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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 systematica...

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Autores principales: Siebecker, Benedikt, Schütze, Tabea, Spohner, Sebastian, Haefner, Stefan, Meyer, Vera
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10588483/
https://www.ncbi.nlm.nih.gov/pubmed/37869709
http://dx.doi.org/10.3389/fbioe.2023.1279146
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author Siebecker, Benedikt
Schütze, Tabea
Spohner, Sebastian
Haefner, Stefan
Meyer, Vera
author_facet Siebecker, Benedikt
Schütze, Tabea
Spohner, Sebastian
Haefner, Stefan
Meyer, Vera
author_sort Siebecker, Benedikt
collection PubMed
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.
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spelling pubmed-105884832023-10-21 Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus Siebecker, Benedikt Schütze, Tabea Spohner, Sebastian Haefner, Stefan Meyer, Vera Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2023-10-06 /pmc/articles/PMC10588483/ /pubmed/37869709 http://dx.doi.org/10.3389/fbioe.2023.1279146 Text en Copyright © 2023 Siebecker, Schütze, Spohner, Haefner and Meyer. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Siebecker, Benedikt
Schütze, Tabea
Spohner, Sebastian
Haefner, Stefan
Meyer, Vera
Transcriptomic insights into the roles of the transcription factors Clr1, Clr2 and Clr4 in lignocellulose degradation of the thermophilic fungal platform Thermothelomyces thermophilus
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 Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10588483/
https://www.ncbi.nlm.nih.gov/pubmed/37869709
http://dx.doi.org/10.3389/fbioe.2023.1279146
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