Unlocking the endoplasmic reticulum ATP importer TrAXER as a new genetic engineering target Enhances Trichoderma reesei cellulase production and cellulose saccharification

Trichoderma reesei has long been established as a prominent industrial platform for cellulase production. However, functionally validated targets for iteratively engineering T. reesei to boost cellulase yield remain insufficient. Herein, the T. reesei orthologue of the human endoplasmic reticulum (ER) ATP importer, designated TrAXER, was identified. TrAXER absence caused defects in fungal tip growth and resulted in a more than 95% reduction in cellulase production, together with a pronounced activation of ER stress response. Appropriate overexpression of TrAXER led to a 22%-38% increase in extracellular cellulase activity. Notably, combinatorial overexpression of TrAXER and the master transcriptional activator Xyr1 not only mitigated the adverse growth phenotypes and ER stress triggered by standalone overexpression of Xyr1, but also enhanced cellulase biosynthesis with a 203% increase in the final extracellular filter paper hydrolase activity at 96 h relative to the parental strain. Transcriptomic profiling revealed that while the main cellulase and hemicellulase genes exhibited significantly downregulated expression in ΔTraxer, Traxer absence led to an upregulation of genes mainly enriched in categories of vesicle-mediated transport, vesicles and response to ER stress. In contrast, TrAXER overexpression in the OExyr1 strain further increased expression of cellulase and hemicellulase genes, but decreased those involved in proteasome assembly and ubiquitin-dependent protein catabolic process. Moreover, the cellulase cocktail derived from the dual engineered strain exhibited greater saccharification capacity on cellulose substrates. These findings highlight the functional significance of AXER orthologue in filamentous fungi, and also established TrAXER as an effective engineering target for construction of cellulase hyperproduction strains.