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Corynebacterium glutamicumにおけるグルタミン酸生産時の代謝フラックス変化とアシル化修飾の関連

庄司(永野), 愛 東京大学 DOI:10.15083/0002002709

2021.10.27

概要

Corynebactrium glutamicumはTween40添加等の刺激によりグルタミン酸を過剰生産する。グルタミン酸生産時には中央代謝経路のフラックスが大きく変化するが、TCA回路とグルタミン酸生成の代謝分岐にあたる2-オキソグルタル酸デヒドロゲナーゼ(ODH)の活性調節以外に、代謝フラックス変化に関わる因子やフラックス変化の詳細なメカニズムはこれまで明らかにされていない。C. glutamicumATCC13032株を対象に実施されたアシローム解析により、グルタミン酸生産条件では中央代謝経路の多くの酵素においてアセチル化が抑制されスクシニル化が促進することが見出された。アセチル化やスクシニル化などのアシル化修飾は、生物種を超えて保存された翻訳後修飾として注目されており、アシル化修飾による代謝酵素の活性調節の例が知られている。そこで、C. glutamicumのグルタミン酸生産時に見出されたアシル化修飾の変化が、代謝フラックスの変化やグルタミン酸生産に影響を及ぼす可能性に着目し、アシル化修飾の制御機構とグルタミン酸生産と関連の深いタンパク質のアシル化修飾の機能解明を本研究の目的とした。
 本論文は、第1章の「序論」を含め5章から構成される。第2章の「C. glutamicumにおけるアシル化修飾の制御機構の解析」では、アシル化修飾に用いられる基質の供給経路に着目し、これまで知られていなかったC. glutamicumにおけるアシル化修飾の制御機構の一端を解明した。第3章の「ホスホエノールピルビン酸カルボキシラーゼ(PEPC)におけるアセチル化修飾の機能解析」では、グルタミン酸生産に重要なオキサロ酢酸供給の補充経路酵素であるPEPCのアシル化修飾に着目し、アセチル化によるPEPC活性の制御機構を明らかにした。第4章の「OdhIにおけるスクシニル化修飾の機能解析」では、ODH活性の調節因子であるOdhIのアシル化修飾に着目し、スクシニル化によるOdhI活性制御の存在を明らかにした。最後に、第5章の「総括と今後の展望」において本論文の内容の総括を行い、C. glutamicumのグルタミン酸生産におけるアシル化修飾の意義について考察した。

C. glutamicumにおけるアシル化修飾の制御機構の解析(第2章)
 本章では、アセチル化・スクシニル化基質の供給に関わる酵素や脱アセチル化酵素(KDAC)ホモログに着目して、C. glutamicumにおけるアシル化修飾の制御機構の解明を目指した。非酵素的アセチル化の基質となるアセチルリン酸の代謝には、phosphotransacetylase(Pta)と、acetate kinase(Ack)が関与している。アセチルCoAからのアセチルリン酸生成反応を担うptaの破壊株でタンパク質全体のアセチル化レベルが減少したことから、アセチルリン酸による非酵素的アセチル化が働いていることが示唆された。また、Pta-Ack経路酵素の破壊によるアセチル化レベルへの影響が、グルタミン酸非生産条件でのみ見られたことから、グルタミン酸生産条件ではPta-Ack経路の代謝フラックスが減少している可能性が示された。続いて、アセチル化ライゼートを基質にしたin vitroでの検討により、C. glutamicumが有する2つのSirtuin型KDACホモログのうち、NCgl0616が脱アセチル化活性を有することを確認した。また、グルタミン酸生産条件でスクシニル化レベルが増加するメカニズムとしてグリオキシル酸経路の関与を検討したが、グリオキシル酸経路酵素の破壊はスクシニル化修飾レベルに影響を与えなかった。

PEPCにおけるアセチル化修飾の機能解析(第3章)
 グルタミン酸生産に重要なオキサロ酢酸供給の補充経路酵素に着目し、PEPCのアセチル化の役割とその制御機構、グルタミン酸生産における意義について調べた。本研究で用いたC. glutamicumATCC13032株は、オキサロ酢酸供給の補充経路としてPEPCとピルビン酸カルボキシラーゼ(PC)を保有しているが、そのうちPEPCがグルタミン酸生産時の主要な補充経路酵素として働くことを明らかにした。グルタミン酸生産時に修飾量が変動するPEPCのアシル化修飾部位のうち、グルタミン酸生産条件でアセチル化修飾量が減少する653番目のLys残基(K653)に着目した。PEPC-K653のアセチル化修飾模倣変異株(K653Q)におけるグルタミン酸生産は、非アシル化修飾模倣変異株(K653R)と比較して著しく低下したことから、K653のアセチル化はグルタミン酸生産に負の影響を与える可能性が示唆された。グルタミン酸生産の結果と対応して、K653Q変異酵素の触媒効率(kcat/Km)は、K653R変異酵素と比べて10分の1以下に低下していた。遺伝暗号を拡張したin vitro翻訳系を用いて653部位特異的にアセチルリジンを導入したPEPC(PEPC-K653Ac)を調製し活性測定を行ったところ、PEPC-K653Acの比活性はアセチルリジンを含まないPEPC-K653と比較して25分の1まで低下した。これらの結果から、K653のアセチル化はPEPC活性を阻害すると結論づけた。また、Sirtuin型KDACであるNCgl0616がPEPC-K653Acに対して脱アセチル化活性を示したことから、NCgl0616によるK653の脱アセチル化がPEPC活性化のメカニズムに関与する可能性が示唆された。NCgl0616によるPEPC-K653脱アセチル化の意義を明らかにするため、野生株、PEPC-K653R変異株、KDAC破壊株を用いてグルタミン酸非生産条件・生産条件におけるPEPC比活性を調べた。その結果、野生株ではグルタミン酸生産時のPEPC比活性は非生産条件と比較して増加を示したのに対し、PEPC-K653R変異株及びKDAC破壊株では比活性の増加は見られなかった。以上より、グルタミン酸生産時に見られるPEPC比活性の増加にNCgl0616によるK653の脱アセチル化が寄与していることが示された。最後に、グルタミン酸生産におけるアセチル化を介したPEPC活性調節の意義を考察した。

OdhIにおけるスクシニル化修飾の機能解析(第4章)
 グルタミン酸生産に重要なODH活性の制御因子OdhIにおけるスクシニル化の機能について解析した。グルタミン酸生産はodhI破壊株では顕著に低下し、odhI過剰発現株では増加することから、OdhIはグルタミン酸生産に重要な因子であると考えられる。OdhIのN末端ドメインにある14番目のThr残基(T14)のリン酸化は、OdhIのフォールディング変化を引き起こしOdhAサブユニットに対するOdhIの結合能を低下させることでODH活性の阻害効果を弱めることが報告されている。アシル化修飾模倣変異体を用いた検討から、OdhIの132番目のリジン残基(K132)のスクシニル化もまたOdhAサブユニットに対するOdhIの結合能を低下させ、ODH活性の阻害効果を弱めることが示唆されていた。そこでin vitroでスクシニル化したOdhIを用いて、模倣変異で示唆されたスクシニル化の役割を明らかにすることを目指した。その結果、OdhI-K132はスクシニルCoAによって非酵素的にスクシニル化されること、またスクシニル化依存的にOdhI活性が抑制されること、K132R変異によってスクシニル化によるOdhIの活性抑制が見られなくなることを示した。これらの結果から、模倣変異を用いた解析から示唆されたように、K132のスクシニル化はOdhIのOdhAへの結合を阻害することでODH活性を維持し、グルタミン酸生産に負の影響を与えると考えられた。また、K132がスクシニル化されていてもリン酸化によるOdhIの活性抑制には影響がなかったことから、OdhIの制御においてスクシニル化はリン酸化による制御に干渉しないことが示唆された。最後に、OdhIが2つの翻訳後修飾によって制御される意義として、非リン酸化型OdhIタンパク質が多量に存在するグルタミン酸生産条件において代謝状態を反映したスクシニル化による制御の必要性について考察した。

総括と今後の展望
 本研究により、アセチル化やスクシニル化がPEPCやOdhIの機能調節を介して、グルタミン酸生産に影響を及ぼすことを明らかにした。また、アセチル化基質の供給に関わるPta-Ack経路酵素やPEPCの破壊がアセチル化レベルに及ぼした影響から、PEPを起点とした代謝フラックスの切り替えが起きていることを見出した。グルタミン酸生産時に観察されるアセチル化修飾の変化は、PEPを起点としたフラックス変化を反映したものであると考えられる。これまでにグルタミン酸生産条件で補充経路の代謝フラックスが上昇することは示唆されていたが、本研究はこれをアセチル化の視点から捉えなおしたと言える。PEPC-K653の脱アセチル化はグルタミン酸生産時のPEPCフラックスの維持に寄与していると考えられるが、PEPC-K653の脱アセチル化がフラックス切り替えのトリガーであるかどうかは不明であり、今後の課題である。本研究は、グルタミン酸生産だけでなく他の有用物質生産においてもアシル化修飾を標的とした代謝改変の意義について重要な知見を与えることが期待される。

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