Dairi Tohru

Faculty of Engineering Applied Chemistry BiotechnologyProfessor
Last Updated :2025/07/05

■Researcher basic information

Degree

  • Agriculture

Researchmap personal page

Researcher number

  • 70264679

Research Keyword

  • 生合成
  • 天然物
  • Natural product biosynthesis

Research Field

  • Life sciences, Applied microbiology
  • Life sciences, Bioorganic chemistry

Educational Organization

■Career

Career

  • Apr. 2010 - Present
    Hokkaido University, Faculty of Engineering
  • Apr. 1994 - Mar. 2010
    Toyama Prefectural University
  • Apr. 1994 - Mar. 2010
    - Biotechnology Research Center, Toyama Prefectural University
  • 1985 - 1994
    協和発酵工業(株)東京研究所
  • 1985 - 1994
    Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.

Educational Background

  • 1985, Nagoya University, 農学研究科, 農芸化学(発酵), Japan
  • 1985, Nagoya University, Graduate School, Division of Agriculture
  • 1983, Nagoya University, School of Agricultural Sciences, 農芸化学(発酵), Japan
  • 1983, Nagoya University, Faculty of Agriculture

Committee Memberships

  • 2017 - Present
    日本農芸化学会, 創立100周年記念事業組織委員会特別委員会 委員, Society
  • Apr. 2016 - Present
    バイオインダストリィー協会, B&I 編集幹事, Society
  • Apr. 2020 - Mar. 2024
    日本放線菌学会, 学会長, Society
  • May 2019 - Apr. 2021
    日本生物工学会, 理事, Society
  • Feb. 2019 - Feb. 2021
    日本農芸化学会, 広報委員長, Society
  • 2017 - 2018
    日本放線菌学会, 副会長, Society
  • 2015 - 2018
    日本農芸化学会, 理事, Society

Position History

  • 教育研究評議会評議員, 2020年4月1日 - 2022年3月31日
  • 大学院総合化学院長, 2020年4月1日 - 2022年3月31日

■Research activity information

Awards

  • Mar. 2023, Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA), JSBBA Award               
    Studies on biosynthetic enzymes leading structural and functional diversities to microbial natural products.
  • Mar. 2022, 日本農芸化学会, トピックス賞               
    微細藻類由来DHA合成酵素の炭素鎖伸長反応の解析
    仲間 陸;小林 飛悠;大塚 慎;佐藤 康治;小笠原 泰志;大利 徹
  • Mar. 2021, 日本農芸化学会, トピックス賞               
    ポリグルタミン酸生合成におけるエピメリ化酵素の同定
    加藤 陽菜多、小笠原 泰志、大利 徹
  • Mar. 2019, 日本農芸化学会, トピックス賞               
    in vitro解析による多価不飽和脂肪酸生合成酵素の炭素鎖長制御機構の解明
    林 祥平;小笠原 泰志;佐藤 康治;丸山 千登勢;濱野 吉十;氏原 哲朗;大利 徹
  • Mar. 2017, 日本農芸化学会, トピックス賞               
    An unprecedented glutamate epimerase for bacterial peptidoglycan biosynthesis
    Ruoyin FENG;Yasuharu Satoh;Yasushi Ogasawara;Tohru Yoshimura;Tohru Dairi
  • Jun. 2015, 日本生物工学会, 第23回生物工学論文賞               
    New gene responsible for para-aminobenzoate biosynthesis
    Y. Satoh;M. Kuratsu;D. Kobayashi;T. Dairi
  • 2012, 酵素応用シンポジウム運営委員会, 酵素応用シンポジウム奨励賞               
    大利 徹
  • 2011, 長瀬財団, Nagase Foundation Award               
    大利 徹
  • 2010, 日本放線菌学会, 日本放線菌学会 学会賞               
    大利 徹
  • 2004, 住木・梅澤記念賞               
    Japan
  • 2004, Sumiki & Umezawa Award from Japan Antibiotic Research Association               
  • 2002, 日本農芸化学会論文賞               
    Japan
  • 2000, 日本農芸化学会奨励賞               
    Japan
  • 2000, Encouragement Award from Japan Society for Bioscience, Biotechnology, and Agrochemistry               
  • 1999, とやま賞               
    Japan
  • 1999, TOYAMA Award from Toyama Hitodukuri Foundation               
  • 1998, 日本放線菌学会浜田賞               
    Japan
  • 1998, Encouragement Award from The Society for Actinomycetes Japan               

Papers

Other Activities and Achievements

Books and other publications

  • Isoprenoid Synthesis in Plants and Microorganisms               
    DAIRI Tohru, Biosynthetic genes and enzymes of isoprenoids produced by actinomycetes
    Springer, 2013, [Contributor]
  • Comprehensive Natural Products II Chemistry and BiologyMander, L., Lui, H.-W., Eds.volume 1, pp. 789–814.               
    Comprehensive Natural Products II Chemistry and BiologyMander, L., Lui, H.-W., Eds.volume 1, pp. 789–814.
    Elsevier: Oxford,, 2010

Courses

  • 生合成工学               
    北海道大学
  • 遺伝子工学特論               
    富山県立大学
  • 生物工学関連法規               
    富山県立大学
  • 技術英語               
    富山県立大学
  • 生物情報学概論               
    富山県立大学
  • 生物工学概論               
    富山県立大学
  • 生物工学実験               
    富山県立大学
  • 分子生物学演習               
    富山県立大学
  • 分子生物学               
    富山県立大学
  • 生化学               
    北海道大学

Affiliated academic society

  • 日本生物工学会               
  • 日本放線菌学会               
  • 日本農芸化学会               

Research Themes

  • Dissecting biosynthetic machineries of natural peptides leading to structural and functional diversities.
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)
    27 Apr. 2022 - 31 Mar. 2027
    大利 徹, 小笠原 泰志, 森田 洋行, 濱野 吉十
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (S), Hokkaido University, 22H04976
  • Dissecting reaction mechanisms of novel peptide epimerases
    Grants-in-Aid for Scientific Research
    01 Apr. 2018 - 31 Mar. 2022
    Dairi Tohru
    (1) MurL catalyzing the epimerization of the terminus L-Glu of UDP-MurNAc-L-Ala-L-Glu, an intermediate in the alternative biosynthetic pathway of peptidoglycan, was shown to activate the substrate by adenylation of α-carbonyl of the L-Glu. By crystal structure analysis, we were unable to determine the active amino acid residues of MurL, and co-crystal of MurL with a mimic of adenylated substrate was thought to be essential for estimation of the active residues and the reaction mechanism. (2) MslH catalyzed the epimerization of C-terminal L-Trp in a ribosomal peptide MS-271. By crystal structure analysis, active amino acid residues, divalent cation trapping residues, and several substrate recognition residues were identified. (3) Polyglutamate contains D-Glu besides L-Glu. PgsA, a polyglutamate biosynthetic enzyme, was suggested to be the epimerase catalyzing isomerization of terminal L-Glu that is just ligated to the elongating polyglutamate substrate.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 18H03937
  • Redesigning Biosynthetic Machineries (Project Team)
    Grants-in-Aid for Scientific Research
    30 Jun. 2016 - 31 Mar. 2021
    Abe Ikuro
    In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. Based on the detailed analyses of the structural diversity of natural products from the viewpoint of genes, enzymes, and reactions, we established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to facilitate the research plan, the project team formulated a research policy and provided a forum to actively promote exchanges, collaborations, and joint research among individual research promoters. Researchers from various fields collaborated and complemented each other in their areas of expertise to conduct joint research and create a new academic field.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), The University of Tokyo, 16H06442
  • Studies on biosynthetic machinery of biologically active natural products
    Grants-in-Aid for Scientific Research
    30 Jun. 2016 - 31 Mar. 2021
    Dairi Tohru
    Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a DHA synthase into an EPA synthase by one amino acid substitution.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 16H06452
  • Redesigning Biosynthetic Machineries (International Activity Support Team)
    Grants-in-Aid for Scientific Research
    30 Jun. 2016 - 31 Mar. 2021
    Abe Ikuro
    In this project, we have made a dramatic development from simply learning biosynthetic machineries to designing new blueprints for producing desired natural products. We established a new world-leading technology platform for creation of complex functional molecules by rational reconstruction of biosynthetic machineries as a new academic field. In order to strongly promote joint research with foreign countries, the International Activity Support Group invited outstanding foreign researchers and held lecture meetings on a regular basis. Furthermore, international symposiums were held with the aim of strengthening cooperation and exchange with the U.S., Germany, China, and other countries. We strategically promoted joint research with foreign countries, focusing on medium-term support for young researchers. Many of the results of joint research with foreign countries resulted in excellent co-authored papers.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), The University of Tokyo, 16K21725
  • Biosynthesis of the Carbonylmethylene Structure Found in a Class of Pseudotripeptides, Ketomemicins
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    01 Apr. 2015 - 31 Mar. 2018
    Dairi Tohru, SATOH YASUHARU, OGASAWARA YASUSHI, MORITA HIROYUKI
    We recently discovered novel pseudotripeptides, ketomemicins, which possess a C-terminal pseudodipeptide connected with a carbonyl methylene instead of an amide bond. The carbonyl methylene structure is stable than amide bond and its biological significance has been shown in several natural and synthetic compounds. Despite the biological significances of these compounds, little is known about its biosynthetic machinery. We therefore examined it by in vitro studies with recombinant enzymes. Consequently, an aldolase, dehydratase, PLP-dependent glycine-C-acetyltransferase, and dehydrogenase were revealed to be involved in the formation of the pseudodipeptide with malonyl-CoA and phenylpyruvate as starter substrates.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 15H03110
  • Biosynthetic machinery: Deciphering and regulating the system for creating structural diversity of baioactive metabolites
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
    01 Apr. 2010 - 31 Mar. 2016
    OIKAWA Hideaki, EGUCHI Tadashi, ABE Ikuro, KUZUYAMA Tomohisa, DAIRI Tohru, IKEDA Haruo, GOMI Katsuya, SAITO Kazuki, KANAYA Shigehiko, ISHIKAWA Jun
    新学術領域領域代表者と8名の計画研究代表者(江口、大利、阿部、池田、五味、斉藤、金谷、石川)、5名の評価委員の意見を取りまとめ、新学術領域研究「生合成マシナリー」の成果報告シンポジウムを、聴衆が期待できる週末のアクセスが良い場所という観点から、東京都港区のコクヨホールに決定した。6月21日の日曜日には、100名近い企業からの出席者や一般参加者に対し、5年間の研究成果を、A01,A02,A03班の班長および指名された顕著な成果を挙げた合計7名の講演者が、判りやすく説明した。


    最終の文部科学省へ提出する成果報告書は、3班構成の各班長が、班員個人より事務局に提出された5年間の領域活動で得られた成果に関する資料をもとにデータを整理し、論文発表、学会発表、シンポジウムや講演会での依頼講演、新聞・テレビなどのメディアでの公表状況、各賞の受賞状況、さらには領域内での共同研究など各班の成果を取りまとめた後、事後評価ヒアリング、さらには一般公開用資料としてデータファイルを作成した。これをベースに冊子体も作成し、公的な研究機関や関連研究者に送付した。


    6月から10月オープンキャンパスや夏休み期間中には、領域代表が所属する北海道大学や班員が所属する研究機関で、中高生を対象にオープンラボなどの企画で、研究活動を疑似体験してもらうために、実験を撮影したビデオの上映、さらに実験室の見学を行なった。さらに11月14-15日(土・日)、東京都江東区にある日本科学未来館で開かれた大規模なサイエンスコミュニケーションのイベント、サイエンスアゴラに出展し、総括班でアウトリーチ用に作成した領域活動の広報用アニメーションやクイズ形式の説明用プレゼンファイルを利用して、来場者に研究成果を説明した。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 22108001
  • Peptide synthesis cooperatively achieved by peptide ligase and ribosomes
    Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    01 Apr. 2013 - 31 Mar. 2015
    DAIRI Tohru
    Pheganomycin (PGM) (1) consists of a nonproteinogenic amino acid, (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid (2) at the N-terminus and a proteinogenic core peptide derived from NVKDGPT or NVKDR. The biosynthetic gene cluster was identified in Streptomyces cirratus to contain a gene encoding a precursor peptide, which included both the core peptides, and several genes plausibly encoding enzymes for 2 biosynthesis. We identified a gene (pgm1) responsible for the peptide bond formation between 2 and the peptides in the cluster. A pgm1-disruptant lost 1 productivity and recombinant PGM1 catalyzed the ATP-dependent peptide bond formation. This is the first example of cooperative peptide synthesis achieved by ribosome and peptide ligase using a peptide as a nucleophile. PGM1 accepted a variety of peptides as the nucleophile and the flexibility was comprehended by the crystal structure of PGM1 and the mutagenesis analyses.
    Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 25560397
  • Studies on biosynthetic machineries for cyclic terpenoids and nucleoside antibiotics
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
    01 Apr. 2011 - 31 Mar. 2015
    DAIRI Tohru, KATO Nobuo, SATOH Yasuharu, ARAI Ryouichi
    1. Fusicoccin A is a diterpene glucoside produced by the fungus Phomopsis amygdali and its derivative lacking the OH group at the 12-position is revealed to be a potential anti-cancer drug. We therefore constructed a mutant producing the desirable intermediate by biosynthetic engineering. 2. Three fungal prenyltransferases, PaxC, PaxD, and AtmD, all of which are responsible for biosynthesis of indole diterpene compounds, were characterized. 3. Aminodeoxyfutalosine is the first intermediate in the new menaquinone biosynthetic pathway. We demonstrated that a radical S-adenosyl methionine enzyme (MqnE) catalyzed the addition of the adenosyl radical to the double bond of 3-[ (1-carboxyvinyl)oxy]benzoic acid derived from chorismate by MqnA. 4. During the biosynthetic studies of pheganomycin, we identified a new ATP-grasp enzyme, which phosphorylated the non-proteogenic amino acids with ATP and the successive nucleophilic attack of the peptides.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 23108101
  • Study on a novel folate biosynthesis pathway on micoorganism
    Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    01 Apr. 2012 - 31 Mar. 2014
    SATOH Yasuharu, DAIRI Tohru, KURATSU Masahiro, OBAYASHI Daiki
    Folate is an essential cofactor in all living cells. para-Aminobenzoate (pABA), a building block of folate, is usually derived from chorismate in the shikimate pathway by reactions of PabABC. We previously suggested that an alternative pathway for pABA biosynthesis would operate in some microorganisms such as Lactobacillus fermentum and Nitrosomonas europaea since these bacteria showed a prototrophic phenotype to pABA despite the fact that there are no orthologs of pabABC in their genome databases. In this study, a gene of unknown function, NE1434, was obtained from N. europaea by shotgun cloning using a pABA-auxotrophic Escherichia coli mutant (dpabABC) as a host. A tracer experiment using [U-13C6]glucose suggested that pABA was de novo synthesized in the transformant. An E. coli dpabABCdaroB mutant carrying the NE1434 gene exhibited a prototrophic phenotype to pABA, suggesting that compounds in the shikimate pathway including chorismate were not utilized as substrates by NE1434.
    Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 24710239
  • Estimation of an alternative biosynthetic pathway for co-factors by close investigation of genome databases.
    Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    2012 - 2012
    DAIRI Tohru, SATOH Yasuharu
    The invovment of new enzymes for co-factors biosynthesis was examined. pABA; A function unknown gene, NE1434, was obtained from Nitrosomonas europaea by shotgun cloning experiment using pABA-auxotrophic E. coli mutant (△pabABC) as a host. Glutathione; Streptomyces strains do not use glutathione. However, SCO0910 was confirmed to be glutamate-cysteine ligase and was essential for ergothioneine biosynthesis. Taurine; SCO3035 in S. coelicolor A3(2) was reported to be the first enzyme in taurine biosynthesis (J. Bacteriol. 188, 5561, 2006). To investigate taurine biosynthesis in S. coelicolor A3(2), SCO3416, 2782, and 2017, putative enzymes catalyzing thesecond reaction, were used for in vitro assay. However, all enzymes did not show the expected activity.
    Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 24651235
  • Screening of natural products which inhibits a new menaquinone biosynthetic pathway
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    2010 - 2012
    DAIRI Tohru
    The futalosine pathway is operating in some bacteria for the biosynthesis of menaquinone. Futalosine is converted into dehypoxanthinyl futalosine (DHFL) by MqnB.In this study, three routes to the formation of DHFL were suggested. DHFL may have been directly formed by MqnB in Thermus thermophilus. In Streptomyces coelicolor, aminodeoxy FL (AFL) was converted to FL by deaminases, then to DHFL. In contrast, MqnB of Helicobacter pylori directly converted AFL into DHFL.
    To identify compounds that specifically inhibit the futalosin pathway, we used B. halodurans that has FL pathway as a test strain. Finally, two candidate fungal culture broths were obtained.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 22310139
  • 微生物が生産する環状テルペノイド生合成マシナリーの解明と再構築
    科学研究費助成事業 新学術領域研究(研究領域提案型)
    2011 - 2011
    大利 徹
    日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 23108501
  • 病原菌が持つメナキノン新規生合成経路の全容解明と経路特異的阻害剤の探索
    科学研究費助成事業 特定領域研究
    2008 - 2009
    大利 徹
    メナキノン(ビタミンK)は、微生物にとって電子伝達系成分として生育に必須である。筆者は、Helicobacter属(ピロリ菌)、Campylobacter属、Wolinella属などの病原微生物や、放線菌Streptomyces属などの微生物では、今まで知られていた経路とは全く異なるフタロシン経路で生合成されることを見出した。今年度、本経路の2番目の反応を触媒するfutalosine hydrolase(MqnB、最近EC3.2.2.26が付与された)の諸性質を高度好熱菌Thermus thermophilusの組換え酵素を用いて検討した。その結果、以下の性質を示した。(1)Futalosineのみが基質となり他の核酸類縁体は基質にならないこと、(2)4量体を形成、(3)至的pHは4.5、(4)至的温度は80度、(5)Km値154.0±5.3μM、kcat1.02/s、(6)hypoxanthineにより弱く阻害されること(Ki値1.1mM)。本酵素がhypoxanthineにより弱く阻害されたことから、hypoxanthine誘導体を合成し、本酵素特異的阻害剤を探索することにより、抗ピロリ菌剤の開発が可能になると期待された。
    また天然物からの抗ピロリ菌リード化合物の探索も行った。大学設備ではピロリ菌を培養できないため、2種類のBacillus属細菌を用いた系で一次スクリーニングを行った。同じBacillus属に属しながら、Bacillus haloduransがメナキノン生合成の際、新規経路を使うのに対し、Bacillus subtilisは既知経路を使う。そこで、(1)B.haloduransに対し抗菌作用を示すが、B.subtilisには影響を及ぼさない化合物を放線菌・カビの培養液中に探索した。次に、(2)B.haloduransに対する生育阻害が、外からメナキノンを添加した際、回復するか検討した。その結果、放線菌の培養中に1つの候補化合物を見出した。現在、本化合物の精製と構造解析を行っている。
    日本学術振興会, 特定領域研究, 富山県立大学, 20018023
  • Studies on diterpene cyclases found in eubacteria
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    2007 - 2009
    大利 徹
    ジテルペン化合物は(炭素数20)、香料、医薬・医薬中間体、植物ホルモンなど重要な化合物を含んでいる。これら化合物の生合成の第一段階は、環化酵素が直鎖状の基質であるゲラニルゲラニル2 リン酸の末端オレフィンをプロトン化、または2 リン酸を脱離することによってカルボカチオンを生成することから始まる。最終的にカルボカチオンが捕捉中性化されるまで、各環化酵素特有のカチオン中間体を経る反応が順次進行し、多種多様なイソプレノイド骨格へと導かれる。反応の第一段階が共通であるにもかかわらず、最終的に多様な骨格を持つ化合物が生成する事実は、環化酵素の特定アミノ酸残基のみが反応に関与するのではなく、酵素全体のアミノ酸残基が種々のカルボカチオン中間体の生成に関与することを示唆する。従って、これら環化酵素の構造機能相関解析を行うことにより、任意の段階でカルボカチオンが捕捉中性化された化合物の生成を制御できる可能性がある。しかし環化酵素に関しては、真核生物を材料に用いた場合、酵素調製が難しいことなどから一部の酵素を除いて殆ど解析が行われていない。このような背景下、筆者は原核生物と真核生物起源のイソプレノイド生合成遺伝子を多数取得しており、今回、(1)お米由来の2 つのent-copalyl diphosphate 生合成酵素、(2)原核生物のNocardia 属放線菌が生産するジテルペン化合物、brasilicardin A 生合成遺伝子クラスターの取得、(3)原核生物のStreptomyces 属放線菌により生産されるテトラテルペン化合物(KS-505a)生合成遺伝子クラスターの取得とこれらの機能解析を行った。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Toyama Prefectural University, 19510220
  • 病原菌が持つメナキノン新規生合成経路の全容解明と経路特異的阻害剤の探索
    科学研究費助成事業 特定領域研究
    2006 - 2007
    大利 徹
    メナキノン(ビタミンK)は,人間にとっては血液凝固に必要なビタミンであり,また微生物では電子伝達系成分として生育に必須である。申請者は,胃潰瘍・胃がんの原因菌として知られているHelicobacter属細菌、食中毒原因菌として知られているCampylobacter属細菌,グラム陽性の土壌細菌であるStreptomyces属細菌などの微生物では,既知の生合成経路として知られているコリスミ酸からメナキノンに至る5ステップの生合成遺伝子群が全く存在しないことに気づいた。そこで,この新規経路の全容解明を行っている。
    1.これまでにStreptomyces属放線菌を材料に用いて,バイオインフォマティクスにより絞り込んだ遺伝子を破壊することにより,7つの遺伝子群が新規経路に関与することを明らかにした(これらの成果については未発表のため,これら遺伝子群を以下men06,men26,men27,men50,men90,men92,men94と略号で記載する)。
    2.また新規経路遺伝子の網羅的取得を目指し,変異剤によるメナキノン生合成欠損株の誘導と相補遺伝子の取得も行った結果,新規経路は既知経路同様コリスミ酸を出発基質とするが,その後全く別経路を経ることも分かった。
    3.上述の破壊株を2つ組み合わせてメナキノン非存在下で混合培養を行った結果,幾つかの菌株の組み合わせで生育が認められた。本現象を利用し,破壊株が関与する生合成上の相対的な位置を(men06)→(men26,men27,men50)→(men90,men92,men94)→メナキノンのように明らかにすることができた。
    4,上述したアッセイ系を用い,破壊株が蓄積する中間体の精製・構造決定を行い,3つの化合物の構造を決定した。
    日本学術振興会, 特定領域研究, 富山県立大学, 18018035
  • Studies on a new biosynthetic pathway for menaquinone (vitamin K)
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    2005 - 2007
    SETO Haruo, SUE Masayuki, DAIRI Toru
    We found that some pathogenic bacteria including Helicobacter pylori and Campylobacter jejuniutilized a new biosynthetic pathway for menaquinone. This pathway is also present in the genus Streptomyces and branches from the well known menaquinone biosynthetic pathway at chorismate. Since this pathway does not present in human, its inhibitors are expected to be useful as antibacterial substances. In order to reveal the details of this pathway, we carried out the following experiments.
    1. At the onset of the experiments, we established unequivocal assignment of 13C-N1VIR spectrum of menaquinone.
    2. Labeling experiments using glucoses labeled by^<13>C at different position proved that menaquinone was formed from erythrose, phosphoenolpyruvate and two different C_2 units originating from C5-C6 of glucose.
    3. Several kinds of blocked mutants of Streptomyces coelicolor that required menaquinone for their growth were prepared.
    4. Based on the results obtained by labeling experiments, 1,4-naphthoquinone-6-carboxylic acid was assumed to be a biosynthetic intermediate of the new pathway. Therefore, we prepared this acid by oxidation of naphthalene-2-carboxylic acid by using Ce(SO_4)_2. This compound enabled the growth of a menaquinone auxotroph of S coelicolor indicating that the synthetic compound is a true intermediate of the new pathway.
    5. By detailed investigation of metabolites accumulated by mutants of S coelicolor a nucleoside compound, futalosine, was isolated. This compound consisted of inosine and a m-substituted benzoic acid derivative, and had been isolated from another strain of Streptomyces as an antitumor substance. This compound was proved to be a biosynthetic intermediate of the new pathway, since it supported the growth of some menaquinone auxotrophs of S coelicolor.
    6. By utilizing other menaquinone auxotrophs of S coelicolor, several intermediates after futalosine were identified.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Tokyo University of Agriculture, 17380075
  • Studies on production and improvement of terpenoids using biosynthetic gene cluster in fungi and plants.
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    2004 - 2006
    SASSA Takeshi, YAMANE Hisakazu, HOSHINO Tsutomu, KATO Nobuo, OIKAWA Hideaki, DAIRI Tohou
    A cDNA encoding fusicoccadiene synthase (PaFS) was isolated from mycelia of fusicoccin-producing fungi. PaFS possesses two domain, terpene cyclase domain at N-termini and prenyltransferase domain at C-termini, and shows multifunctional catalyzation. We have clarified the detailed cyclization mechanism forming fusicoccane skeleton from GGDP, and have succeeded in synthesizing a fucicoccin derivative that has a differentiation inducing activity. Mechanism on construction of molecular skeleton of diterpene, phomaction was investigated by chemical methods. Heterologous expression of the genes responsible for biosynthesis of diterpene, aphidicolin using yeast was explored. A common biosynthetic intermediate of indole diterpenoide was synthesized as non-and deuterium-labeled form. The deuterium-labeled intermediate was converted into paxilline and emindole DA via epoxidation and cyclization in two different fungi in an intact manner. These results give first evidence that various indole diterpenoide are biosynthesized from a common intermediate. We have cloned three diterpene cyclase genes and one tetraterpene cyclase gene from actinomycetes. Of the four cyclases, enzymatic properties of the two diterpene cyclases were investigated with recombinant enzymes. The functional analyses were performed in respect to Rv3377c and Rv3378c involved in Mycobacterium tuberculosis H37 strain. These genes encode diterpene synthases for producing halimane skeleton. Studies on squalene cyclase were also conducted to gain more insights into the catalytic mechanism. Successful site-specific incorporation of fluorophenylalanines into the catalytic sites demonstrated the importance of cation-pi interaction. In vitro translation system revealed the function of a diterpene cyclase gene from the moss Physcomoitrella patens. This system was also available for determination of production and localization of cytochrome P450 monooxygenase proteins. The function of P450 monooxygenases responsible for biosynthesis of plant hormones, brassinosteroid and gibberellin, was revealed by heterologous expression in Pichia pastoris. We identified 6 cDNAs encoding diterpene cyclases responsible for phytoalexin bisynthesis in rice. We indicated strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis. The presence of phytocassane biosynthesis gene cluster on chromosome 2 was also suggested.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Yamagata University, 16208012
  • Studies on isoprenoid biosynthetic genes isolated from prokaryotes
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    2004 - 2006
    DAIRI Tohru, MATSUURA Nobuyasu
    We have cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp.strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-copalyl diphosphate(CDP) synthase and a geranylgeranyl diphosphate(GGDP) synthase, respectively, by experiments using recombinant enzymes. ORF3,that did not show similarities with any other known proteins was also expressed in E.coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into pimara-9(11),15-diene. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg^<2+> and Zn^<2+> for its activity. The optimum pH and temperature were 5.5 and 35℃. The Km value was calculated to be 13.7±1.0μM for GGDP and the kcat value was 3.3 x 10^<-2>/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30℃. The Km value for ent-CDP was estimated to be 2.6±0.2μM and the kcat value was 1.4 x 10^<-3>/sec.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Toyama Prefectural University, 16310153
  • Studies on diterpene cydases found in actinomycetes.
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    2002 - 2003
    DAIRI Tohru
    Fubacterial diterpene cyclase genes have been doned from a diterpenoid-antibiotic, terpentecin producer. Their products, ORF11 and ORF12, were confirmed to be essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same -basic skeleton as terpentecm. Functional analyses of these two enzymes were also performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cydized intermediate (terpentedienol diphosphate, TDP), and then it was transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly reacted with GGDP and converted GGDP into three olefinic compounds. Moreover, the ORF12 product reacted even with farnesyl diphosphate (FDP) giving three olefinic compounds, which had the same structures as those formed from GGDP except for the chain-lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the reaction by an ionization-initiated reaction of substrates to an allylic carbocation followed by deprotonation to the olefin. Kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of FDP and that the relative activity of the reaction converting TDP into TTE. was highest among the reactions using TDP, GGDP, or FDP as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Toyama Prefectural University, 14560074
  • Scientific research on elucidation of terpene-cyclic reactions in the biosynthesis of bioactive microbial terpenoids.
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    2001 - 2003
    SASSA Takeshi, OIKAWA Hideaki, KATO Nobuo, HOSHINO Tsutomu, TOYOMASU Tomonobu, DAIRI Tohru
    (1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Yamagata University, 13306009
  • Studies on the biosynthesis of terpenoids produced by streptomycetes.
    Grants-in-Aid for Scientific Research
    2000 - 2001
    DAIRI Tohru
    Two gene clusters containing the mevalonate pathway genes and the terpentecin (TP) biosynthetic genes were cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, TP producer. In the former cluster, seven genes enecoding geranylgerairyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarbosylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in Ε. coli and Streptomyces Iividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate paftway were involved in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in Ε. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. In the latter cluster, two ORFs, ORF11 and ORF12 that encode proteins showing similarities to eucaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were found. The two cyclase genes were expressed in Streptomyces Iividans. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has a common basic skeleton as does TP. The two enzymes were overproduced in Ε. coli and purified to homogeneity. The recombinant ORF11 product converted GGDP into an intermediate with diphosphate, and then it was transformed into terpenteteiene by the recombinant ORF12 product. To the best of our knowledge, this is the first report about a eubacterial DC.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Toyama Prefectural University, 12660083
  • Cloning and Analysis of Tautomycin Biosynthetic Genes
    Grants-in-Aid for Scientific Research
    1996 - 1998
    UBUKATA Makoto, MATSUURA Nobuyasu, DAIRI Tohru
    Tautomycin (TM) isolated from a culture broth of Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Since TM and its analogues are expected to be useful probes for elucidating the signal trasduction of mammalian cells, we studied on the absolute structure, conformational analysis, structure-activity relationship, and biosynthesis of the polyketide compound, TM. In the present study, we have studied on the cloning and analysis of TM biosynthetic genes. Here I report the results of experiments done in 1996, 1997 and 1998.
    1. Development of transformation system: Protoplast formation and regeneration of the TM producer were achieved by using the reported procedure for other Streptomyces sp. In addition to above experiments, the transformation system was established by curing the producer of the cryptic plasmid by a heat shock treatment. The satisfactory efficiency of 2 x 10ィイD15ィエD1 transformants/μgDNA was obtained using the pIJ702 plasmid, with the result that we optimized various conditions of the transformation system.
    2. Cloning of the putative TM biosynthetic gene: The KS and AT primers, which were designed from the published sequences in PKS Type I, were used in PCR strategy to obtain products. The primer design strategy was successful in identifying a fragment from S. spiroverticillatus genomic DNA and a PKS on cosmid pWE15. Sequence analysis of the PCR product strongly suggests that we have cloned a PKS gene flagment in the TM producer.
    3. Gene disruption of the putative TM biosynthetic gene: To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involving in TM biosynthesis, the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kb region into the corresponding region on the chromosome. The resulting 15 strains were fermented and assayed for the presence of TM. Although none of the strains produced TM, an another antibiotic xanthostatin was produced by all strains. Genomic Southern hybridization analyses showed that TM biosynthetic genes in these strains were deleted from the genomic DNA.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Toyama Prefectural University, 08456062
  • Reaction Mechanisms and Application of New Bacterial Enzymes
    Grants-in-Aid for Scientific Research
    1996 - 1997
    ASANO Yasuhisa, KOMEDA Hidenobu, KATO Yasuo, DAIRI Tohru
    (1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed.
    (2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography.
    (3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined.
    (4) Results were presented in some international academic meetings.
    Japan Society for the Promotion of Science, Grant-in-Aid for international Scientific Research, Toyama Prefectural University, 08044218
  • Development of Novel Lyases and Their Application to the Synthesis of Optically Active Compounds
    Grants-in-Aid for Scientific Research
    1995 - 1996
    ASANO Yasuhisa, YAMADA Hideaki, DAIRI Tohru, KATO Yasuo
    First, we screened for microorganisms having new hydro-lyases catalyzing hydration of maleic acid and itaconic acid, respectively, to produce D-malic acid and citramalic acid which are food additives and starting materials in synthetic organic chemistry, etc. The bacterial maleate hydratase from Arthrobacter sp. was purified to homogeneity and characterized. The culture and reaction conditions for the production of optically pure D-malate from maleate were studied. Under an optimum condition, 87 grams/liter of D-malate was produced in 20 h. The yield was 72 mole %. (S)-(+)-Citramalate producing activity from itaconate was also studied. A.denitrificans produced (S)-(+)-Citramalate in a 99.9 % enantiomeric excess. Under an optimum condition, 27 g of (S)-(+)-citramalate per liter was produced in 30 h. The yield was 69 mol%.
    Second, a screening was carried out for new 3-methylaspartate ammonia-lyases which catalyze the synthesis of L-aspartic acid derivatives from fumaric acid derivatives. We discovered for the first time that 3-methylaspartate ammonia-lyase (MAL) producers are relatively widely distributed in the family of facultatively anaerobic Enterobacteriaceae. Crystalline MALs from Citrobacter freundii, C.amalonaticus, and Morganella morganii, were characterized. Using cell-free extracts of the isolates, optically pure (2S, 3S)-3-methylaspartic, (2S, 3S)-3-ethylaspartic, and (2R, 3S)-3-chloroaspartic acids were synthesized from corresponding fumaric acid derivatives.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Toyama Prefectural University, 07660117
  • Studies on biosynthesis of terpenoids produced by microorganisms               
    1996
    Competitive research funding
  • Studies on antibiotic biosynthesis               
    1994
    Competitive research funding
  • Studies on biosynthesis of natural product               
    Competitive research funding
  • Studies on biosynthetic genes and enzymes of isoprenoids produced by microorganisms               
    Competitive research funding

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