[要旨] 発酵産業はビールやヨーグルト, 醤油や食酢といった食品生産だけではなく, 医薬品や工業製品の原材料生産も含む巨大産業である. その近代的な生産現場においては高い生産性を維持するために, 発酵熱による発酵槽の過高温を抑制する大規模な冷却装置が導入され, 徹底した温度管理が行われている. 一方, 伝統的発酵産業では, その過高温が原因となり醸造菌の増殖停止や死滅が発生することもあり, 夏期には醸造を休止するところも多い. 本研究では, 発酵生産時の冷却コスト(つまりはCO_2の排出)の削減と高温発酵による生産効率の向上を目的として, 食酢醸造における優占種であるAcetohacter pasteurianusを利用し, 育種法と細胞融合法を用いて食酢合成能を有する耐熱性酢酸菌株の開発とその耐熱性発酵菌の社会実装にむけた無冷却高温発酵実験を実施した. 育種法にはA.pasteurianus NBRC 3283から分離されたAp32株(食酢合成能あり, 常温性)を用いて, 生育限界温度を超えて増殖が可能な耐熱性育種株MU株を樹立した. さらにMU株を高濃度エタノール存在下で繰り返し静置培養することにより, 高い食酢合成能を有する耐熱性育種株(JK株)の獲得に成功した. 細胞融合法ではAp32株と耐熱性育種株Ap01/42C株(食酢合成能なし)を細胞融合し, 食酢合成能を有する耐熱性細胞融合株(mika株)の樹立に成功した. 酢酸菌を用いた育種法では, 生育限界温度という多因子性形質に対する酢酸菌の高い柔軟性(ゲノム易変異性)が再確認された. 今後はゲノム解析による耐熱性形質を付与する遺伝子群の同定を進める. また, 細胞融合法では遺伝子組換えに頼らない簡便な形質導入法の開発に繋がる可能性を示すことができた. 一細胞ゲノム解析などの技術を利用し, 細胞融合及びゲノム融合の動的解析を進めたい. 発酵槽に冷却装置を設置しない伝統的な食酢醸造を行う田村造酢(株)では, 発酵熱により発酵槽が過高温となるため猛暑期には食酢醸造は行われない. そこで耐熱性発酵菌の社会実装にむけた無冷却高温発酵実験として, 2014年の8月と9月にJK株とmika株を用いた250L大型発酵試験とJK株を用いた日本酒を原料とする320L食酢醸造試験を行った. いずれの発酵実験においても酢酸度は目標となる数値まで上昇した. しかし, 2014年の夏は異常な冷夏であり, 田村造酢(株)の種菌を用いた場合でさえも発酵槽の過高温は発生せず, 耐熱性菌の耐熱性形質が発揮される条件は得られなかった. 耐熱性発酵菌の社会実装にむけて, 耐熱性形質の有効性を確認するための無冷却高温発酵実験を引き続き計画している. [Abstract] In order to realize the continual and steady reduction of greenhouse gas emissions in the mid and long term, we are working to obtain fruitful research leading to green innovation as a team for "Genome-based research and development of thermo-tolerant microbes aiming at low-cost fermentation" (a JST-ALCA project). Microbial fermentation is important in bio-industry such as not only brewing or food production but also a variety and abundant of productions of medicinal and industrial materials. Facing with a global warming and also an electric power crisis, it becomes more requested to keep the process at low temperature to make sure the stable fermentation or high productivities. We aim at developing methods to provide useful thermo-tolerant microbes able to ferment stably above 40℃ through genome engineering on the basis of breeding with adaptive evolution, cell-cell fusion (or bacterial crossbreeding) and genetic manipulation using genes related to this thermo-tolerance.Traditional breeding is suitable to modify microbes regarding to multi-gene phenotypes and most acceptable for the society based on its verified safety and reliability. Compared to the method, artificial genetic manipulation is more efficient to provide aimed functions to microbes and appropriate for maintenance of microbial diversity in many companies, but it is not welcome to the people in many countries. Thus a new method for systematic genomeengineering with advantages of traditional breeding and artificial genetic manipulation is requested. However no systems have been established yet, cell-cell fusion is seemed as a prospected candidate to develop such systems. Here we chose Acetobacter pasteurianus, which is widely used for vinegar production in the world, as a model organism for development of methods to generate thermo-tolerant fermentation microbes. As results, we successfully established two strains with two phenotypes, thermo-tolerant (growing at up to 42℃) and pellicle formation. One of the two strains was established from a strain with pellicle formation and thermo-labile (growing at 39℃ or low) phenotypes based on breeding with adaptive evolution. The other one was generated by cell-cell fusion with a pellicle formation but thermo-labile strain and a non-pellicle formation but thermo-tolerant strain. Using these strains, we attempted to brew vinegar in 250 and 320 liter scales without any cooling system in the middle of this summer as implementation experiments prier to utilization in fermentation companies. Both strains could form stable pellicles and produce acetic acid up to the expected concentrations under a procedure employed during cool seasons in Tamura Zousu Company. Using this procedure, the temperature of fermentation tanks generally rise above one which a thermo-labile strain can survive and ferment vinegar. Unfortunately it was abnormally cold in this summer and the temperatures of tanks did not reach even at 35℃, and thus the test point of thermo-tolerant phenotype was not validated.

Departmental Bulletin Paper

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