Deletion of the small GTPase rac1 in Trichoderma reesei provokes hyperbranching and impacts growth and cellulase production

Abstract

Abstract Background Trichoderma reesei is widely known for its enormous protein secretion capacity and as an industrially relevant producer of cellulases and hemicellulases. Over the last decades, rational strain engineering was applied to further enhance homologous and heterologous enzyme yields. The introduction of hyperbranching is believed to increase protein secretion, since most exocytosis is located at the hyphal apical tip. There are several genetic modifications which can cause hyperbranching, for example the deletion of the small Rho GTPase rac. Rac plays a crucial role in actin dynamics and is involved in polarisation of the cell during germination and apical extension of the hyphae. Results We deleted rac1 in a T. reesei strain with an ectopically overexpressed endoglucanase, CEL12A, under Pcdna1 control. This deletion provoked a hyperbranching phenotype and strong apolar growth during germination and in mature hyphae. The strains displayed dichotomous branching and shorter total mycelium length with a larger hyphal diameter. Δrac1 strains exhibited a decreased radial growth on solid media. Biomass formation in liquid cultures was carbon source dependent; similar to the reference strain during growth on lactose, increased on d-glucose and slightly enhanced on cellulose. While extracellular cellulase activities remained at parental strain levels on d-glucose and cellulose, the specific activity on lactose cultures was increased up to three times at 72 h accompanied by an upregulation of transcription of the main cellulases. Although the morphology of the Δrac1 strains was considerably altered, the viscosity of the culture broth in fed-batch cultivations were not significantly different in comparison to the parental strain. Conclusions Deletion of the small Rho GTPase rac1 changes the morphology of the hyphae and provokes hyperbranching without affecting viscosity, independent of the carbon source. In contrast, biomass formation and cellulase production are altered in a carbon source dependent manner in the Δrac1 strains.

Keywords

• Trichoderma reesei
• Strain engineering
• Hyperbrancher
• Rac
• Rho GTPase
• Apolar growth