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Analysis of an antimicrobial metabolite and its biosynthetic gene of a fungal endophyte

PUREV, Enkhee 名古屋大学

2020.05.14

概要

エンドファイト(内生菌)は植物の組織内部に共生する微生物で、生理活性物質を生産することで宿主植物の耐病性、耐虫性、動物による食害や環境ストレスに対する抵抗性の向上に寄与することが知られている。ニュージーランドでは、家畜毒素生産性を抑え耐虫性を残した牧草が広く牧畜に利用されている。 また、エピクロエ ( Epichloё)属エンドファイトはイネ科植物に共生する子嚢菌門の真菌で、芝の性能向上など商業的に広く利用されている。代表的なエピクロエエンドファイト Epichloё festucae も種々の生理活性物質を生産するが、最近、ある転写因子をコードする遺伝子 vibA を過剰発現させると未知の抗真菌物質の生産が顕著に観測されることが明らかになった。本博士論文研究では、この未知の生理活性物質の化学的解明と生合成機構の解明を目的としている。

まず、先行研究で樹立されていた Epichloё festucae E437 株の vibA 過剰発現変異体 Ptef::VibA (E437)を液体培養し、イネ科植物斑点病菌 Drechslera erythrospila の胞子発芽阻害活性を指標にして、活性を示した培養ろ液を各種クロマトグラフィーにより分画し、活性物質の精製に成功した。NMR や MALDI-MS の解析、および改良 Mosher法による絶対立体配置決定により、活性物質の構造を ε-poly-L-lysine (以下ε-PL)と決定した(右図)。また質量分析の結果より、L-リジン残基の重合度を 28-34 と決定した。この結果は、放線菌由来のε-PL 標品との比較により確定した。

ε-PL の生産性は、E437 野生株の 10 mg/L に対し変異株では 39 mg/mL と、vibA 遺伝子の過剰発現により約4倍に向上した。一方、ε-PL を生合成できない別の菌株 E. festucae Fl1 についても vibA 過剰発現株を樹立したところ、2.7 mg/L の生産性を示した。この結果から、いずれの株もε-PL 生合成能を有しており、転写因子 VibA が何らかの関与によりε-PL 生合成を促進・覚醒していることが示唆された。

次にε-PL 生合成遺伝子 pls の同定を試みた。この遺伝子は放線菌などの細菌では知られているが真菌では報告がなかったので、E. festucae ゲノム配列を使ってホモロジー検索を行ったところ、アミノ酸レベルで 51%の類似性をもつ遺伝子 epls が見い出された。そこで E437 株と Fl1 株の両方についてこの真菌ε-PL 生合成遺伝子 epls の過剰発現株を、vibA 過剰発現株と同様の方法を用いて樹立した。得られた株の生産性を調べた結果、前者由来の変異株 Ptef::Epls (E437)は 70 mg/L、後者由来の変異株 Ptef::Epls (Fl1)は 14 mg/L と、vibA 過剰発現よりさらに高い生産性(それぞれ 1.8 倍、5 倍) を示した。以上の研究により、2種の株それぞれから2種の遺伝子の過剰発現株、すなわち4種の変異株が得られたので、これらが生産するε-PL の重合度を比較したところ、 E437 株は高い重合度( 28-34, 26-33)、Fl1 は低い重合度( 8-18, 8-20)と宿主となる株により大きく異なることが分かった。今のところ、この理由は不明である。

最後に、精製したε-PL の植物病原菌に対する抗真菌活性を評価した。その結果、重合度の違いに関わらず、D. erythrospila に対し 10 g/mL で、Botrytis cinerea および Phytophthora infestans に対し 1 g/mL でその胞子発芽を阻害した。一方、真菌の生育(菌糸成長)に対しては、300 g/disk で D. erythrospila と P. capsici に対し弱い阻害活性を示すのみであった。

以上のように、本研究により真菌のε-PL 生合成酵素 epls が初めて同定され、その過剰発現がε-PL の生産性を大きき向上させること、転写因子 VibA が生産生の促進に未知の経路で強く関与することが明らかとなった。ε-PL は防腐剤として食品添加剤に用いられる安全性の高い化合物であることから、本研究成果は、epls 高発現エピクロエエンドファイトを感染(共生)させることにより耐病性作物を育種するという魅力的な方法論を示唆するものと言える。

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参考文献

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