The mitochondrial states and activities of production yeasts used in the fermentation industry vary according to the availability of oxygen, size of the fermentation tank and temperature of the raw material. However, the involvement of the mitochondrial states of these yeasts in the production profile of organic acids during alcoholic fermentation has not been investigated in detail. In this study, the effects of the mitochondrial state of a sake brewing yeast on the organic acid production profile during an alcoholic fermentation process were investigated. It was elucidated that the mitochondrial state during the propagation stage significantly affected the mitochondrial morphology and the organic acid production profile during the alcoholic fermentation. When yeast mitochondria were active, they were highly branched in the propagation stage, and the yeast cells produced significantly more succinate and less malate. In contrast, when the yeast mitochondria were inactive, they were long and filamentous in appearance, and the yeast produced significantly less succinate and more malate. The change in malic acid content was reversed when an uncoupler of mitochondrial membrane potential, carbonylcyanide p-trifluoromethoxyphenylhydrazone, was added to the culture, indicating that the change in the organic acid production profile could be attributed to mitochondrial activity. Furthermore, the content of malic acid and succinic acid could be converted from a respirative to a fermentative profile by exposing the yeast to a mitochondrion-inactivating environment for 12 or 24 h. Taken together, it was shown that the mitochondrial status of the yeast affects malic acid production during alcoholic fermentation.

Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.

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Reactive oxygen species (ROS) posed a risk for the transition of vertebrates from aquatic to terrestrial life. How ancestral organisms adapted to such ROS exposure has remained a mystery. Here, we show that attenuation of the activity of the ubiquitin ligase CRL3Keap1 for the transcription factor Nrf2 during evolution was key to development of an efficient response to ROS exposure. The Keap1 gene was duplicated in fish to give rise to Keap1A and the only remaining mammalian paralog Keap1B, the latter of which shows a lower affinity for Cul3 and contributes to robust Nrf2 induction in response to ROS exposure. Mutation of mammalian Keap1 to resemble zebrafish Keap1A resulted in an attenuated Nrf2 response, and most knock-in mice expressing such a Keap1 mutant died on exposure as neonates to sunlight-level ultraviolet radiation. Our results suggest that molecular evolution of Keap1 was essential for adaptation to terrestrial life.

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