KIN-1 Function in Neurospora crassa Revealed by Constitutive clr-2 Expression and Cross-Species Complementation

Conventional kinesin contributes to fungal polarized growth, but its role in cellulase production remains unclear. We examined the effects of kin-1 deletion, constitutive clr-2 expression, and complementation with the native gene or a Trichoderma reesei gene in Neurospora crassa. Deletion of kin-1 produced compact colonies, increased branching in the hyphae examined, and reduced extension rates without reducing biomass in sucrose cultures. Under Avicel conditions, the deletion strain showed reduced extracellular protein accumulation and lower cbh-1 and clr-2 transcript levels. Introduction of a clr-2 expression cassette into the deletion background increased the transcript levels of both genes relative to Δkin-1 and increased extracellular protein and cellulase activities under both Avicel and sucrose conditions. At a fixed total protein loading of 0.1 mg per saccharification reaction, performance varied with the carbon source used for enzyme production and the straw substrate. Heterologous complementation alleviated the growth defects. On day 7 of Avicel cultivation, extracellular protein normalized to intracellular protein was lower in the deletion strain and was restored to near-WT levels in both complemented strains. These results link KIN-1-dependent morphogenesis to the production of cellulolytic products and show that constitutive clr-2 expression can alleviate some phenotypes caused by kin-1 deletion. The extent to which the tested T. reesei gene substitutes for the native gene depends on the phenotype. This study does not establish direct cellulase transport by KIN-1, a change in secretion rate per unit biomass, or complete restoration of secretion.