Phenotypic, Genomic, and Transcriptomic Comparison of Industrial Aspergillus oryzae Used in Chinese and Japanese Soy Sauce: Analysis of Key Proteolytic Enzymes Produced by Koji Molds.

Aspergillus oryzae, which generates numerous enzymes for the breakdown of raw materials, is an essential koji mold in soy sauce production. For better soy sauce productivity and flavor quality, China and Japan have developed their own industrial A. oryzae strains at distinct evolutionary branches for use in soy sauce production for decades. However, systematic comparison between the two national industrial strains has been poorly conducted, and thus we have not been able to generate adequate knowledge, especially regarding what are the key hydrolytic enzymes produced by A. oryzae during koji production. This study sequenced and assembled three high-quality genome sequences of industrial A. oryzae originating from China and Japan. Based on the genome sequences, a phylogenetic tree analysis was performed and revealed the evolutional distances between the two national industrial koji molds. Meanwhile, a comparative phenotypic analysis revealed that the two national industrial strains differed in growth and catalytic characteristics, particularly in proteolytic enzyme activities. To investigate the molecular mechanism underlying the phenotypic difference, we conducted systematic comparative genome and transcriptome investigations. We found minor differences in the quantity and diversity of proteolytic enzyme genes between Chinese and Japanese koji molds, while the protease secretion ratio and transcriptional level were dissimilar. We identified 58 potential important enzymes associated with high protein breakdown efficiency during industrial koji fermentation by combining comparative phenotypic and transcriptome data. More research is required to confirm the function of these putative key hydrolytic enzymes. Aspergillus oryzae is widely used as an industrial koji mold for soy sauce brewing due to its powerful raw material decomposition capability. Although various proteases in A. oryzae have been identified, it remains a challenge to find essential enzymes involved in soy sauce production. Generally, the industrial A. oryzae used in soy sauce brewing has excellent proteolytic activity. Based on this, we analyzed key proteolytic enzymes according to a comparison of the genome and transcriptome between three industrial strains. This study found little difference in gene numbers and mutations of proteolytic enzymes between three industrial A. oryzae strains. However, variations in protease secretion ratio and transcriptome were discovered between industrial strains. Based on that, we generated 58 candidate key proteolytic enzymes. This work comprehensively analyzed three industrial koji molds, revealing genome development under separate artificial domestication and helping in the study of key proteolytic enzymes during soy sauce production.