Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening

Background: Lignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei, but the underlying mechanism associated with this process is not well understood. Although previous comparative genomic studies have revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations compared with its parental strain QM6a, how these mutations actually contribute to the hypersecretion phenotype remains to be elucidated. Results: In this study, we systematically screened gene knockout (KO) mutants in the cellulolytic fungus Neurospora crassa, which contains orthologs of potentially defective T. reesei RUT-C30 mutated genes. Of the 86 deletion mutants screened in N. crassa, 12 exhibited lignocellulase production more than 25% higher than in the wild-type (WT) strain and 4 showed nearly 25% lower secretion. We observed that the deletion of Ncap3m (NCU03998), which encodes the mu subunit of the adaptor protein 3 (AP-3) complex in N. crassa, led to the most significant increase in lignocellulase secretion under both Avicel and xylan culture conditions. Moreover, strains lacking the beta subunit of the AP-3 complex, encoded by Ncap3b (NCU06569), had a similar phenotype to Delta Ncap3m, suggesting that the AP-3 complex is involved in lignocellulase secretion in N. crassa. We also found that the transcriptional abundance of major lignocellulase genes in Delta Ncap3m was maintained at a relatively higher level during the late stage of fermentation compared with the WT, which might add to the hypersecretion phenotype. Finally, we found that importation of the T. reesei ap3m ortholog Trap3m into Delta Ncap3m can genetically restore secretion of lignocellulases to normal levels, which suggests that the effect of the AP-3 complex on lignocellulase secretion is conserved in cellulolytic ascomycetes. Conclusions: Using the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Through systematic genetic screening of 86 corresponding orthologous KO mutants in N. crassa, we identified several genes, particularly those encoding the AP-3 complex that contribute to lignocellulase secretion. These findings will be useful for strain improvement in future lignocellulase and biomass-based chemical production.