I studied at the Sleep and Circadian Neurobiology Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, from April 2018 to March 2020. At Stanford University, I mainly researched the following themes: (1) sleep basic research using mice (administering compounds with sleep or wakefulness effects to mice and examining their effects), and (2) research on circadian rhythm disorders. There are only a few institutions in the world that can conduct sleep basic research using mice, and Stanford University is a wonderful environment to immerse yourself in research, as it is home to not only psychiatrists but also neurologists and many basic researchers. In this article, I would like to review the experiments I conducted during my study abroad, using mice to verify the effects of natural compounds on wakefulness or sleep. In one study, we evaluated the effects of ginkgolides (A, B, and C) and bilobalide on arousal, locomotion, and core body temperature. The results showed that only ginkgolide B dose-dependently increased the amount of arousal and decreased the amount of NREM sleep in the physiological sleep-wake cycle of mice. In another study, we tested the sleep-inducing effects of sake yeast in mice under an acute insomnia model. We showed that sake yeast dose-dependently increased REM and non-REM sleep, decreased arousal within 6 hours after oral administration of sake yeast, and decreased locomotion and core body temperature in a new cage.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Modification of the genetic background and, in some cases, the introduction of targeted mutations can play a critical role in producing trait characteristics during the breeding of crops, livestock, and microorganisms. However, the question of how similar trait characteristics emerge when the same target mutation is introduced into different genetic backgrounds is unclear. In a previous study, we performed genome editing of AWA1, CAR1, MDE1, and FAS2 on the standard sake yeast strain Kyokai No. 7 to breed a sake yeast with multiple excellent brewing characteristics. By introducing the same targeted mutations into other pedigreed sake yeast strains, such as Kyokai strains No. 6, No. 9, and No. 10, we were able to create sake yeasts with the same excellent brewing characteristics. However, we found that other components of sake made by the genome-edited yeast strains did not change in the exact same way. For example, amino acid and isobutanol contents differed among the strain backgrounds. We also showed that changes in yeast cell morphology induced by the targeted mutations also differed depending on the strain backgrounds. The number of commonly changed morphological parameters was limited. Thus, divergent characteristics were produced by the targeted mutations in pedigreed sake yeast strains, suggesting a breeding strategy to generate a variety of sake yeasts with excellent brewing characteristics.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Sake yeast is one of the important factors that characterize the aroma and taste of sake. To obtain sake yeast strains with different metabolic capabilities from other strains, breeding of a sake yeast is an effective way. In this study, sake yeast strain Y5201 was mutagenized by synchrotron light irradiation to obtain the mutant strains showing different brewing characteristics from parental strain Y5201, and comparative genome analysis between strain Y5201 and mutant strains was performed to identify mutation points and patterns induced by synchrotron light irradiation. Screening with the drug-resistant and fermentation tests selected the nine mutants (C18, C19, C29, C50, C51, C52, C54, T25, and T49) from the mutagenized Y5201 cells. Principal component analysis results based on the analysis of the small-scale brewing test metabolites showed that the mutant strain C19 was different from other strains, which had higher productivity of ethyl caproate and isoamyl acetate than those of the Y5201. Comparative genome analysis revealed that mutants by synchrotron light irradiation had a higher diversity of single nucleotide substitutions and a higher frequency of Indel (insertion/deletion) in these DNA than ethyl methanesulfonate and UV irradiation. These results suggest that synchrotron light irradiation is an effective and unique mutagen for yeast breeding.

Sake yeast is mostly diploid, so the introduction of recessive mutations to improve brewing characteristics requires considerable effort. To construct sake yeast with multiple excellent brewing characteristics, we used an evidence-based approach that exploits genome editing technology. Our breeding targeted the AWA1, CAR1, MDE1, and FAS2 genes. We introduced eight mutations into standard sake yeast to construct a non-foam-forming strain that makes sake without producing carcinogens or an unpleasant odor, while producing a sweet ginjo aroma. Small-scale fermentation tests showed that the desired sake could be brewed with our genome-edited strains. The existence of a few unexpected genetic perturbations introduced during breeding proved that genome editing technology is extremely effective for the serial breeding of sake yeast.

The mechanism of pyruvate-underproduction of aneuploid sake yeast was investigated in this study. In our previous report, we revealed that an increase in chromosome XI decreases pyruvate productivity of sake yeast. In this report, we found that increased copy number of CCP1, which is located on chromosome XI and encodes cytochrome-c peroxidase, decreased the pyruvate productivity of sake yeasts. Introducing an extra copy of CCP1 activated respiratory metabolism governed by Hap4 and increased reactive oxygen species. Therefore, it was concluded that increased copy number of CCP1 on chromosome XI activated respiratory metabolism and decreased pyruvate levels in an aneuploid sake yeast. This is the first report that describes a mechanism underlying the improvement of brewery yeast by chromosomal aneuploidy.

We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4-5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Sake is the national alcoholic beverage of Japan, and its history can be traced back more than 1300 years. With the development and maturity of the sake-brewing technique, a unique flavor and taste gradually formed, which led to its wide use in Japan and internationally. This paper reviews and discusses the research advances of sake rice, koji, and sake yeast. The various enzymes and involved genes of microbes in the rice koji, and the separation/breeding of sake yeasts are expounded particularly. Moreover, the fields where further research is required are presented. Therefore, this review presents recent comprehensive research details of sake\'s ingredients and the involved study perspectives.