Effects of Aspergillus oryzae -derived rice- koji protein on the sake metabolome

Rice- koji is produced by cultivating Aspergillus oryzae on steamed rice and plays a central role in the production of traditional Japanese alcoholic beverages such as sake and shochu, as well as fermented seasonings like miso and soy sauce. While some functions of the proteins produced by A. oryzae during rice- koji fermentation have been clarified, the comprehensive protein profile and their specific roles in sake brewing are still not fully understood. We hypothesized that proteins expressed by A. oryzae in rice- koji contribute in diverse ways to shaping the metabolic characteristics of sake. In this study, we performed proteomic analyses on four types of rice- koji to explore rice- koji proteins (RKPs) and identified 159 rkp genes. From this set, selected targets were used to construct single-gene disruptants (Δ rkp ), resulting in the generation of 85 distinct Δ rkp strains. Sake brewing experiments using these disruptants were carried out to assess the impact of individual rkp gene deletions. The results revealed alterations in the mycelial biomass of A. oryzae in rice- koji and showed clear associations with variations in multiple sake components. Further metabolomic analysis demonstrated that disruption of 57 rkp genes led to notable changes in at least one sake-derived metabolite peak. Overall, our findings indicate that RKPs identified through proteomic analysis of rice- koji exert a wide range of effects on both the growth of A. oryzae and the metabolite composition of sake. These insights are expected to advance our understanding of how RKPs influence sake brewing and the broader biological roles of A. oryzae . IMPORTANCE Rice-koji plays a crucial role in sake brewing. Although some functions of Aspergillus oryzae –derived proteins have been elucidated, most remain poorly understood. In this study, we identified rice-koji proteins (RKPs) via proteomic analysis and investigated their effects on sake composition using gene disruption strains. Analyses of samples from rice-koji preparation and small-scale sake brewing showed that disruption of rkp genes altered A. oryzae growth and modified multiple sake component profiles. Notably, rkp genes that influenced fungal biomass in rice-koji were also associated with significant changes in sake composition. These findings reveal the diverse contributions of A. oryzae to sake brewing and enhance our understanding of the fermentation potential of filamentous fungi, with potential applications in other fermentation-based industries.