Transcription factor MtCLR-2 regulates cellulase production via direct modulation of Mtegl2 and Mtbgl1 expression in Myceliophthora thermophila

The thermophilic fungus Myceliophthora thermophila can secrete large amounts of lignocellulolytic enzymes, such as cellulases and xylanases, which are regulated by multiple transcription factors. However, the understanding of the regulatory mechanism of cellulase gene expression in M. thermophila is limited. Here, we characterized the function of MtCLR-2, a M. thermophila ortholog of CLR-2, a key cellulolytic transcriptional regulator initially identified in Neurospora crassa. Deletion of Mtclr-2 significantly reduced cellulase activities, particularly affecting endoglucanase production, whereas overexpression of Mtclr-2 led to elevation in cellulase secretion when M. thermophila was grown on Avicel. Subcellular localization assay of MtCLR-2 fused to green fluorescent protein (GFP) indicated that MtCLR-2 is localized to the nucleus. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis revealed that disruption of Mtclr-2 caused a decrease in transcript levels of the β-glucosidase gene bgl1 (MYCTH₆6804) and the endoglucanase gene egl2 (MYCTH₈6753) throughout the stages of growth in cellulose medium. Furthermore, electrophoretic mobility shift assays (EMSAs) demonstrated that MtCLR-2 directly binds to the promoter regions of bgl1 and egl2 in a zinc-dependent manner. The comparative transcriptomic analysis also showed that MtCLR-2 positively regulates the expression of ribosomal protein genes under cellulosic conditions. These findings contribute to a better understanding of the regulatory network governing cellulase gene expression and provide a potential target for boosting cellulase biosynthesis in M. thermophila.