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.

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.

Project description: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. 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 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. 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. 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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The transcription factor MtCLR-2 regulates cellulase production by directly modulating the expression of Mtegl2 and Mtbgl1 in Myceliophthora thermophila

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