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Modification of the protein secretory system in wood rot fungi

Research Project

Project/Area Number 18H02254
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 40020:Wood science-related
Research InstitutionKyoto University

Principal Investigator

本田 与一  京都大学, 農学研究科, 教授 (70252517)

Co-Investigator(Kenkyū-buntansha) 中沢 威人  京都大学, 農学研究科, 助教 (80608141)
河内 護之  京都大学, 農学研究科, 特定助教 (70771294)
Project Period (FY) 2018-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000)
Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2020: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Fiscal Year 2019: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Fiscal Year 2018: ¥10,270,000 (Direct Cost: ¥7,900,000、Indirect Cost: ¥2,370,000)
Keywords担子菌 / 分泌系 / 木材腐朽菌 / リグニン分解 / タンパク質分泌系 / 細胞内小器官 / 白色腐朽菌 / タンパク質分泌 / 木材分解酵素 / 遺伝子組換え / リグニン分解酵素 / 分泌酵素 / ゲノム編集 / 糖鎖修飾
Outline of Annual Research Achievements

木材腐朽菌による木質分解酵素の生産に関する研究では、従来、遺伝子発現(転写)と、実際に分泌生産される酵素量との間にギャップがあり、タンパク質分泌系 の機能強化が重要であることが指摘されてきた。本研究課題では、申請者らが開発した様々な分子遺伝学研究ツールを用いることで、腐朽菌のタンパク質分泌系のボトルネックとなり得るステップ群) について明らかにし、ゲノム改変によるネックの解消を行って分泌タンパク質の高生産が可能な株の育種を目指す。 リグニン分解酵素の転写量を人為的に高めた場合に、それに見合う酵素活性が見られない理由としてこれまで「翻訳後調節機構」の存在が提案されてきたが、実 際に解析した例はない。本課題ではこれまでに蛍光タンパク質(mCherry)やルシフェラーゼタンパク質をレポーターとして用い、 ゲノムよりサーベイしてきた各細胞内小器官に存在すると考えられるタンパク質のシグナル配列と連結することで、細胞内のタンパク質分泌系路について、蛍光顕微鏡を用いて分子細胞生物学的に解析を行った。また、木材腐朽菌におけるゲノム編集系の開発を進め、任意のゲノム編集を可能とする技術基盤の開発にも取り組んでいる。その結果、モデル木材腐朽菌ヒラタケにおけるCRISPR/Cas9を用いたゲノム編集システムの開発に成功し、さらにin vitroでCas9タンパク質とgRNAを会合させたものを直接プロトプラストに導入する技術の開発に成功した。この結果は、将来の木材腐朽菌によるタンパク質分泌系の解明とその制御を可能にする画期的なものである。

Research Progress Status

令和3年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和3年度が最終年度であるため、記入しない。

Report

(4 results)
  • 2021 Annual Research Report
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • 2018 Annual Research Report

Research Products

(15 results)

All 2022 2021 2020 2019 2018

All Journal Article (2 results) (of which Peer Reviewed: 2 results,  Open Access: 2 results) Presentation (13 results) (of which Int'l Joint Research: 10 results,  Invited: 6 results)

  • [Journal Article] Gene targeting using pre-assembled Cas9 ribonucleoprotein and split-marker recombination in <i>Pleurotus ostreatus</i>2021

    • Author(s)
      Boontawon Tatpong、Nakazawa Takehito、Xu Haibo、Kawauchi Moriyuki、Sakamoto Masahiro、Honda Yoichi
    • Journal Title

      FEMS Microbiology Letters

      Volume: 368 Pages: 1-1

    • DOI

      10.1093/femsle/fnab080

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus2021

    • Author(s)
      Boontawon Tatpong、Nakazawa Takehito、Inoue Chikako、Osakabe Keishi、Kawauchi Moriyuki、Sakamoto Masahiro、Honda Yoichi
    • Journal Title

      AMB Express

      Volume: 11 Pages: 1-1

    • DOI

      10.1186/s13568-021-01193-w

    • NAID

      120006960305

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] Genome editing and functional analysis of wood-rot fungus, Pleurotus ostreatus2022

    • Author(s)
      Yoichi Honda, Iori Yamaguchi, Fuga Yamasaki, Takehito Nakazawa, Moriyuki Kawauchi, Masahiro Sakamoto, Minji Oh
    • Organizer
      ICBE2021
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Genome editing in mushroom-forming fungi with CRISPR/Cas9 technology2022

    • Author(s)
      Yoichi Honda;Fuga Yamasaki;Daishiro Koshi;Takeshi Kobukata;Takehito Nakazawa;Moriyuki Kawauchi;Masahiro Sakamoto;Minji Oh
    • Organizer
      KSM 50th anniversary international meeting
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Genome editing to fight with, not against, post-genomics in mushroom fungi2020

    • Author(s)
      Yoichi Honda
    • Organizer
      IUMS2020
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] CRISPR/Cas9 in mushrooms without integration of ectopic DNA2020

    • Author(s)
      Chikako INOUE, Dong Xuan NGUYEN, Takehito NAKAZAWA, & Masahiro SAKAMOTO Yoichi HONDA
    • Organizer
      ECFG2020
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Distribution and shape of organelles visualized with fluorescent proteins in the agaricomycete Pleurotus ostreatus2019

    • Author(s)
      Shivani, Takehito Nakazawa, Masahiro Sakamoto, Yoichi Honda
    • Organizer
      Asia Mycological Congress
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Distribution and shape of organelles visualized with fluorescent proteins in the agaricomyete Pleurotus ostreatus2019

    • Author(s)
      Shivani, Takehito Nakazawa, Masahiro Sakamoto, Yoichi Honda
    • Organizer
      シンポジウム IFOが繋ぐ京大微生物学のフロントライン
    • Related Report
      2019 Annual Research Report
  • [Presentation] Molecular genetics and genome editing in white rot fungus, Pleurotus ostreatus2018

    • Author(s)
      Yoichi Honda
    • Organizer
      Xiamen Forum on Biomass Frontiers
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Molecular genetic approaches beyond the post-genomic era2018

    • Author(s)
      Yoichi Honda
    • Organizer
      9th International Conference on Mushroom Biology and Mushroom Products
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Genome editing with CRISPR/Cas9 system in Pleurotus ostreatus: toward non-GM molecular breeding2018

    • Author(s)
      Takehito Nakazawa
    • Organizer
      SPIRITS 2018 Workshop on ‘Genetic diversity and molecular breeding in cultivated mushrooms’
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] ヒラタケ及びCeriporiopsis subvermisporaにおけるCRISPR/Cas9によるゲノム編集系の確立2018

    • Author(s)
      井上 智香子,中沢 威人, Dong Xuan NGUYEN,刑部 敬史,坂本 正弘, 本田 与一
    • Organizer
      日本きのこ学会大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] ヒラタケにおける分泌型レポーターアッセイシステムの構築2018

    • Author(s)
      坂 知奈美,中沢 威人,湯村 直樹,竹中 敦紀,Dong Xuan Nguyen, 坂本 正弘,本田 与一
    • Organizer
      日本きのこ学会大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Efficient Gene Disruption with CRISPR/Cas9 system in Pleurotus ostreatus and Ceriporiopsis subvermispora2018

    • Author(s)
      Chikako INOUE, Takehito NAKAZAWA, Xuan Dong NGUYEN, Keishi OSAKABE, Masahiro SAKAMOTO, and Yoichi HONDA
    • Organizer
      9th International Conference on Mushroom Biology and Mushroom Products
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Reporter Assay System for Secretory Pathway in Pleurotus ostreatus2018

    • Author(s)
      Chinami SAKA, Takehito NAKAZAWA, Naoki YUMURA, Atsuki TAKENAKA, Dong Xuan NGUYEN, Masahiro SAKAMOTO and Yoichi HONDA
    • Organizer
      9th International Conference on Mushroom Biology and Mushroom Products
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research

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Published: 2018-04-23   Modified: 2022-12-28  

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