Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, Delta ppoA and Delta ppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the Delta ppoA strain was the same as that produced by the parental strain. In contrast, although the Delta ppoC strain grew on the rice koji, 1-octen-3-ol was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.