Improved recombinant protein production and scale-up fermentation of Aspergillus oryzae hyphal-dispersion hydrophobin-deficient strain

In liquid fermentation of filamentous fungi such as Aspergillus oryzae , increased broth viscosity and biomass adhesion to bioreactor surfaces remain major challenges. We previously developed a hyphal dispersion mutant lacking two hyphal adhesion factors, namely, cell wall α-1,3-glucan (AG) and biofilm galactosaminogalactan (GAG) (AGΔ-GAGΔ strain). The culture broth of the AGΔ-GAGΔ strain has low viscosity, which improves mixing and enzyme production. However, mycelia still extensively attach to bioreactor walls and downstream equipment, which impairs mixing and reduces product recovery. The hydrophobin RolA, a surface-active protein of A . oryzae , densely coats conidia and hyphae and contributes to cell surface hydrophobicity. In this study, we disrupted the rolA gene in AGΔ-GAGΔ (AGΔ-GAGΔ-Δ rolA strain) and evaluated the effects of this disruption on hyphal adhesion to the walls of culture vessels, enzyme production, and bioreactor performance. At the flask scale, the adhesion to glass surfaces was significantly reduced and activity of recombinant xylanase was increased by 14%. Improved culture recovery at the end of fermentation further increased total enzyme yield. In a lab-scale stirred-tank bioreactor (4 L), both growth and enzyme production were increased. In a 200-L bioreactor, xylanase activity was also higher in AGΔ-GAGΔ-Δ rolA and computational fluid dynamics (CFD) analysis predicted improved hydrodynamic properties at equivalent volumetric power consumption. Fermentation of AGΔ-GAGΔ-Δ rolA was successfully scaled up to a 3000-L bioreactor. Overall, the AGΔ-GAGΔ-Δ rolA strain has increased enzyme production and scalability, supporting its suitability for industrial applications.