Functional engineering of the ACE3 C-terminal residue S705 improves cellulase production in Trichoderma reesei.

Improving cellulase production in Trichoderma reesei remains essential for reducing enzyme costs in lignocellulosic biorefineries, whereas effective rational point engineering of ACE3, an essential transcription factor for cellulase production, remains largely unexplored. In this study, we identified serine 705 (S705) in the C-terminal region of ACE3 as an effective engineering site for enhancing cellulase production. Acidic substitutions at S705, such as S705E, severely reduced pNPCase and FPase activities and markedly repressed cellulase-related gene expression. Further analysis showed that the S705E mutation markedly reduced ACE3 protein accumulation, decreased ACE3 occupancy at the cbh1 promoter, and impaired RNA polymerase II recruitment, consistent with an inhibitory effect of a negative charge at this position on ACE3-mediated cellulase activation. In contrast, charge-reversal substitutions with basic amino acids substantially improved cellulase production. S705H/K/R mutations further increased cellulase activities based on the hypercellulolytic ACE3 truncation, with the S705R mutant showing the strongest enhancement. These positive substitutions strengthened ACE3-mediated transcriptional activation, leading to increased xyr1 expression and broad upregulation of cellulase- and hemicellulase-related genes. Electrostatic surface analysis showed that S705 substitutions markedly altered the local surface charge of ACE3.Importantly, this strategy was transferable to industrial T. reesei strains in a background-dependent manner. The optimized SSII-S705H strain showed robust cellulase production in a 30-L fermenter, with extracellular protein concentration reaching approximately 125 g/L. Overall, these results demonstrate that charge-based engineering of ACE3 S705 is an effective strategy for enhancing industrial cellulase-production performance in T. reesei.