Transfer of nitrogen fixing ability to plants: Model experiments in cyanobacteria

• Project/Area Number: 15H04387
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Plant molecular biology/Plant physiology
• Research Institution: Nagoya University
• Principal Investigator: FUJITA Yuichi 名古屋大学, 生命農学研究科, 教授 (80222264)
• Co-Investigator(Renkei-kenkyūsha): SUGITA Mamoru 名古屋大学, 遺伝子実験施設, 教授 (70154474)
• Research Collaborator: YAMAKAWA Hisanori ; TSUJIMOTO Ryoma ; IHARA Kunio ; UESAKA Kazuma ; KOTANI Hiroya ; TOMATSU Chie ; YOKOMIZO Konomi ; YAMAMOTO Haruki
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥18,070,000 (Direct Cost: ¥13,900,000、Indirect Cost: ¥4,170,000); Fiscal Year 2017: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2016: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000); Fiscal Year 2015: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000)
• Keywords: 窒素固定 / ニトロゲナーゼ / シアノバクテリア / トランスポゾンタギング / 光合成 / 合成生物学 / 転写制御タンパク質 / 応用微生物 / ゲノム / 酵素 / 遺伝子 / ゲノムリシーケンス / 転写制御

Outline of Final Research Achievements

We established transposon-tagging mutagenesis in the nonheterocystous cyanobacterium Leptolyngbya boryana. Using this technique, we identified one gene that is involved in mechanism for coexisting nitrogenase and photosynthesis. To identify a gene set for transfer of nitrogen fixing ability to plants we isolated transformants in which the 20-kb nif gene cluster from L. boryana was integrated into a genome neutral site of the cyanobacterium Synechocystis sp. PCC 6803. To activate the expression of the nif genes the cnfR gene encoding the transcriptional activator for the nif genes was also integrated into another genome neutral site. A low but significant activity of nitrogenase was successfully detected in the transformants. This is the first example for functional expression of nitrogenase in oxygenic photosynthetic organisms.

Research Products

• [Journal Article] Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism (2018)
  • Author(s): Tsujimoto, R., Kotani, H., Yokomizo, K., Yamakawa, H., Nonaka, A., and Fujita, Y.
  • Journal Title: Scientific Reports Volume: 印刷中
  • Peer Reviewed / Open Access
• [Journal Article] Biochemistry of chlorophyll biosynthesis in photosynthetic prokaryotes. (2017)
  • Author(s): Fujita, Y., Yamakawa, H.
  • Journal Title: Modern Topics in the Phototrophic Prokaryotes-Metabolism, Bioenergetics, and Omics, Hallenbeck, P. ed. Volume: - Pages: 67-122
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Identification of a cis-element in nitrogen fixation genes recognized by CnfR in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana. (2016)
  • Author(s): Tsujimoto, R., Kamiya, N., and Fujita, Y.
  • Journal Title: Molecular Microbiology Volume: 101 Issue: 3 Pages: 411-424
  • DOI: 10.1111/mmi.13402
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] 酸素パラドクスの統御で収斂するクロロフィル生合成と窒素固定 (2016)
  • Author(s): 藤田祐一、辻本良真
  • Journal Title: 光合成研究 Volume: 26 Pages: 204-215
  • Peer Reviewed / Open Access
• [Journal Article] Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus (2016)
  • Author(s): Nomata, J., Terauchi, K. and Fujita, Y.
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 470 Issue: 3 Pages: 704-709
  • DOI: 10.1016/j.bbrc.2016.01.070
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Transformation of the cyanobacterium Leptolyngbya boryana by electroporation. (2015)
  • Author(s): Tsujimoto, R., Kotani, H., Nonaka, A., Miyahara, Y., Hiraide, Y. and Fujita, Y.
  • Journal Title: Bio-protocol Volume: 5
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX mono methyl ester cyclase in cyanobacteria (2015)
  • Author(s): Yamanashi, K., Minamizaki, K. and Fujita, Y.
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 463 Issue: 4 Pages: 1328-1333
  • DOI: 10.1016/j.bbrc.2015.06.124
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 非窒素固定性シアノバクテリアSynechocystis sp. PCC 6803への窒素固定能付与に向けた試み (2018)
  • Author(s): 横溝この実・小谷弘哉・辻本良真・山川壽伯・藤田祐一
  • Organizer: 第59回日本植物生理学会年会
• [Presentation] Cyanobacterial low oxygen adaptation mechanisms resolving the oxygen paradox between photosynthesis and nitrogen fixation. (2017)
  • Author(s): Fujita, Y.
  • Organizer: Universidad Politecnica de Madrid, CBGP Seminar
  • Place of Presentation: Universidad Politecnica de Madrid, Spain
  • Year and Date: 2017-03-31
  • Int'l Joint Research / Invited
• [Presentation] 非窒素固定性シアノバクテリアSynechocystis sp. PCC 6803への窒素固定移入の試み (2017)
  • Author(s): 山川壽伯、小谷弘哉、辻本良真、上坂一馬、井原邦夫、藤田祐一
  • Organizer: 第58回日本植物生理学会年会
  • Place of Presentation: 鹿児島大学
  • Year and Date: 2017-03-17
• [Presentation] ヘテロシスト非形成型シアノバクテリアLeptolyngbya boryanaにおけるトランスポゾン変異導入による窒素固定欠損変異株の単離 (2017)
  • Author(s): 戸松千映、上坂一馬、井原邦夫、藤田祐一
  • Organizer: 第58回日本植物生理学会年会
  • Place of Presentation: 鹿児島大学
  • Year and Date: 2017-03-16
• [Presentation] 光合成と窒素固定の酸素パラドクスを統御する低酸素適応機構 (2017)
  • Author(s): 藤田祐一、山川壽伯、辻本良真
  • Organizer: 第19回植物オルガネラワークショップ「植物オルガネラの進化と機能、そして可能性」
  • Place of Presentation: 鹿児島大学
  • Year and Date: 2017-03-15
  • Invited
• [Presentation] Characterization of the transcriptional regulator CnfR for the nif genes in nitrogen fixing cyanobacteria (2017)
  • Author(s): Fujita, Y.
  • Organizer: 20th International Congress on Nitrogen Fixation
  • Int'l Joint Research / Invited
• [Presentation] 光合成生物における窒素固定のモデルシアノバクテリア：Leptolyngbya boryana (2017)
  • Author(s): 藤田祐一
  • Organizer: 藍藻の分子生物学 2018
  • Invited
• [Presentation] Synechocystis sp. PCC 6803への窒素固定能付与に向けた試み (2017)
  • Author(s): 横溝この実・小谷弘哉・辻本良真・藤田祐一
  • Organizer: 藍藻の分子生物学 2018
• [Presentation] 光合成と窒素固定の酸素パラドクスの統御機構 (2016)
  • Author(s): 藤田祐一
  • Organizer: 第27回フォーラム・イン・ドージン「低酸素応答の光と影～エリスロポエチン純化から40年～」
  • Place of Presentation: 熊本ホテルキャッスル
  • Year and Date: 2016-10-28
  • Invited
• [Presentation] Transfer of cyanobacterial nitrogen fixation genes into the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803. (2016)
  • Author(s): Fujita, Y., Yamakawa, H., Tsujimoto, R., Kotani, H.
  • Organizer: 4th Asian Conference on Plant-Microbe Symbiosis and Nitrogen Fixation
  • Place of Presentation: Penang, Malaysia
  • Year and Date: 2016-10-17
  • Int'l Joint Research
• [Presentation] シアノバクテリアの低酸素適応機構と酸素依存的緑化 (2016)
  • Author(s): 藤田祐一
  • Organizer: 第7回光合成学会年会およびシンポジウム
  • Place of Presentation: 東京理科大
  • Year and Date: 2016-05-28
  • Invited
• [Presentation] 窒素固定性シアノバクテリアLeptolyngbya boryanaにおける窒素固定と光合成の両立に関わる遺伝子の探索 (2016)
  • Author(s): 山川壽伯、小谷弘哉、野中葵、辻本良真、藤田祐一
  • Organizer: 第7回光合成学会年会およびシンポジウム
  • Place of Presentation: 東京理科大
  • Year and Date: 2016-05-27
• [Presentation] 非窒素固定性シアノバクテリアへの窒素固定能付与の試み (2016)
  • Author(s): 小谷弘哉・辻本良真・藤田祐一
  • Organizer: 第57回日本植物生理学会年会
  • Place of Presentation: 盛岡
  • Year and Date: 2016-03-19
• [Presentation] 非窒素固定性シアノバクテリアにおけるCnfR制御を利用した窒素固定遺伝子群の発現 (2016)
  • Author(s): 辻本良真・小谷弘哉・藤田祐一
  • Organizer: 第10回日本ゲノム微生物学会年会
  • Place of Presentation: 東京
  • Year and Date: 2016-03-04
• [Presentation] Nitrogenase-like reductases in bacteriochlorophyll biosynthesis: Reaction mechanism and plasticity. (2016)
  • Author(s): Fujita, Y.
  • Organizer: The 5th International Conference on MEXT Project of Integrated Research on Chemical Synthesis “Chemical Science for Future Societies”
  • Place of Presentation: 名古屋
  • Year and Date: 2016-01-29
  • Int'l Joint Research / Invited
• [Presentation] Coexistence of nitrogen fixation and photosynthesis in the cyanobacterium Leptolyngbya boryana. (2016)
  • Author(s): Kotani, H., and Fujita, Y.
  • Organizer: The 5th International Conference on MEXT Project of Integrated Research on Chemical Synthesis “Chemical Science for Future Societies”
  • Place of Presentation: 名古屋
  • Year and Date: 2016-01-29
  • Int'l Joint Research
• [Presentation] Reaction mechanism of nitrogenase-like protochlorophyllide reductase. (2015)
  • Author(s): Fujita, Y.
  • Organizer: 19th International Congress on Nitrogen Fixation
  • Place of Presentation: Pacific Grove, CA, USA
  • Year and Date: 2015-10-04
  • Int'l Joint Research / Invited
• [Presentation] Loss of the cytM encoding cytochrome cM stimulates dark heterotrophic growth of cyanobacteria. (2015)
  • Author(s): Hiraide, Y., Uesaka, K., Ihara, K. and Fujita, Y.
  • Organizer: International Symposium on Photosynthetic Prokaryotes 2015
  • Place of Presentation: Tubingen, Germany
  • Year and Date: 2015-08-05
  • Int'l Joint Research