Elucidation of the mechanism of excessive heat generation duringkoji making
Osamu MIZUTANIFaculty of Agriculture, University of the Ryukyus
Research objectiveDuring koji fermentation, the koji molds, Aspergillus oryzae and Aspergillus
luchuensis, degrade the starch in steamed rice by producing large amounts of amylolytic
enzymes, and these molds use the degraded products as an energy source. It is generally known
that koji molds emit a large amount of fermentation heat during the rice degradation process.
Although temperature control of koji is important for koji mold growth and enzyme production,
prolonged high temperatures are disadvantageous for koji mold growth. If the fermentation
heat of koji molds is not controlled, excessive temperatures may be reached at which kojimolds cannot grow. Despite this evidence, the mechanism of excessive koji mold heatgeneration is as yet unknown. In this study, to identify the gene responsible for triggering
excessive fermentation heat during koji making, we focused on an alternative oxidase (Aox),
reported to be involved in the generation of fever in plants such as Symplocarpus foetidus (Fig.
1A), and conducted an investigation to clarify the relationship between Aox in koji molds andfermentation heat production.
Methods
Using the known aoxA in Aspergillus niger as a template, we searched the homologous
genes in the genomes of A. oryzae and A. luchuensis. We named the genes AoaoxA and AlaoxA,respectively. These AoaoxA and AlaoxA disruptants were constructed using the Ao∆ligD1) andAl∆ligD2) strains as hosts. We also constructed an AoaoxA-overexpressing strain. To reveal therelationship between Aox and fermentation heat, we made koji using these disruptants and theoverexpressing strain. The fermentation heat generated during koji making was determinedusing a non-airflow box3), which produced uniform conditions in the culture substrate withhigh reproducibility and enabled the control of favorable conditions without agitation of thesubstrate during culture.
Results
1. Construction of AoaoxA and AlaoxA disruptants and observation of these phenotypes
To investigate whether the alternative oxidase, Aox, in A. oryzae and A. luchuensis isinvolved in the production of fermentation heat, we constructed AoaoxA and AlaoxA
disruptants and an AoaoxA-overexpressing strain. Thereafter, we observed the growth
response of the phenotypes of the AoaoxA and AlaoxA disruptants to inhibitors of electrontransport chain complex proteins, such as cyanide. The AoaoxA and AlaoxA disruptants weresensitive to sodium azide, which is an inhibitor of complex protein IV (Fig. 1B). This result
suggests that the AoaoxA and AlaoxA genes encode an alternative oxidase. In contrast, nodifference was observed in the growth of the AoaoxA disruptant compared to the parent strain
under ROS stress and temperature stress.
2. The examination of fermentation heat using AoaoxA and AlaoxA disruptants
To investigate whether AoxA in A. oryzae and A. luchuensis is involved in theproduction of fermentation heat, we made koji using AoaoxA and AlaoxA disruptants and an
AoaoxA-overexpressing strain. The AoaoxA disruptant and the overexpressing strain showeda similar fermentation temperature course compared to that of the parent strain. The amountof fermentation heat produced by the AlaoxA disruptant was initially slightly higher than that
produced by the parent strain; however, after 48 h of incubation, the fermentation heatproduced by the AlaoxA disruptant and the parent strain was similar (Fig. 1C). In addition,
there was no difference in the activity of enzymes such as alpha-amylase and alphaglucosidase in the koji produced by each of the constructed strains compared to that in theparent strain koji.
Conclusion
At present, we have not obtained any data that indicate that the aoxA genes of A. oryzaeand A. luchuensis are involved in the production of fermentation heat. These findings suggestthat factors other than AoxA in A. oryzae and A. luchuensis may be involved in fermentation
heat generation, although AoxA is involved in cyanide-resistant respiration. We will conduct
further research to identify the genes involved in fermentation heat generation in A. oryzaeand A. luchuensis.
References
1) Mizutani, O., et al. (2008) A defect of LigD (human Lig4 homolog) for nonhomologous
end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae.
Fungal Genet. Biol. 45: 878-889.
2) Takahashi, T., Mizutani, O., Shiraishi, Y., and Yamada, O. (2011) Development of anefficient gene-targeting system in Aspergillus luchuensis by deletion of the nonhomologous end joining system. J. Biosci. Bioeng. 112: 529-534.
3) Ito, K., Gomi, K., Kariyama, M., and Miyake, T. (2017) Quantitative evaluation of haze

formation of koji and progression of internal haze by drying of koji during koji making. J.
Biosci. Bioeng. 124: 62-70.
Acknowledgment
We would like to thank Dr. Kazunari Ito for providing the ePTFE membrane.

Fig. 1 (A) Schematic representation of the localization and role of alternative oxidase (Aox)in mitochondria. (B) The phenotype of the AoaoxA disruptant (∆AoaoxA) and the AoaoxAoverexpressing strain (OEAoaoxA) under sodium azide treatment. (C) Fermentation heatgeneration using the AlaoxA disruptant (∆AlaoxA) during koji making.