and more general transcription factor. Key words: carbon catabolite repression, ascomycetes, Zn fingers.

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Carbon catabolite repression of xylanase I (xyn1) gene expression in Trichoderma reesei

Molecular Microbiology ◽

10.1046/j.1365-2958.1996.00094.x ◽

1996 ◽

Vol 21 (6) ◽

pp. 1273-1281 ◽

Cited By ~ 121

Author(s):

Robert L. Mach ◽

Joseph Strauss ◽

Susanne Zeilinger ◽

Martin Schindler ◽

Christian P. Kubicek

Keyword(s):

Gene Expression ◽

Trichoderma Reesei ◽

Catabolite Repression ◽

Carbon Catabolite Repression

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Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis

Fungal Genetics and Biology ◽

10.1016/j.fgb.2014.10.009 ◽

2014 ◽

Vol 73 ◽

pp. 93-103 ◽

Cited By ~ 36

Author(s):

Amanda Cristina Campos Antoniêto ◽

Lílian dos Santos Castro ◽

Rafael Silva-Rocha ◽

Gabriela Felix Persinoti ◽

Roberto Nascimento Silva

Keyword(s):

Trichoderma Reesei ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Rna Seq ◽

Genome Wide

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The facC Gene of Aspergillus nidulans Encodes an Acetate-Inducible Carnitine Acetyltransferase

Journal of Bacteriology ◽

10.1128/jb.180.23.6242-6251.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6242-6251 ◽

Cited By ~ 25

Author(s):

Christopher J. Stemple ◽

Meryl A. Davis ◽

Michael J. Hynes

Keyword(s):

Fusion Protein ◽

Aspergillus Nidulans ◽

Catabolite Repression ◽

Sole Carbon Source ◽

Carbon Catabolite Repression ◽

Regulatory Gene ◽

Translation Product ◽

Carnitine Acetyltransferase ◽

Significant Similarity ◽

Intracellular Compartments

ABSTRACT Mutations in the facC gene of Aspergillus nidulans result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the facC gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in facC mutants, while fatty acid-inducible activity was absent in an acuJ mutant. Acetate induction offacC expression was dependent on the facBregulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′ facC sequences. Carbon catabolite repression of facC expression was affected by mutations in thecreA gene and a CreA fusion protein bound to 5′facC sequences. Mutations in the acuJ gene led to increased acetate induction of facC expression and also of an amdS-lacZ reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of facB. A model is presented in which facCencodes a cytosolic CAT enzyme, while a different CAT enzyme, which isacuJ dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments.

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Carbon catabolite repression involves physical interaction of the transcription factor CRE1/CreA and the Tup1–Cyc8 complex in Penicillium oxalicum and Trichoderma reesei

Biotechnology for Biofuels ◽

10.1186/s13068-021-02092-9 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yueyan Hu ◽

Mengxue Li ◽

Zhongjiao Liu ◽

Xin Song ◽

Yinbo Qu ◽

...

Keyword(s):

Gene Expression ◽

Filamentous Fungi ◽

Transcriptional Repression ◽

Catabolite Repression ◽

Enzyme Production ◽

Carbon Catabolite Repression ◽

Penicillium Oxalicum ◽

Physical Interaction ◽

Corepressor Complex ◽

Encoding Gene

Abstract Background Cellulolytic 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. Results The 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. Conclusion This 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.

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Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis

Microbiology ◽

10.1099/mic.0.26326-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2345-2355 ◽

Cited By ~ 25

Author(s):

José Manuel Inácio ◽

Carla Costa ◽

Isabel de Sá-Nogueira

Keyword(s):

Bacillus Subtilis ◽

Catabolite Repression ◽

Molecular Mechanisms ◽

Northern Blot Analysis ◽

Carbon Catabolite Repression ◽

Glucose Repression ◽

Regulatory Gene ◽

Site Directed Mutagenesis ◽

Transcriptional Level ◽

Fusion Analysis

The Bacillus subtilis proteins involved in the utilization of l-arabinose are encoded by the araABDLMNPQ–abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by l-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription ‘roadblocking’ of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.

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