Perturbing glycosylphosphatidylinositol (GPI)-anchor biosynthesis alters cell wall architecture and modulates fungal morphology

Filamentous fungi are widely used for industrial protein production but their tendency to form dense mycelial pellets limits nutrient transfer, oxygen uptake and fermentation efficiency. While cell wall components are known to influence fungal aggregation, the molecular mechanisms linking cell wall biosynthesis to macroscopic morphology remain poorly defined. Using Aspergillus oryzae as a model, we show that perturbation of glycosylphosphatidylinositol (GPI)-anchored protein biosynthesis influences fungal morphology by altering cell wall composition. Disruption of the GPI ethanolamine phosphate transferase 1 (mcd4) or inhibition of glucosaminyl phosphatidylinositol acyltransferase Gwt1 using antifungal drug manogepix (MGX) induced hyper-branching and weakened the cell wall. Notably, chemical inhibition of Gwt1 by MGX causes a marked transition from pelleted to dispersed mycelial growth. Solid-state NMR (ssNMR) analysis revealed reorganisation of galactosaminogalactan (GAG) and galactomannan (GM), including complete loss of cationic galactosamine (GalN), a key determinant of hyphal adhesion. Transcriptomic analysis revealed downregulation of genes involved in somatic cell fusion, linking altered wall composition to impaired germling aggregation. Strikingly, MGX treatment induced distinct morphological outcomes in other industrially relevant fungi, indicating species-specific cell- wall dependencies. Together, these findings identify GPI-anchored proteins as key regulators of fungal morphology and provide a framework for rational morphology engineering to improve industrial fermentation.