Lai, YapengWang, JuanXie, NingLiu, GangLacap-Bugler, Donnabella Castillo2025-02-232025-02-232025-02-14Microbiological Research, ISSN: 0944-5013 (Print); 1618-0623 (Online), Elsevier, 294, 128097-128097. doi: 10.1016/j.micres.2025.1280970944-50131618-0623http://hdl.handle.net/10292/18736Myceliophthora thermophila is a thermophilic fungus, known to produce industrially important enzymes in biorefineries. The mechanism underlying cellulase and xylanase expression in filamentous fungi is a complex regulatory network controlled by numerous transcription factors (TFs). These TFs in M. thermophila remain unclear. Here, we identified and characterised a novel cellulase and xylanase regulator MtFKH1 in M. thermophila through comparative transcriptomic and genetic analyses. Five of the eight potential TFs, which showed differential expression levels when grown on Avicel and glucose, were successfully deleted using the newly designed CRISPR/Cas9 system. This system identified the forkhead TF MtFKH1. The disruption of Mtfkh1 elevated the 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. The loss of Mtfkh1 also exhibited an impairment in sporulation in M. thermophila. Real-time quantitative reverse transcription PCR (RT-qPCR) and the electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 regulates the gene expression and specifically bind to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050), respectively. Furthermore, DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, with 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 expand our knowledge on how forkhead transcription factor regulates lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulase and xylanase.© 2025 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).http://creativecommons.org/licenses/by/4.0/CellulaseComparative transcriptomeCRISPR/Cas9Forkhead transcription factorMyceliophthora thermophilaXylanaseCRISPR/Cas9CellulaseComparative transcriptomeForkhead transcription factorMyceliophthora thermophilaXylanase31 Biological Sciences3106 Industrial BiotechnologyBiotechnologyHuman GenomeGenetics2.1 Biological and endogenous factors0605 Microbiology1108 Medical MicrobiologyMicrobiology3107 MicrobiologyJournal ArticleOpenAccess10.1016/j.micres.2025.128097