Identification of a novel forkhead transcription factor MtFKH1 for cellulase and xylanase gene expression in Myceliophthora thermophila ATCC 42464

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. Transcription factors (TFs) play a pivotal regulatory role in fugal lignocellulolytic enzymes synthesis. We speculated that TFs may be responsible for significantly improved cellulase and xylanase activities of M. thermophila that observed under Avicel culture compared to glucose condition. Therefore, the transcriptomes of M. thermophila wild type (WT) cultured on Avicel (A_RNA) and Glucose (G_RNA) at 36 hours were screened for potential regulators of cellulolytic and xylanolytic genes expression. All RNA sequencing data were generated from three biological replicates.