Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus
Introduction:Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, is used in industry to produce lignocellulolytic enzymes and heterologous proteins. However, the transcriptional network driving the expression of these proteins remains elusive. As a first step to systematical...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-10-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1279146/full |
| _version_ | 1797665180586344448 |
|---|---|
| author | Benedikt Siebecker Tabea Schütze Sebastian Spohner Stefan Haefner Vera Meyer |
| author_facet | Benedikt Siebecker Tabea Schütze Sebastian Spohner Stefan Haefner Vera Meyer |
| author_sort | Benedikt Siebecker |
| collection | DOAJ |
| description | Introduction:Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, is used in industry to produce lignocellulolytic enzymes and heterologous proteins. However, the transcriptional network driving the expression of these proteins remains elusive. As a first step to systematically uncover this network, we investigated growth, protein secretion, and transcriptomic fingerprints of strains deficient in the cellulolytic transcriptional regulators Clr1, Clr2, and Clr4, respectively.Methods: The genes encoding Clr1, Clr2, and Clr4 were individually deleted using split marker or the CRISPR/Cas12a technology and the resulting strains as well as the parental strain were cultivated in bioreactors under chemostat conditions using glucose as the carbon source. During steady state conditions, cellulose was added instead of glucose to study the genetic and cellular responses in all four strains to the shift in carbon source availability.Results: Notably, the clr1 and clr2 deletion strains were unable to continue to grow on cellulose, demonstrating a key role of both regulators in cellulose catabolism. Their transcriptomic fingerprints uncovered not only a lack of cellulase gene expression but also reduced expression of genes predicted to encode hemicellulases, pectinases, and esterases. In contrast, the growth of the clr4 deletion strain was very similar compared to the parental strain. However, a much stronger expression of cellulases, hemicellulases, pectinases, and esterases was observed.Discussion: The data gained in this study suggest that both transcriptional regulators Clr1 and Clr2 activate the expression of genes predicted to encode cellulases as well as hemicellulases, pectinases, and esterases. They further suggest that Clr1 controls the basal expression of cellulases and initiates the main lignocellulolytic response to cellulose via induction of clr2 expression. In contrast, Clr4 seems to act as a repressor of the lignocellulolytic response presumably via controlling clr2 expression. Comparative transcriptomics in all four strains revealed potentially new regulators in carbohydrate catabolism and lignocellulolytic enzyme expression that define a candidate gene list for future analyses. |
| first_indexed | 2024-03-11T19:41:11Z |
| format | Article |
| id | doaj.art-1644373512184f59994ff722b3ddbbe2 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-11T19:41:11Z |
| publishDate | 2023-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-1644373512184f59994ff722b3ddbbe22023-10-06T10:00:58ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-10-011110.3389/fbioe.2023.12791461279146Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilusBenedikt Siebecker0Tabea Schütze1Sebastian Spohner2Stefan Haefner3Vera Meyer4Chair of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, GermanyChair of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, GermanyBASF SE, Ludwigshafen, GermanyBASF SE, Ludwigshafen, GermanyChair of Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Berlin, GermanyIntroduction:Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, is used in industry to produce lignocellulolytic enzymes and heterologous proteins. However, the transcriptional network driving the expression of these proteins remains elusive. As a first step to systematically uncover this network, we investigated growth, protein secretion, and transcriptomic fingerprints of strains deficient in the cellulolytic transcriptional regulators Clr1, Clr2, and Clr4, respectively.Methods: The genes encoding Clr1, Clr2, and Clr4 were individually deleted using split marker or the CRISPR/Cas12a technology and the resulting strains as well as the parental strain were cultivated in bioreactors under chemostat conditions using glucose as the carbon source. During steady state conditions, cellulose was added instead of glucose to study the genetic and cellular responses in all four strains to the shift in carbon source availability.Results: Notably, the clr1 and clr2 deletion strains were unable to continue to grow on cellulose, demonstrating a key role of both regulators in cellulose catabolism. Their transcriptomic fingerprints uncovered not only a lack of cellulase gene expression but also reduced expression of genes predicted to encode hemicellulases, pectinases, and esterases. In contrast, the growth of the clr4 deletion strain was very similar compared to the parental strain. However, a much stronger expression of cellulases, hemicellulases, pectinases, and esterases was observed.Discussion: The data gained in this study suggest that both transcriptional regulators Clr1 and Clr2 activate the expression of genes predicted to encode cellulases as well as hemicellulases, pectinases, and esterases. They further suggest that Clr1 controls the basal expression of cellulases and initiates the main lignocellulolytic response to cellulose via induction of clr2 expression. In contrast, Clr4 seems to act as a repressor of the lignocellulolytic response presumably via controlling clr2 expression. Comparative transcriptomics in all four strains revealed potentially new regulators in carbohydrate catabolism and lignocellulolytic enzyme expression that define a candidate gene list for future analyses.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1279146/fullThermothelomyces thermophilusMyceliophthora thermophilacellulaseslignocellulolytic enzymestranscription factorstranscriptomics |
| spellingShingle | Benedikt Siebecker Tabea Schütze Sebastian Spohner Stefan Haefner Vera Meyer Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus Frontiers in Bioengineering and Biotechnology Thermothelomyces thermophilus Myceliophthora thermophila cellulases lignocellulolytic enzymes transcription factors transcriptomics |
| title | Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus |
| title_full | Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus |
| title_fullStr | Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus |
| title_full_unstemmed | Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus |
| title_short | Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus |
| title_sort | transcriptomic insights into the roles of the transcription factors clr1 clr2 and clr4 in lignocellulose degradation of the thermophilic fungal platform thermothelomyces thermophilus |
| topic | Thermothelomyces thermophilus Myceliophthora thermophila cellulases lignocellulolytic enzymes transcription factors transcriptomics |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1279146/full |
| work_keys_str_mv | AT benediktsiebecker transcriptomicinsightsintotherolesofthetranscriptionfactorsclr1clr2andclr4inlignocellulosedegradationofthethermophilicfungalplatformthermothelomycesthermophilus AT tabeaschutze transcriptomicinsightsintotherolesofthetranscriptionfactorsclr1clr2andclr4inlignocellulosedegradationofthethermophilicfungalplatformthermothelomycesthermophilus AT sebastianspohner transcriptomicinsightsintotherolesofthetranscriptionfactorsclr1clr2andclr4inlignocellulosedegradationofthethermophilicfungalplatformthermothelomycesthermophilus AT stefanhaefner transcriptomicinsightsintotherolesofthetranscriptionfactorsclr1clr2andclr4inlignocellulosedegradationofthethermophilicfungalplatformthermothelomycesthermophilus AT verameyer transcriptomicinsightsintotherolesofthetranscriptionfactorsclr1clr2andclr4inlignocellulosedegradationofthethermophilicfungalplatformthermothelomycesthermophilus |