G-protein signaling components differentially regulate the activity of multiple cellulase enzyme classes in Neurospora crassa.

Heterotrimeric G-protein subunits, adenylyl cyclase, and Regulators of G-protein Signaling (RGS) proteins are required for the conversion of cellulose to glucose in the model filamentous fungus Neurospora crassa. Transcriptomics has revealed roles for several of these genes in the expression of specific cellulase enzyme classes, including endo-β-1,4-glucanases (endoglucanase), exo-β-1,4-glucanases (cellobiohydrolases), β-1,4-glucosidases (β-glucosidases), polysaccharide monooxygenases and cellobiose dehydrogenases. In this study, we measured endoglucanase, cellobiohydrolase and β-glucosidase enzyme activities in secretomes from all G-protein subunit and RGS single mutants, Gα multiple mutants, and the adenylyl cyclase mutant Δcr-1 after transfer from glucose to cellulose medium. We observed that the Gα subunits gna-1 and gna-3 together are required for endoglucanase activity. cr-1 is essential, while gna-2 synergizes with gna-3 to regulate cellobiohydrolase activity. Multiple G-protein genes impacted β-glucosidase activity to some extent, but none were essential for activity. cAMP supplementation of the G protein mutants resulted in the greatest increase in endoglucanase and cellobiohydrolase activity, suggesting an important role for cAMP levels in the activity of these enzyme classes. Among the RGS mutants, the Δrgs-2 strain has greatly reduced activity for all three enzyme classes, like the strain expressing the constitutively active gna-3Q208L allele. In contrast, the Δrgs-1 mutant and the gna-1Q204L strain showed significantly higher endoglucanase activity as compared to wild type. Taken together, our results indicate that G protein signaling components differentially impact the activity of three major cellulase enzyme classes in N. crassa. Our findings will inform future strain engineering approaches to increase production of specific cellulase activities in culture supernatants of filamentous fungi.