Using pure oxygen aeration to increase recombinant protein production by an Aspergillus oryzae hyphal dispersion...

A high growth rate is essential for increasing protein production efficiency in liquid fermentation of filamentous fungi, such as Aspergillus oryzae. However, the increase in culture viscosity due to fungal growth constrains the overall yield. We have demonstrated that culture viscosity is lower in A. oryzae AGΔ-GAGΔ strains, which are deficient in the cell surface polysaccharides α-1,3-glucan (AG) and galactosaminogalactan (GAG), than in the wild-type (WT) strains. Nevertheless, even in aerated fermentation, an increase in AGΔ-GAGΔ viscosity results in oxygen depletion, which limits fungal growth and enzyme production. In this study, we investigated viscosity dynamics and protein production during high-cell-density fermentation of AGΔ-GAGΔ under pure oxygen aeration. Fed-batch cultivation of the WT and AGΔ-GAGΔ strains, expressing recombinant xylanase (XynF1), was used to compare the effects of air and pure oxygen aeration at the same flow rate. At 60 h, AGΔ-GAGΔ under pure oxygen aeration showed higher cell density (1.2× WT under pure oxygen aeration, 2.1× AGΔ-GAGΔ under air aeration) and XynF1 activity (1.8× WT under pure oxygen aeration, 2.3× AGΔ-GAGΔ under air aeration). Under pure oxygen aeration, AGΔ-GAGΔ showed lower viscosity (0.32×) and mixing time (0.50×) than WT. At 60 h, fine mycelial pellets (micropellets; 200-700 μm) were clearly observed in AGΔ-GAGΔ under pure oxygen but not under air aeration. These findings suggest that oxygen enrichment during AGΔ-GAGΔ cultivation mitigated the increase in viscosity, thereby promoting higher cell density and protein production. The formation of micropellets in AGΔ-GAGΔ likely contributed to a reduction in culture viscosity.