Isolation and characterization of koji mold (Aspergillus oryzae) from nature in Niigata

Koji mold (Aspergillus oryzae) is a key microorganism in brewing and fermentation in Japan. We isolated koji molds from the environment in Niigata Prefecture. Eighty-one environmental samples were placed on isolation medium made from steamed rice with wood ash and 36 Aspergillus section Flavi-like strains were obtained. Of those, 26 strains did not produce aflatoxin. We studied their morphology, sequence of ITS region, calmodulin gene, aflatoxin biosynthetic homologous gene cluster and α-amylase gene and fermentation-related enzyme activities. Furthermore, DNA-seq analysis of 14 strains from 26 non-aflatoxin producing strains were conducted and compared the three mycotoxin biosynthetic gene clusters (aflatoxin, cyclopiazonic acid, and aflatrem) and fermentation-related genes against those of reference strain A. oryzae RIB40. In some strains, gene sequences confirmed the absence of mycotoxin production, but differences in fermentation-related enzyme activities could not be explained well by amino acid substitutions. We classified the 26 isolates into 6 morphology types based on the appearance of colonies and mating types, and it was found that strains of the same morphology type had similar enzymatic profiles and gene sequences. Our results show that koji molds with various properties occur in the environment, and it will expand the possibilities of koji mold in industrial use.