Discovery of Putative Phosphorylation Sites in Cre1 and Ace1 as Engineering Targets for Enhanced Cellulase Production in Trichoderma reesei

In the filamentous fungus Trichoderma reesei, cellulase production is subject to carbon catabolite repression (CCR) mediated by the key repressors Cre1 and Ace1. In this study, we identified two previously unreported phosphorylation sites-S204 in Cre1 and S145 in Ace1-through phosphoproteomic profiling. Site-directed mutagenesis revealed that dephosphorylation-mimicking mutations (Cre1S204A and Ace1S145A) significantly relieved CCR and promoted cellulase biosynthesis, while phosphomimetic mutations exerted opposite effects. Notably, the Cre1S204A/Ace1S145A double mutant exhibited a combined enhancement in cellulase activity, achieving a 0.7-fold increase in pNPCase activity compared to the parental strain, along with substantially upregulated cellulase gene transcription and increased extracellular CBH1 accumulation. These results demonstrate that S204 and S145 are functionally relevant phosphorylation sites that negatively modulate cellulase production. Our findings provide novel engineering targets for strain improvement and, more broadly, open new avenues for investigating the phosphorylation-based regulatory mechanisms of Cre1 and Ace1 in future studies. From an applied perspective, the work offers a straightforward and effective genetic strategy for the rational design of high-performance fungal strains for lignocellulose bioconversion.