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Development of novel multi-copy gene integration method in genetic engineering of filamentous fungi

Research Project

Project/Area Number 16K14887
Research Category

Grant-in-Aid for Challenging Exploratory Research

Allocation TypeMulti-year Fund
Research Field Applied microbiology
Research InstitutionKobe University

Principal Investigator

Wakai Satoshi  神戸大学, 科学技術イノベーション研究科, 特命准教授 (50545225)

Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
KeywordsAspergillus oryze / ナノポアシーケンス / ゲノム構造 / 遺伝子組換え / タンデムリピート / 長鎖DNA合成 / 麹菌 / マルチコピー導入 / ナノポアシーケンサ / MinION / 分子生物学 / 糸状菌 / マルチコピー / 次世代シーケンス / 長鎖DNA / 多重遺伝子導入 / 多重遺伝子破壊 / セルラーゼ / 遺伝子破壊
Outline of Final Research Achievements

Filamentous fungus Aspergillus oryzae can be genetically engineered by co-transformation. In co-transformation, external gene fragments are integrated at random and at multiple copy on the chromosome. Therefore, positions and copy number of integrated genes are unknown and uncontrollable. In this study, I revealed that copy numbers of integrated genes in the co-transformation was altered depending on the edge sequences of integrated gene fragment. Furthermore, I successfully determined the position and copy number of integrated genes on the chromosome of some genetically engineered strains using nanopore sequencer MinION, and firstly detected long tandem repeat structure of integrated genes at length in over 50~100 kb. In the future, this result will contribute to develop in vivo construction method of artificially designed long chain DNA fragment in eukaryotic cell.

Academic Significance and Societal Importance of the Research Achievements

近年、持続可能な社会の形成を目指して、合成生物学の分野の研究が進められている。合成生物学分野では、種々の遺伝子組み換え技術を用いて、有用で安全な微生物をさらに有用な微生物へと育種し、様々な有用物質を生産することが求められている。本研究では、日本において1000年以上の食文化での使用実績がある麹菌の安全性とポテンシャルに可能性を見出し、複雑な遺伝子改変を小さな労力で達成し、迅速に有用な遺伝子組換え麹菌を作成する技術の作成を目指した。結果として、コピー数多型をコントロールする技術や生体内で数10kbを越える長鎖DNAを合成する技術の開発に成功した。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Research-status Report
  • 2016 Research-status Report
  • Research Products

    (16 results)

All 2019 2018 2017 2016

All Journal Article (2 results) (of which Peer Reviewed: 2 results) Presentation (14 results) (of which Int'l Joint Research: 5 results,  Invited: 1 results)

  • [Journal Article] Modified expression of multi-cellulases in a filamentous fungus Aspergillus oryzae2019

    • Author(s)
      Wakai Satoshi、Nakashima Nanami、Ogino Chiaki、Tsutsumi Hiroko、Hata Yoji、Kondo Akihiko
    • Journal Title

      Bioresource Technology

      Volume: 276 Pages: 146-153

    • DOI

      10.1016/j.biortech.2018.12.117

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Future insights in fungal metabolic engineering2017

    • Author(s)
      Wakai Satoshi、Arazoe Takayoshi、Ogino Chiaki、Kondo Akihiko
    • Journal Title

      Bioresource Technology

      Volume: 245 Pages: 1314-1326

    • DOI

      10.1016/j.biortech.2017.04.095

    • Related Report
      2017 Research-status Report 2016 Research-status Report
    • Peer Reviewed
  • [Presentation] Comparative genome analysis of genetically engineered Aspergillus oryzae strains by long-read sequencing using nanopore sequencer2019

    • Author(s)
      Satoshi Wakai, Silai Zhang, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo
    • Organizer
      10th International symposium of Innovative BioProduction Kobe
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] ナノポアシーケンサを用いた遺伝子組換え麹菌のゲノミクスおよびトランスクリプトミクス2019

    • Author(s)
      若井暁、張斯来、荻野千秋、堤浩子、秦洋二、近藤昭彦
    • Organizer
      日本農芸化学会2019年度大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 2)Tandem-repeat structure of integrated genes on the genomes of genetically engineered Aspergillus oryzae strains2019

    • Author(s)
      Satoshi Wakai, Silai Zhang, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, Akihiko Kondo
    • Organizer
      ASM Microbe 2019
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] ナノポアシーケンサを用いた遺伝子組換え麹菌ゲノム上の長鎖タンデムリピート構造の解析2018

    • Author(s)
      若井暁、荻野千秋、堤浩子、秦洋二、近藤昭彦
    • Organizer
      日本生物工学会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 多彩なアウトプット能を示すAspergillus細胞工場2018

    • Author(s)
      若井暁
    • Organizer
      第8回合成生物工学シンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] マルチコピー遺伝子導入黄麹菌のゲノム構造解析2018

    • Author(s)
      若井暁、荻野千秋、堤浩子、秦洋二、近藤昭彦
    • Organizer
      第18回糸状菌分子生物学コンファレンス
    • Related Report
      2018 Annual Research Report
  • [Presentation] 遺伝子組み換え麹菌培養上清カクテルを用いた希少糖高生産プロセスの開発2017

    • Author(s)
      若井 暁、浅井 菜々実、荻野 千秋、堤 浩子、秦 洋二、矢追 克郎、松沢 智彦、近藤 昭彦
    • Organizer
      日本農芸化学会2017年度大会
    • Place of Presentation
      京都女子大(京都)
    • Year and Date
      2017-03-17
    • Related Report
      2016 Research-status Report
  • [Presentation] Construction of multi-cellulases expressing Aspergillus oryzae as host microorganism for bioproduction from cellulase biomass2017

    • Author(s)
      Satoshi Wakai, Nanami Asai, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, and Akihiko Kondo
    • Organizer
      The 8th international symposium of innovative BioProduction Kobe
    • Place of Presentation
      Kobe Univ. (Kobe)
    • Year and Date
      2017-02-03
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research
  • [Presentation] 麹菌のco-transformation時のコピー数多型性に関する解析2017

    • Author(s)
      若井暁、荻野千秋、堤浩子、秦洋二、近藤昭彦
    • Organizer
      第17回糸状菌分子生物学コンファレンス
    • Related Report
      2017 Research-status Report
  • [Presentation] バイオマスの効率的な分解を志向したマルチセルラーゼ高発現麹菌の開発2017

    • Author(s)
      片山 周平、若井 暁、浅井 菜々実、荻野 千秋、堤 浩子、秦 洋二、近藤 昭彦
    • Organizer
      第19回化学工学会学生発表会(大阪大会)
    • Place of Presentation
      大阪大学(大阪)
    • Related Report
      2016 Research-status Report
  • [Presentation] マルチセルラーゼ発現黄麹菌の培養条件によるセルラーゼ生産性の違い2016

    • Author(s)
      片山 周平、若井 暁、浅井 菜々実、荻野 千秋、堤 浩子、秦 洋二、近藤 昭彦
    • Organizer
      第16回糸状菌分子生物学コンファレンス
    • Place of Presentation
      京都大学(京都)
    • Year and Date
      2016-11-17
    • Related Report
      2016 Research-status Report
  • [Presentation] 三種セルラーゼ共・強発現麹菌によるセルロースからのコウジ酸生産2016

    • Author(s)
      若井 暁、浅井 菜々実、荻野 千秋、堤 浩子、秦 洋二、近藤 昭彦
    • Organizer
      第68回日本生物工学会大会
    • Place of Presentation
      富山国際会議場(富山)
    • Year and Date
      2016-09-28
    • Related Report
      2016 Research-status Report
  • [Presentation] Direct Fermentation of L-Lactic Acid from Starch by Genetically Engineered Aspergillus oryzae2016

    • Author(s)
      Satoshi Wakai, Nanami Asai, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, and Akihiko Kondo
    • Organizer
      Metabolic Engineering 11
    • Place of Presentation
      Awaji Yumebutai International Conference Center (Hyogo)
    • Year and Date
      2016-06-26
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research
  • [Presentation] Simultaneous Saccharification and Fermentation of L-Lactic Acid Production from Starch by Genetically Engineered Aspergillus oryzae2016

    • Author(s)
      Satoshi Wakai, Nanami Asai, Chiaki Ogino, Hiroko Tsutsumi, Yoji Hata, and Akihiko Kondo
    • Organizer
      ASM Microbe 2016
    • Place of Presentation
      Boston (USA)
    • Year and Date
      2016-06-16
    • Related Report
      2016 Research-status Report
    • Int'l Joint Research

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Published: 2016-04-21   Modified: 2020-03-30  

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