Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed with the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20degreesC for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20degreesC or 30degreesC for 63 d. In the sensory evaluation of the sake stored at 20degreesC or 30degreesC for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.