Functional characterization of CEL3C reveals its critical role in regulating cellulase gene expression in Trichoderma reesei Rut C30

The nuclear-localized beta-glucosidase CEL3C in Trichoderma reesei plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain T. reesei Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28% (p < 0.05), with beta-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97%, 19.40%, and 28.99%, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and beta-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6% increase), ACE3 (75.49% increase), and RXE1 (161.56% increase), suppression of repressors RCE1 (65.86% decrease) and RCE2 (65.23% decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54% decrease). Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering T. reesei with optimized industrial cellulase yields.