JP2023514216A - L-グルタミン酸の生産能が向上したコリネバクテリウムグルタミクム変異株およびこれを用いたl-グルタミン酸の生産方法 - Google Patents
L-グルタミン酸の生産能が向上したコリネバクテリウムグルタミクム変異株およびこれを用いたl-グルタミン酸の生産方法 Download PDFInfo
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Abstract
Description
acterium renale)、コリネバクテリウムポルティソリ(Corynebacterium pollutisoli)、コリネバクテリウムイミタンス(Corynebacterium imitans)、コリネバクテリウムカスピウム(Corynebacterium caspium)、コリネバクテリウムテスツディノリス(Corynebacterium testudinoris)、コリネバクテリウムシュードペラルギ(Corynebacaterium pseudopelargi)、またはコリネバクテリウムフラベセンス(Corynebacterium flavescens)であってもよい。
1-1.コリネバクテリウムデゼルティ(C.deserti)由来の機械感受性イオンチャネル遺伝子が導入されたベクターの製造
機械感受性イオンチャネル遺伝子mscS1を導入するために、コリネバクテリウムデゼルティ(C.deserti GIMN1.010)から染色体DNAを分離、精製した後、鋳型として用いて、下記表1のプライマー3および4を用いてPCR(95℃で30秒、58℃で30秒、72℃で2分、30回繰り返し)で増幅した。
前記1-1のコリネバクテリウムデゼルティの代わりにコリネバクテリウムクルディラクティス(C.crudilactis strain JZ16)を用い、下記表2のプライマーを用いたことを除けば、同様に製造してmscS2遺伝子導入用pKmscS2ベクターを製造した。
前記1-1のコリネバクテリウムデゼルティの代わりにコリネバクテリウムカルナエ(C.callunae DSM 20147)を用い、下記表3のプライマーを用いたことを除けば、同様に製造してmscS3遺伝子導入用pKmscS3ベクターを製造した。
機械感受性イオンチャネル遺伝子mscS3の107番目のアミノ酸残基を置換して導入するために、コリネバクテリウムカルナエ(C.callunae DSM 20147)から染色体DNAを分離、精製した後、鋳型として用いて、下記表4のプライマー1および2、プライマー3および4を用いてPCR(95℃で30秒、58℃で30秒、72℃で2分、30回繰り返し)で増幅した。
前記1-4のプライマーの代わりに下記表5のプライマーを用いたことを除けば、同様に製造してmscS3遺伝子の107番目のロイシン残基をバリンに置換する変異導入用pKmscS3-L107Vベクターを製造した。
コリネバクテリウムグルタミクムKCTC 11558BP菌株の形質転換のための方法としてvan der Restなどの方法に基づいて修飾したエレクトロコンピテントセル(electrocompetent cell)の製造法を使用した。
から成長性のある菌株を選別した。得られたコロニーを前記表1~3の各プライマー7および8を用いてmscS1、mscS2、mscS3遺伝子がそれぞれ導入されたコリネバクテリウムグルタミクム変異株(IS1、IS2、IS3)を確認し、前記表4および5の各プライマー5および6を用いてmscS3-L107A、mscS3-L107V遺伝子がそれぞれ導入されたコリネバクテリウムグルタミクム変異株(IS3-A、IS3-V)を確認した。
実施例1で製造されたmscS1、mscS2、mscS3遺伝子導入変異株(IS1、IS2、IS3)と、親菌株であるコリネバクテリウムグルタミクムKCTC 11558BP菌株に対して、L-グルタミン酸の生産性を比較した。
実施例1で製造されたmscS3、mscS3-L107A、mscS3-L107V遺伝子導入変異株(IS3、IS3-A、IS3-V)と、親菌株であるコリネバクテリウムグルタミクムKCTC 11558BP菌株に対して、L-グルタミン酸の生産性を比較した。
これまで本発明についてその好ましい実施例を中心に説明した。本発明の属する技術分野における通常の知識を有する者は、本発明が本発明の本質的な特性を逸脱しない範囲で変形された形態で実現できることを理解するであろう。そのため、開示された実施例は、限定的な観点ではなく、説明的な観点で考慮されなければならない。本発明の範囲は上述した説明ではなく、特許請求の範囲に示されており、それと同等の範囲内にあるすべての差異は本発明に含まれたものと解釈されなければならない。
Claims (5)
- コリネバクテリウム属(Corynebacterium sp.)菌株由来の機械感受性イオンチャネル遺伝子(mechanosensitive ion channel)を含み、L-グルタミン酸の生産能が向上したコリネバクテリウムグルタミクム(Corynebacterium glutamicum)変異株。
- 前記コリネバクテリウム属菌株は、コリネバクテリウムデゼルティ(Corynebacterium deserti)、コリネバクテリウムクルディラクティス(Corynebacterium crudilactis)およびコリネバクテリウムカルナエ(Corynebacterium callunae)からなる群より選択された1種以上である、請求項1に記載のコリネバクテリウムグルタミクム変異株。
- 前記機械感受性イオンチャネル遺伝子は、配列番号1~5の塩基配列のいずれか1つに暗号化されたものである、請求項1に記載のコリネバクテリウムグルタミクム変異株。
- コリネバクテリウム属(Corynebacterium sp.)菌株由来の機械感受性イオンチャネル遺伝子を導入するステップを含む、請求項1に記載のコリネバクテリウムグルタミクム変異株の製造方法。
- i)請求項1に記載のコリネバクテリウムグルタミクム変異株をL-グルタミン酸生産培地で培養するステップと、
ii)前記変異株または変異株が培養された培養液からL-グルタミン酸を回収するステップと
を含むL-グルタミン酸の生産方法。
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- 2020-08-26 CN CN202080096445.8A patent/CN115135767B/zh active Active
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EP4105331A4 (en) | 2024-03-06 |
JP2024096439A (ja) | 2024-07-12 |
CN115135767A (zh) | 2022-09-30 |
WO2021162189A1 (ko) | 2021-08-19 |
CN115135767B (zh) | 2024-03-15 |
US20230136217A1 (en) | 2023-05-04 |
KR102269639B1 (ko) | 2021-06-25 |
EP4105331A1 (en) | 2022-12-21 |
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