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 -1,3-glucan (AG) and biofilm galactosaminogalactan (GAG) (AG{Delta}-GAG{Delta} strain). The culture broth of the AG{Delta}-GAG{Delta} 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 AG{Delta}-GAG{Delta} (AG{Delta}-GAG{Delta}-{Delta}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 recombinant enzyme activity was increased by 10%. Improved culture recovery at the end of fermentation further increased total enzyme yield. In a lab-scale stirred-tank bioreactor, both growth and enzyme production were increased. Scaling-up to a 200-L bioreactor showed reduced agitation power consumption while improving hydrodynamic properties. Fermentation of AG{Delta}-GAG{Delta}-{Delta}rolA was successfully scaled up to a 3000-L bioreactor; consistent enzyme activity and improved flow circulation in the bioreactors were confirmed by computational fluid dynamics analysis. Overall, the AG{Delta}-GAG{Delta}-{Delta}rolA strain has increased enzyme production and scalability, supporting its suitability for industrial applications.