Identification of an NDT80/PhoG transcription factor TrNf1 and boosting cellulase expression by combining its deletion with xyr1overexpression in Trichoderma reesei

The filamentous fungusTrichoderma reeseiserves as an industrial workhorse for production of cellulolytic enzymes. However, the regulatory network governing cellulase biosynthesis inT. reeseiremains incompletely understood, which limits rational engineering towards obtaining hyper-producing strains. Herein, a previously uncharacterized NDT80/PhoG family transcription factor (TrNf1) was identified and characterized as a negative regulator of cellulase expression. Deletion of TrNf1led to increases in the concentration of total secreted protein and overall cellulase activity from 0.017 mg/ml to 0.071 mg/ml and from 0.15 U/ml to 0.46 U/ml, corresponding to 4.2-fold and 3.1-fold, respectively. Deletion of TrNf1 induced global transcriptional reprogramming by activating a large array of carbohydrate-active enzyme (CAZyme) genes, remodeling the transcriptional regulatory network, and upregulating key activators such as xyr1 and ace3. Overexpressingxyr1in theTrNf1-deletion strain substantially improved overall cellulase production from 0.46 U/mL to 1.46 U/mL, which corresponded to a 3.2-fold increase compared with ΔTrNf1 and a 9.7-fold increase compared with TU-6. Importantly, the repressive function is evolutionarily conserved inAspergillus nidulans, where the homolog of TrNf1, i.e. AnNf1, similarly suppresses cellulase production. Collectively, our findings uncover a novel negative regulatory role for an NDT80/PhoG protein in fungal cellulase production and provide a conserved candidate target that can be tried in future strain engineering.