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Affiliation |
Faculty of Agriculture Department of Biochemistry and Applied Biosciences |
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Assistant Professor |
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External Link |
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KIYOSHI Keiji
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Degree 【 display / non-display 】
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醸造学 ( 2016.3 東京農業大学 )
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Fermentation technology ( 2012.3 Tokyo University of Agriculture )
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Fermentation technology ( 2010.3 Tokyo University of Agriculture )
Research Areas 【 display / non-display 】
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Life Science / Applied microbiology
Papers 【 display / non-display 】
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Physiological role of the EHL gene in sake yeast and its effects on quality of sake. Reviewed
Tomonaga K, Tanaka J, Kiyoshi K, Akao T, Watanabe K, Kadokura T, Nakayama S
Journal of bioscience and bioengineering 137 ( 3 ) 195 - 203 2024.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Bioscience and Bioengineering
The EHL1/2/3 genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast Saccharomyces cerevisiae. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed ehl1/2/3 mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the EHL1/2/3 genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the EHL1/2/3 genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the ehl1/2/3 mutant was comparable with that of strain H3, but succinate production was decreased in the ehl1/2/3 mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the ehl1/2/3 mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the EHL1/2/3 genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.
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Furuya K, Kiyoshi K, Punjuy C, Yoshida N, Maruyama R, Yasuda T, Watanabe K, Kadokura T, Nakayama S
Journal of bioscience and bioengineering 136 ( 3 ) 198 - 204 2023.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Bioscience and Bioengineering
Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564) is a butanol-producing strain suitable for application to butanol production from cellulosic materials by co-culture with cellulolytic and thermophilic species, such as Hungateiclostridium thermocellum (synonym: Clostridium thermocellum). The optimal temperature for butanol production by strain N1-4 is 30 °C, and the strain is sensitive to a high culture temperature of 37 °C. Given that spore formation is observed at high frequency when strain N1-4 is cultivated at 37 °C, we assumed in a previous study that the initiation of sporulation is related to a decrease in butanol production. Therefore, to investigate the relationship between butanol production and spore formation, we generated strain N1-4 isolates in which genes related to spore formation were disrupted. The sporulation-related gene disruptants of spo0A, sigE, sigG, and sigK lost the ability to produce heat-resistant spores, irrespective of the culture temperature. Among the gene disruptants produced, only the spo0A disruptant lost butanol-producing ability when cultivated at 30 °C. Interestingly, the sigE disruptant maintained butanol productivity similar to that observed at 30 °C, even when cultivated at 37 °C. In addition, the sigE disruptant successfully produced butanol from Avicel cellulose by co-culture with H. thermocellum at a fermentation temperature of 37 °C.
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Takase S, Tomonaga K, Tanaka J, Moriya C, Kiyoshi K, Akao T, Watanabe K, Kadokura T, Nakayama S
Journal of bioscience and bioengineering 136 ( 1 ) 44 - 50 2023.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Bioscience and Bioengineering
Biotin is an essential coenzyme that is bound to carboxylases and participates in fatty acid synthesis. The fact that sake yeast exhibit biotin prototrophy while almost all other Saccharomyces cerevisiae strains exhibit biotin auxotrophy, implies that biotin prototrophy is an important factor in sake brewing. In this study, we inserted a stop codon into the biotin biosynthetic BIO3 gene (cording for 7,8-diamino-pelargonic acid aminotransferase) of a haploid sake yeast strain using the marker-removable plasmid pAUR135 and investigated the fermentation profile of the resulting bio3 mutant. Ethanol production was not altered when the bio3 mutant was cultured in Yeast Malt (YM) medium containing 10% glucose at 15 °C and 30 °C. Interestingly, ethanol production was also not changed during the sake brewing process. On the other hand, the levels of organic acids in the bio3 mutant were altered after culturing in YM medium and during sake brewing. In addition, ethyl hexanoate and isoamyl acetate levels decreased in the bio3 mutant during sake brewing. Metabolome analysis revealed that the decreased levels of fatty acids in the bio3 mutant were attributed to the decreased levels of ethyl hexanoate. Further, the transcription level of genes related to the synthesis of ethyl hexanoate and isoamyl acetate were significantly reduced. The findings indicated that although the decrease in biotin biosynthesis did not affect ethanol production, it did affect the synthesis of components such as organic acids and aromatic compounds. Biotin biosynthesis ability is thus a key factor in sake brewing.
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Alawi A., Kiyoshi K., Matsumoto M., Yamaguchi T., Narita T., Morita T., Suzuki T., Nakajima-Kambe T.
Bioresource Technology Reports 20 2022.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Bioresource Technology Reports
In this study, a promising bacterial strain for the bio-production of 1,3-propandeiol was isolated as a contaminant from a pilot-scale ethanol-producing bioreactor that used glycerol as a substrate. The isolated strain was identified as Citrobacter braakii and named TB-96. As TB-96 showed a fast growth rate, had strong antibiotic resistance, and could assimilate glycerol to produce high concentrations of 1,3-propanediol, it was used for further investigation to promote the biorefining of waste glycerol generated from biodiesel fuel production. First, to increase the 1,3-propanediol productivity of TB-96, the whole genome was sequenced, and the metabolic pathways were investigated. Next, the synthesis pathways of the main by-products—lactate and formate—were disrupted to construct the strain TB-96-dLF. In fed-batch experiments, TB-96-dLF produced up to 60 g/L 1,3-propanediol using only corn steep liquor as a low-cost nitrogen source.
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醸造における木桶の温故知新 Invited
清 啓自
生物工学会誌 99 ( 12 ) 639 - 639 2021.12
Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人 日本生物工学会
Presentations 【 display / non-display 】
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Isolation of ammonia gas obligately requiring actinomycete, Nocardiopsis sp., and its growth property
Naoto Yoshida, Keiji Kiyoshi
2024.3.25
Event date： 2024.3.24 - 2024.3.27
Language：Japanese Presentation type：Oral presentation (general)
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Effect of electro-fermentation on Citrobacter braakii TB-96 strain and construction of new culture system through application
Takuma Yanase, Kengo Inoue, Naoto Yoshida, Toshiaki Nakjima-Kanbe, Keiji Kiyoshi
2024.3.25
Event date： 2024.3.24 - 2024.3.27
Language：Japanese Presentation type：Oral presentation (general)
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High-expression and stable promoter selection in the presence of glycerol in Citrobacter braakii strain TB-96
Takezo Kimura, Rei Sameshima1, Takuma Yanase, Naoto Yoshida, Toshiaki Nakajima-Kambe, Keiji Kiyoshi
2024.3.26
Event date： 2024.3.24 - 2024.3.27
Language：Japanese Presentation type：Oral presentation (general)
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Citrobacter braakii TB-96株におけるグリセロール存在下での 高発現プロモーターの選抜
木村 武蔵, 鮫島 礼 柳瀬 卓馬, 吉田 ナオト, 清 啓自
第29回九州支部 福岡大会 2023.12.3
Event date： 2023.12.2 - 2023.12.3
Language：Japanese Presentation type：Oral presentation (general)
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醤油醸造における木桶仕込みよる菌叢と呈味・香気成分への影響
吉田 栞, 森田 浩平, 坂口直人, 吉田ナオト, 清 啓自
令和5年度日本醸造学会大会 2023.10.5
Event date： 2023.10.4 - 2023.10.5
Language：Japanese Presentation type：Oral presentation (general)
Grant-in-Aid for Scientific Research 【 display / non-display 】
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微生物の発酵をコントロールする栄養たち
Grant number：23HT0192 2023.04 - 2024.03
独立行政法人日本学術振興会 科学研究費補助金 ひらめきときめきｻｲｴﾝｽ
Authorship：Principal investigator
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核酸代謝の増強が環境ストレスへの対応に及ぼす影響
Grant number：21K14773 2021.04 - 2024.03
独立行政法人日本学術振興会 科学研究費補助金 若手研究
Authorship：Principal investigator
全ての生物は自身の周辺環境から受けるストレスに適応しながら活動、生存し繁殖していく。環境から受けるストレスとは避けられないものであり、そこへ如何に適応できるのかが結果として生物の
種の強さを決定づけているといえる。これは単細胞生物である微生物においては尚更であり、特に発酵に用いられる微生物では如何にこのストレスによる発酵の低下を避けるかは生産性を左右する極めて重要な課題である。外的ストレスが過剰であると微生物は休眠化あるいは細胞死が生じる。
本研究では発酵性の微生物において申請者がこれまでに発見した培地による核酸供給による
熱ストレス耐性の知見を基に、ストレス環境下における核酸などの主要代謝物の外的な供給および生合成の強化によって、いかなるストレスに対してもストレス耐性を付与することが可能であるかを分子生物学的アプローチによって試みる。 -
バイオ燃料廃棄物系バイオマスからポリマー原料への微生物転換
Grant number：99999999 2017.04 - 2019.03
科学研究費補助金 科学技術振興機構 先端的技術開発
Authorship：Coinvestigator(s)
Available Technology 【 display / non-display 】
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農業廃棄物からのブタノール生産
バイオディーゼル燃料の副産物からのアルコール生産
木質が微生物発酵に及ぼす影響の解明Home Page： 微生物機能開発学研究室：
Related fields where technical consultation is available：酒類や発酵食品の醸造技術、様々な原料の微生物への発酵利用
Message：微生物の発酵技術は、様々な物質生産へと応用できます。食品の産業廃棄物など現在利用されていない資源の有効活用法などで微生物の活用を検討の際にお気軽にご相談ください。(清: kiyoshi.keiji.p8@cc.miyazaki-u.ac.jp)