Deletion of homologs of the SREBP pathway results in hyper-production of cellulases in Neurospora crassa and Trichoderma reesei

The filamentous fungus Neurospora crassa efficiently utilizes plant biomass and is a model organism for genetic, molecular and cellular biology studies. Here, a set of 567 single-gene deletion strains was assessed for cellulolytic activity as compared to the wild-type parental strain. Mutant strains included were those carrying a deletion in: (1) genes encoding proteins homologous to those implicated in the Saccharomyces cerevisiae secretion apparatus; (2) genes that are homologous to those known to differ between the Trichoderma reesei hyper-secreting strain RUT-C30 and its ancestral wild-type strain; (3) genes encoding proteins identified in the secretome of N. crassa when cultured on plant biomass and (4) genes encoding proteins predicted to traverse the secretory pathway.

These studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.

The 567 single-gene deletion collection was cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild-type strain resulted in the identification of seven hyper-production and 18 hypo-production strains. Some of these deleted genes encoded proteins that are likely to act in transcription, protein synthesis and intracellular trafficking, but many encoded fungal-specific proteins of undetermined function. Characterization of several mutants peripherally linked to protein processing or secretion showed that the hyper- or hypo-production phenotypes were primarily a response to cellulose. The altered secretome of these strains was not limited to the production of cellulolytic enzymes, yet was part of the cellulosic response driven by the cellulase transcription factor CLR-2. Mutants implicated the loss of the SREBP pathway, which has been found to regulate ergosterol biosynthesis genes in response to hypoxic conditions, resulted in a hyper-production phenotype. Deletion of two SREBP pathway components in T. reesei also conferred a hyper-production phenotype under cellulolytic conditions. Conclusions: These studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.