Induction of CtsR-regulated chaperones improves Xylanase production in Bacillus subtilis
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ABSTRACT:
PROVIDER: PRJNA901390 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22273579.fastq.gz | Fastqsanger.gz | |||
| SRR22273580.fastq.gz | Fastqsanger.gz | |||
| SRR22273581.fastq.gz | Fastqsanger.gz | |||
| SRR22273582.fastq.gz | Fastqsanger.gz | |||
| SRR22273583.fastq.gz | Fastqsanger.gz |
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Project description:We examined the effect of high-level expression of the commercially important endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq. Rather unexpectedly, we found a reduced expression of several protein chaperones, including ClpC, ClpE and ClpX, was downregulated when XynA was overproduced. Expression of these proteins is controlled by the transcriptional repressor CtsR. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. Preventing this downregulation by knocking out the involved transcriptional repressor CtsR resulted in increased XynA production by more than 25 %.
2023-02-21 | GSE217916 | GEO
Project description:In a previous study we used RNA-seq to identify cellular stresses related to the overexpression of xylanase XynA, and found that upregulation of the CtsR regulon improves the yield of XynA production in B. subtilis. In this study, we compared the transcriptomes of B. subtilis cells overexpressing either the xylanase XynA or the heterologous amylase AmyM, to identify general and enzyme-specific stress responses. In addition, we further compared the translational profiles of cells overexpressing XynA and AmyM using ribosome profiling.
2024-08-30 | GSE270692 | GEO
Project description:BackgroundThe bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq.ResultsUsing the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%.ConclusionsOverproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.
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Project description:Background: Filamentous fungi produce large quantities of extracellular enzymes, such as cellulase and xylanase, to degrade polysaccharides derived from plant biomass. This process is controlled by a complex network of transcription factors (TFs). However, the regulatory mechanisms of cellulase and xylanase gene expression are poorly understood. Results: In this study, we reveal that the bZIP transcription factor Mthac-1 exerts dual regulatory effects on the production of cellulase and xylanase in the filamentous fungus Myceliophthora thermophila. The absence of Mthac-1 caused a reduction in cellulase and xylanase activities, as well as decreased protein secretion during the early phase of cultivation in Avicel medium, compared with the wild-type (WT) strain. However, in the middle and late stages, enzyme activities and protein secretion were noticeably enhanced by the deletion of Mthac-1. Additionally, the loss of Mthac-1 led to severe growth defects on sloid media containing various carbon sources, characterized by few hyphae branching and reduced conidiation. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis showed that Mthac-1 dynamically regulates the expression of major cellulase genes. Furthermore, electrophoretic mobility shift assays (EMSAs) demonstrated that Mthac-1 directly binds to the promoter regions of the β-glucosidase gene bgl1 (MYCTH_66804), cellobiohydrolase gene cbh1 (MYCTH_109566), endoglucanase gene egl2 (MYCTH_86753), xylanase gene xyn1 (MYCTH_112050), and the crucial regulatory gene xyr1 (MYCTH_2310145), exhibiting higher binding affinity for xyn1 and xyr1. Notably, comparative transcriptomic analysis indicated that Mthac-1 also plays an important role in the expression of 26S proteasome-encoding genes under cellulolytic conditions. Conclusions: Mthac-1 functions as a dual regulator of cellulase and xylanase production by regulating the expression of major cellulase and xylanase genes, as well as the key transactivator gene Mtxyr1, via direct promoter binding. Our work provides new insights into the regulatory mechanisms underlying cellulase and xylanase gene expression and has potential applications in fungal strain engineering in biorefinery industries.
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Project description:In this study,comparative genomic, transcriptomic and secretomic profilings of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators for cellulase and xylanase gene expression.
2016-08-31 | GSE80076 | GEO
Project description:Myceliophthora thermophila, a thermophilic fungus, has been utilized to produce industrially important enzymes in biorefineries. In filamentous fungi, the mechanisms underlying cellulase and xylanase expression have been explored, which uncovered the complex networks controlled by numerous transcription factors (TFs). However, the TFs regulating cellulase and xylanase gene expression and production remain unclear in M. thermophila. Here, we identified and characterized a novel cellulase and xylanase regulator MtFKH1 (MYCTH_2307931) in M. thermophila through comparative transcriptomic and genetic analyses. Comparative transcriptome analyses of M. thermophila grown on Avicel and glucose screened eight potential transcription factor encoding genes and five of them were successfully deleted by newly developed CRISPR/Cas9 system, which caused the identification of a forkhead TF MtFKH1. The disruption of Mtfkh1 remarkably elevated cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. Loss of Mtfkh1 also exhibited an impairment in sporulation but normal mycelia growth compared with WT. Real-time quantitative reverse transcription PCR (RT-qPCR) showed that MtFKH1 regulated the expression of essential cellulase and xylanase genes, and electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 could specifically bound to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050). Further DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, which exhibited strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings enlarge our knowledge of how forkhead transcription factor regulate lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulases and xylanases.
2025-04-14 | GSE274798 | GEO
Project description:Myceliophthora thermophila, a thermophilic fungus, has been utilized to produce industrially important enzymes in biorefineries. In filamentous fungi, the mechanisms underlying cellulase and xylanase expression have been explored, which uncovered the complex networks controlled by numerous transcription factors (TFs). However, the TFs regulating cellulase and xylanase gene expression and production remain unclear in M. thermophila. Here, we identified and characterized a novel cellulase and xylanase regulator MtFKH1 (MYCTH_2307931) in M. thermophila through comparative transcriptomic and genetic analyses. Comparative transcriptome analyses of M. thermophila grown on Avicel and glucose screened eight potential transcription factor encoding genes and five of them were successfully deleted by newly developed CRISPR/Cas9 system, which caused the identification of a forkhead TF MtFKH1. The disruption of Mtfkh1 remarkably elevated cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. Loss of Mtfkh1 also exhibited an impairment in sporulation but normal mycelia growth compared with WT. Real-time quantitative reverse transcription PCR (RT-qPCR) showed that MtFKH1 regulated the expression of essential cellulase and xylanase genes, and electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 could specifically bound to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050). Further DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, which exhibited strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings enlarge our knowledge of how forkhead transcription factor regulate lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulases and xylanases.
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Project description:Redox regulation of stress proteins, such as molecular chaperones, guarantees an immediate response to oxidative stress conditions. This review focuses on the two major classes of redox-regulated chaperones, Hsp33 in bacteria and typical 2-Cys peroxiredoxins in eukaryotes. Both proteins employ redox-sensitive cysteines, whose oxidation status directly controls their affinity for unfolding proteins and therefore their chaperone function. We will first discuss Hsp33, whose oxidative stress-induced disulfide bond formation triggers the partial unfolding of the chaperone, which, in turn, leads to the exposure of a high-affinity binding site for unfolded proteins. This rapid mode of activation makes Hsp33 essential for protecting bacteria against severe oxidative stress conditions, such as hypochlorite (i.e., bleach) treatment, which leads to widespread protein unfolding and aggregation. We will compare Hsp33 to the highly abundant eukaryotic typical 2-Cys peroxiredoxin, whose oxidative stress-induced sulfinic acid formation turns the peroxidase into a molecular chaperone in vitro and presumably in vivo. These examples illustrate how proteins use reversible cysteine modifications to rapidly adjust to oxidative stress conditions and demonstrate that redox regulation plays a vital role in protecting organisms against reactive oxygen species-mediated cell death.
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