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麹菌Aspergillus oryzaeにおける新規二次代謝制御因子KpeAの機能解析

荒川 弦矢 東京農業大学

2020.05.12

概要

本研究では,まず初めに麹菌 Aspergillus oryzae における新規二次代謝制御因子の探索を目的としてコウジ酸の生産量を指標としたスクリーニングを行い,破壊するとコウジ酸生産が増加する遺伝子 kpeA を見出した。KpeA はZn(II)2-Cys6 型の制御因子であったが,通常 N 末端に存在するモチーフがアミノ酸配列の中央に存在する,ユニークな構造をしていた。また,KpeA は子嚢菌類の内の糸状菌に広く保存された制御因子であったが,子嚢菌類でも酵母や担子菌類,接合菌類には保存されていなかった。

KpeA は二次代謝の他に分生子形成も制御していた。kpeA 破壊株の分生子数と頂のう数が減少することからKpeA が分生子形成に関わることが明らかになり,さらに転写解析から KpeA が BrlA を介して分生子形成を制御することが明らかになった。

次に kpeA 破壊株のコウジ酸生産量の定量を行ったところ,kpeA 破壊株はE-F1 株の 6 倍のコウジ酸生産を示し,さらに転写解析から KpeA がコウジ酸生産を転写レベルで抑制していることが明らかになった。また,KpeA はコウジ酸だけでなく,ペニシリンやシクロピアゾン酸の生産制御にも関わることが明らかになった。

KpeA はモチーフの位置が特徴的な Zn(II)2-Cys6 型制御因子であったが,KpeA のような構造の制御因子は 186 個推定されているZn(II)2-Cys6 型制御因子の内に 4 個しかなく,KpeA のモチーフが偶然生じた機能しない配列であることが懸念された。そこで,モチーフ内の Cys 残基を Ala 残基に置換したAla 置換株を作製し,その形質を比較した。その結果,Ala 置換株は kpeA破壊株と同様の表現型を示し,KpeA のZn(II)2-Cys6 モチーフが機能していることが明らかになった。

醸造現場では麹菌を蒸米上で培養する。このような培養を固体培養と呼ぶが,固体培養における二次代謝や分生子形成の分子レベルの研究はされていない。そこで,液体培養や平板培養において二次代謝と分生子形成の制御に重要な役割を果たしていることが明らかになった KpeA について解析を行った。kpeA 破壊株と高発現株を用いた解析から,KpeA が種麹製造及び製麹において分生子形成を促進していることが明らかになり,転写解析から KpeA が固体培養においても BrlA を介して分生子形成を制御していることが明らかになった。また,米麹に含まれるコウジ酸の正確な量は報告されていなかったが,本研究で HPLC による解析から E-F1 株の米麹に 27.8 mg/kg·koji のコウジ酸が含まれていることが明らかになった。kpeA 破壊株の米麹にはE-F1 株の米麹の 4 倍以上のコウジ酸が含まれており,KpeA が製麹中にコウジ酸生産を制御していることが明らかになった。さらに,KpeA と褐変について検討した。褐変の代替指標として米麹抽出液の褐変を調べたところ,kpeA 破壊株の米麹抽出液が褐変しなかった。そこで,各株のチロシナーゼ活性を測定したが,kpeA 破壊株のチロシナーゼ活性はE-F1 株よりも高かった。 kpeA 破壊株の米麹抽出液が褐変しなかった現象の原因が米麹に含まれるコウジ酸であると考え,E-F1 株の米麹抽出液に kpeA 破壊株と同濃度になるようにコウジ酸を添加したところ,E-F1株の米麹抽出液が褐変しなかった。この結果から,米麹中のコウジ酸が褐変や黒粕を抑制することが示唆された。

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