Enhancing cellulase production in Neurospora crassa through combined deletion of the phospholipase D-encoding gene pla-7 and modulation of transcription factor CLR-2 expression.

Neurospora crassa, a saprophytic fungus, naturally secretes plant cell wall-degrading enzymes, demonstrating strong cellulases production. Despite its century-long use as a model organism, its industrial applications are underexplored. We compared N. crassa with Trichoderma reesei, an industrial workhorse, for cellulases production and lignocellulose degradation. The extracellular protein secretion level of N. crassa WT is significantly higher than that of T. reesei QM6a, indicating industrial potential. However, its mycelial morphology and dependence on insoluble substrates like lignocellulose pose bioreactor challenges. Deleting the phospholipase D gene pla-7 in N. crassa resulted in shorter aerial hyphae, increased branching, and improved biomass on sucrose. Although pla-7 deletion hindered cellulase induction on cellulose in shake flasks, mis-expressing clr-2 restored cellulase production in Δpla-7 strains. Additionally, protein secretion levels in Δpla-7::Mclr-2 strains were approximately doubled on both sucrose and cellulose carbon sources compared to WT::Mclr-2 strains in shake flasks. Furthermore, Δpla-7::Mclr-2 strains demonstrated enhanced fermentation properties in bioreactors using sucrose. These results highlight N. crassa' s industrial promise and provide insights for enhancing production of cellulases in other fungi.