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

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.

Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion

“…When racA was deleted, the mycelial tip increased by about 20%, the number of secreted vesicles increased, and the secretion of glucoamylase increased 4 times as compared to the wild type strain (Fiedler et al, 2018). Similarly, deletion of racA resulted in a hyperbranched phenotype and three folds increase of cellulase activity in T. reesei (Fitz et al, 2019).…”

Morphological and Metabolic Engineering of Yarrowia lipolytica to Increase β-Carotene Production

“…lipolytica, can switch to either yeast form or pseudohyphae and hyphae when exposed to various extracellular environmental stresses (e.g., temperature, pH, mechanical stress, osmotic pressure, carbon, and nitrogen source). − Industrial fermentations often run under stress conditions, including high temperatures and osmotic pressures or nutrient deficiency, which may induce the switch to hyphal growth . The mycelial morphology affects the viscosity and efficiency of mass and oxygen transfer in the fermentation broth. − Compared with pseudohyphae and hyphae, single yeast forms are easy to manipulate and are metabolically more active than mycelia in submerged cultures . Therefore, cell morphological changes are highly unfavorable for industrial applications.…”

The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

“…Our ndings demonstrated that the Ras small GTPase RSR1 negatively regulates cellulase expression through the ACY1-cAMP-PKA pathway and transcription factors, including cre1, ace3, and xyr1, and that the deletion of RSR1 can increase cellulase production. Previous studies have shown that Ras2 promotes the expression of cellulase genes [37], while rac1 represses it [51], suggesting that the pathway of action of small GTPases is a multichannel circuit. In the future, overexpression of the Ras2 gene or knockout of rac1 in the RSR1 deletion strain will be conducted to determine whether there are interactions between three small GTPases.…”