Isolation of a deuA-deficient strain of Aspergillus oryzae and its application to soy sauce fermentation

The thermostable protease DeuA from Aspergillus oryzae contributes to undesirable proteolysis in processed foods prepared with heated soy sauce. To obtain a deuA-deficient A. oryzae strain without recombinant DNA techniques or genome editing, we developed a high-throughput screening method utilizing the bacteriostatic effect of protamine on Latilactobacillus sakei. Loss of DeuA activity reduced protamine degradation, thereby suppressing bacterial acid production and enabling rapid identification of mutants. Screening of approximately 20,000 UV-mutagenized colonies from the industrial strain A. oryzae No. 110 yielded four candidates, of which strain M4 was selected because it retained growth and neutral protease activity comparable to those of the wild-type strain while showing markedly reduced thermostable protease activity. DNA sequence analysis of M4 revealed a 4-bp deletion in deuA, which introduced a frameshift and a premature stop codon. In a soy sauce-marinated egg white model, samples treated with M4-derived soy sauce showed markedly higher hardness than those treated with soy sauce brewed with the wild-type strain. Sensory evaluation also showed a clear preference for samples treated with M4-derived soy sauce. Soy sauce brewed with M4 showed general chemical properties comparable to those of soy sauce brewed with the wild-type strain. These findings demonstrate that DeuA strongly influences texture deterioration in processed foods and that the developed screening strategy provides an effective conventional mutagenesis-based approach for producing soy sauce with reduced proteolytic activity.