Presentation Information
[2Brew-13-KL]Isolation, identification, and comparative analysis of Aspergillus oryzae and its related species, Aspergillus flavus, from the nature environment.
○Kanae Sakai1, Ken-Ichi Kusumoto1 (1. The University of Osaka (Japan))
Keywords:
Aspergillus oryzae,Aspergillus flavus,aflatoxin,α-amylase
Aspergillus oryzae is a filament fungi widely used in the brewing industry such as sake, miso, soy sauce. The safety of A. oryzae is well-established through long history of consumption and genome analysis, but very closely related fungus, Aspergillus flavus, is known as aflatoxin producer. A. oryzae and A. flavus share high homology not only in appearance but also in genome sequence, making them indistinguishable by general classification methods such as ITS region sequences. However, in recent years, several efforts have been made to produce fermented foods by newly collecting A. oryzae from nature. Therefore, a simple method for distinguishing between A. oryzae and A. flavus is needed to ensure the safety in the brewing industry in the future. Furthermore, comparing the newly isolated A. oryzae strains from nature with industrial strains is expected to reveal aspects of the domestication process.
In this study, A. oryzae/A. flavus like strains were isolated from soil and rice ears collected in various locations of Japan. Isolated strains were identified using a method newly developed to distinguish between A. oryzae and A. flavus. As a result, 46 A. oryzae strain and 114 A. flavus strains were obtained from nature. In some cases, A. oryzae and A. flavus were isolated from same environmental sample, highlighting the importance of accurate species identification. Fifteen of isolated strains were further confirmed through genome sequencing analysis to verify the reliability of new identification method. From the phylogenetic tree based on the aflatoxin biosynthetic gene cluster homolog sequence, it was found that several A. oryzae strains isolated from nature were located in different clade than the industrial strains. When the copy number of α-amylase gene, which is known to have multiple copies in industrial strains, were examined in isolated strains, most of them had only one copy and the enzymatic activity was low. This suggests the possibility that strains before domestication were exist in nature, and it is expected to provide insights into what happened during domestication process that made the strain suitable for brewing.
In this study, A. oryzae/A. flavus like strains were isolated from soil and rice ears collected in various locations of Japan. Isolated strains were identified using a method newly developed to distinguish between A. oryzae and A. flavus. As a result, 46 A. oryzae strain and 114 A. flavus strains were obtained from nature. In some cases, A. oryzae and A. flavus were isolated from same environmental sample, highlighting the importance of accurate species identification. Fifteen of isolated strains were further confirmed through genome sequencing analysis to verify the reliability of new identification method. From the phylogenetic tree based on the aflatoxin biosynthetic gene cluster homolog sequence, it was found that several A. oryzae strains isolated from nature were located in different clade than the industrial strains. When the copy number of α-amylase gene, which is known to have multiple copies in industrial strains, were examined in isolated strains, most of them had only one copy and the enzymatic activity was low. This suggests the possibility that strains before domestication were exist in nature, and it is expected to provide insights into what happened during domestication process that made the strain suitable for brewing.
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