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Inducer Free Cellulase Secretion in Neurospora Crassa and comparitive analysis of cellulase induction in Aspergillus nidulans

ABSTRACT: Purpose: To explore conservation of gene regulation by the transcription factor clr-2/clrB in Neurospora crassa and Aspergillus nidulans Methods: mRNA from wild type and clr-2/clrB mutants were collected after a culture shift from sucrose/glucose to Avicel (crystaline cellulose) or no carbon media Results: We show that N. crassa and A. nidulans have similair global transcriptional responses to Avicel, with several hundred genes showing specific induction, though the induced genes are more specifically targeted at cellulose for N. crassa and more targeted at hemicellulose and pectin for A. nidulans. clr-2/clrB has a conserved fundamental function in cellulose induction, though the mechanism has diverged. Misexpression of clr-2 is sufficeint for inducer free cellulase secretion in N. crassa, but neither clrB or heterologous clr-2 is sufficient for inducer free cellulase secretion in A. nidulans. Conclusions: Our study demonstrates a conserved and essential role in cellulose utilization for the transcription factor clr-2 in filamentous ascomycetes and demonstrates that manipulation of clr-2 expression can be used to control cellulase expression in some species. Biological triplicates of liquid culture N. crassa and A. nidulans were harvested at 4 hours and 6 hours, respectively, after a switch to media of interest. Global mRNA abundances from liquid cultures of N. crassa and A. nidulans were measured by sequencing on the Illumina Genome Analyzer IIx and HiSeq2000 platforms.

ORGANISM(S): Aspergillus nidulans FGSC A4

SUBMITTER: Samuel Coradetti

PROVIDER: E-GEOD-44100 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Inducer Free Cellulase Secretion in Neurospora Crassa and comparitive analysis of cellulase induction in Aspergillus nidulans

Project description:Purpose: To explore conservation of gene regulation by the transcription factor clr-2/clrB in Neurospora crassa and Aspergillus nidulans Methods: mRNA from wild type and clr-2/clrB mutants were collected after a culture shift from sucrose/glucose to Avicel (crystaline cellulose) or no carbon media Results: We show that N. crassa and A. nidulans have similair global transcriptional responses to Avicel, with several hundred genes showing specific induction, though the induced genes are more specifically targeted at cellulose for N. crassa and more targeted at hemicellulose and pectin for A. nidulans. clr-2/clrB has a conserved fundamental function in cellulose induction, though the mechanism has diverged. Misexpression of clr-2 is sufficeint for inducer free cellulase secretion in N. crassa, but neither clrB or heterologous clr-2 is sufficient for inducer free cellulase secretion in A. nidulans. Conclusions: Our study demonstrates a conserved and essential role in cellulose utilization for the transcription factor clr-2 in filamentous ascomycetes and demonstrates that manipulation of clr-2 expression can be used to control cellulase expression in some species.

2013-09-01 | GSE44100 | GEO

Conserved and Essential Transcription Factors for Cellulase Gene Expression in Ascomycete Fungi.

Project description:Transcriptional profiling with next-generation sequencing methods refined our understanding of the N. crassa transcriptional response to cellulose and demonstrated that the newly characterized transcription factors clr-1 and clr-2 were required for the bulk of that response including induction all major cellulase and some major hemicellulase genes. N. crassa pregrown in Sucrose and transferred to Avicel (cellulose), Sucrose or media with no carbon added. Biological triplicates used to identify differentially expressed genes in WT. Single libraries for mutant strains identify which genes show deficient regulation in response to Avicel. Note: Samples named "cdr1" and "cdr2" correspond to the genes clr-1 and clr-2 respectively.

2012-04-23 | E-GEOD-35227 | biostudies-arrayexpress

VIB1, a link between glucose signaling and carbon catabolite repression, is essential for plant cell wall degradation by Neurospora crassa

Project description:Purpose: To explore the function of VIB1 in regulating cellulase production in Neurospora crassa. Method: mRNA from vib-1 mutants were collected after a culture shift from sucrose to Avicel (crystaline cellulose) or carbon-free media. Expression profiles of vib-1 mutants were compared with published profiles of wild type created under the same conditions. Results: We found that many genes that specifically upregulated in wild type upon exposure to Avicel were expressed at low levels in M-bM-^HM-^Fvib-1 and many other genes involved in metabolism and energy were expressed at high levels compared to wild type. Conclusions: Our study shows that VIB1 is required for suppression of glucose response and carbon catabolite repression to allow proper expression of clr-2 and subsequent cellulase production in response to cellulosic induction. Biological triplicates of liquid culture N. crassa were harvested at 4 hours after a switch from sucrose media to media of interest. Global mRNA abundances were measured by sequencing on the Illumina Genome Analyzer Iix and HiSeq2000 platforms.

2014-07-08 | E-GEOD-52316 | biostudies-arrayexpress

Conserved and Essential Transcription Factors for Cellulase Gene Expression in Ascomycete Fungi.

2012-04-24 | GSE35227 | GEO

QUANTITATIVE PROTEOME PROFILING OF NEUROSPORA CRASSA REVEALS A CELLOBIOSE-DEPENDENT NETWORK COORDINATING LIGNOCELLULOLYTIC RESPONSES

Project description:Cellulose, particularly the major cellulolytic product cellobiose, can induce the production of enzymes associated with deconstruction of lignocellulose in filamentous fungi. However, the detailed mechanisms underlying this biotechnologically important process remain to be disclosed. Here, the proteome response to cellobiose, crystalline cellulose (Avicel), and carbon starvation of a Neurospora crassa triple β-glucosidase mutant were compared using tandem mass tag (TMT)-based proteome quantification. Improved quantification accuracy was achieved with synchronous precursor selection (SPS)-based MS3 technology compared to MS2 using a high resolution tribrid mass spectrometer. Exposure to carbon starvation, cellobiose or Avicel induced the production of cellulase and lytic polysaccharide monooxygenase enzymes in N. crassa, as well as a cellobionic acid transporter, indicating their functional roles in the early adaptation to plant cell wall. In particular, cellobiose specifically induced the production of proteins in the functional categories of protein processing and export as well as cell wall organization. The data presented here integrates the signaling pathway associated with cellobiose transporters CDT-1 and/or CDT-2 with the direct targets of the transcription factors CLR-1, CLR-2, and XLR-1, the unfolded protein response (UPR) mediated by Ire-1/Hac-1, as well as calcium homeostasis and cell wall organization. The cellobiose-dependent response network will be useful for rational strain improvement to facilitate the production of lignocellulases in filamentous fungi and plant biomass-based products.

2020-05-15 | PXD011598 | Pride

A novel conserved transcription factor CLR-4 regulates cellulase genes expression through controlling the intracellular cAMP level in ascomycete fungi

Project description:Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and plays key roles in biological carbon cycle. Although cellulases induction recently described in some saprobic filamentous fungi, regulation of cellulase transcription has not been studied thoroughly. Here, we identified and characterized the novel cellulase regulation factors clr-4 in Neurospora crassa and its ortholog Mtclr-4 in Myceliophthora thermophila. Deletion of clr-4 and Mtclr-4 displayed similarly defective phenotypes in cellulolytic enzymes production and activities. Transcriptomics analysis of Δclr-4/ΔMtclr-4 revealed down-regulation of not only encoding genes of (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1), but also the key genes of cAMP signaling pathway such as adenylate cyclase cr-1. Consistently, the significant decreased levels of intracellular cAMP were observed in Δclr-4/ΔMtclr-4 compared to wild-type during cellulose utilization. Electrophoretic mobility shift assays (EMSA) verified that CLR-4 could directly bind to the promoter regions of adenylyl cyclase (Nccr-1) and cellulose regulator clr-1, while MtCLR-4 bind to upstream regions of adenylyl cyclase Mtcr-1 and biomass deconstruction regulators Mtclr-2 and Mtxyr-1. Concluded, the novel cellulase expression regulators (CLR-4/MtCLR-4) findings here significantly enrich our understanding of the regulatory network of cellulose degradation and provide new targets for industrial fungi strain engineering for plant biomass deconstruction in biorefinery.

2021-03-18 | GSE111986 | GEO

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa

Project description:Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, CLP1 (NCU05853), a putative cellodextrin transporter-like protein, that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain M-NM-^T3M-NM-2G formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with M-NM-^T3M-NM-2G. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. N. crassa was pregrown in sucrose and transferred to cellobiose media. Up regulated and down regulated genes expressions were compared with M-NM-^T3M-NM-2G and M-NM-^T3M-NM-2GM-NM-^Tclp1 strain.

2014-11-13 | E-GEOD-60004 | biostudies-arrayexpress

Evidence of a critical role for cellodextrin transporter 2 (CDT-2) in both cellulose and hemicellulose degradation and utilization in Neurospora crassa

Project description:CDT-1 and CDT-2 are two cellodextrin transporters discovered in the filamentous fungus Neurospora crassa. Previous studies focused on characterizing the role of these transporters in only a few conditions, including cellulose degradation, and the function of these two transporters is not yet completely understood. In this study, we show that deletion of cdt-2, but not cdt-1, results in growth defects not only on Avicel but also on xylan. cdt-2 can be highly induced by xylan, and this mutant has a xylodextrin consumption defect. Transcriptomic analysis of the cdt-2 deletion strain on Avicel and xylan showed that major cellulase and hemicellulase genes were significantly down-regulated in the cdt-2 deletion strain and artificial over expression of cdt-2 in N. crassa increased cellulase and hemicellulase production. Together, these data clearly show that CDT-2 plays a critical role in hemicellulose sensing and utilization. This is the first time a sugar transporter has been assigned a function in the hemicellulose degradation pathway. Furthermore, we found that the transcription factor XLR-1 is the major regulator of cdt-2, while cdt-1 is primarily regulated by CLR-1. These results deepen our understanding of the functions of both cellodextrin transporters, particularly for CDT-2. Our study also provides novel insight into the mechanisms for hemicellulose sensing and utilization in N. crassa, and may be applicable to other cellulolytic filamentous fungi. N. crassa was pregrown in Sucrose and transferred to Avicel (cellulose) or Xylan(hemicellulose) media. Up regulated and down regulated genes expressions were compared with wild type strain on two conditions (Avicel and xylan) respectively.

2014-09-25 | E-GEOD-44673 | biostudies-arrayexpress

The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei

Project description:The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes and also serves as a model for investigations on these enzymes and their genes. Most of them are obligatorily dependent on the Zn(II)Cys(VI) transcriptional activator XYR1. XYR1 is constitutively expressed at a low basal, but induced in the presence of cellulase- and hemicellulose inducers, and transported into the nucleus. The factors mediating its import and export across the nuclear pore complexes (karyopherins) are therefore expected to play a key role in its function. We identified 14 karyopherins in T. reesei, of which 8 were predicted to be involved nuclear protein import. Their systematic knock-out and testing for cellulase formation identified KAP8 (an orthologue of A. nidulans KapI, and Saccharomyces cerevisiae Kap121/Pse1p) to be essential for cellulase gene expression, and for the appearance of GFP-XYR1 in the nucleus. Transcriptomic analysis of Δkap8 and a retransformant under cellulase-inducing conditions revealed the downregulation of 64 Cazymes in the Δkap8 strain under inducing conditions, including all cellulases and hemicellulases known to be under XYR1 control, and of 12 transcription factors of which 2 were known to be associated with cellulase regulation (ACE3, CLR2). Together, these new observations underscore the role of nuclear transport of XYR1 in the regulation of cellulase and hemicellulose gene expression in T. reesei, and identify KAP8 as the major karyopherin involved in this process. Two strains were used: T. reesei ku70 (pyr4-), in which the kap8 (=kapI/Pse1/Kap121) reading frame has been replaced by the T. reesei pyr4 gene; and a kap8-retransformant of the same strain and two time points for each strain T0h and T3h. All samples are in tricplicate.

2015-03-03 | E-GEOD-59600 | biostudies-arrayexpress

Network of nutrient sensing pathways and a conserved kinase cascade integrates osmolarity and carbon sensing in Neurospora crassa

Project description:Identifying nutrients available in the environment and utilizing them in the most efficient manner is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of carbohydrates, from simple sugars to the complex carbohydrates found in plant cell walls. The zinc binuclear cluster transcription factor CLR-1 is necessary for utilization of cellulose, a major, recalcitrant component of the plant cell wall; however, expression of clr-1 in the absence of an inducer is not sufficient to induce cellulase gene expression. We performed a screen for unidentified actors in the cellulose-response pathway and identified a gene encoding a hypothetical protein (clr-3) that is required for repression of CLR-1 activity in the absence of an inducer. Using clr-3 mutants, we implicated the hyperosmotic-response pathway in the tunable regulation of glycosyl hydrolase production in response to changes in osmolarity. The role of the hyperosmotic-response pathway in nutrient sensing may indicate that cells use osmolarity as a proxy for the presence of free sugar in their environment. These signaling pathways form a nutrient-sensing network that allows N. crassa cells to tightly regulate gene expression in response to environmental conditions.

2017-09-25 | GSE95681 | GEO

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