JP7475409B2 - L-アルギニンまたはl-シトルリン生産能が向上したコリネバクテリウム属微生物およびこれを用いたl-アルギニンまたはl-シトルリンの生産方法 - Google Patents
L-アルギニンまたはl-シトルリン生産能が向上したコリネバクテリウム属微生物およびこれを用いたl-アルギニンまたはl-シトルリンの生産方法 Download PDFInfo
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- JP7475409B2 JP7475409B2 JP2022162572A JP2022162572A JP7475409B2 JP 7475409 B2 JP7475409 B2 JP 7475409B2 JP 2022162572 A JP2022162572 A JP 2022162572A JP 2022162572 A JP2022162572 A JP 2022162572A JP 7475409 B2 JP7475409 B2 JP 7475409B2
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- arginine
- corynebacterium
- citrulline
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Description
ynebacterium 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)であってもよいが、これに限定されるものではない。
アセチルグルタメートキナーゼの活性が強化されたコリネバクテリウムグルタミカム変異株を製造するために、L-アルギニン生産菌株であるコリネバクテリウムグルタミカム14GR(KCCM13219P)菌株およびE.coli DH5a(HIT Competent cellsTM、Cat No.RH618)を使用した。
菌株に生合成経路を強化させるためにアセチルグルタメートキナーゼに変異を誘導した。本実施例で用いた方法は、アセチルグルタメートキナーゼを暗号化しているargB遺伝子の発現を増加するために、当該遺伝子の特定位置に変異を誘発した。argB遺伝子の258番目のアミノ酸をグルタミンからロイシンに置換し、コリネバクテリウムグルタミカムゲノム上でargB遺伝子の真ん中を中心に左アーム1614bp部分と右アーム854bp部分をPCRで増幅して、オーバーラップPCR(overlap PCR)方法で連結した後、pk19msbベクターにクローニングした。前記プラスミドをpk19msb+argB-E258Lと名付けた(図1参照)。前記プラスミドを作製するためにそれぞれの遺伝子断片を増幅するのに下記表1のプライマーを使用した。
クローニングベクターを用いて、変異菌株であるAB1菌株を製造した。クローニングベクターの最終濃度が1μg/μl以上となるように用意して、コリネバクテリウムグルタミカム14GRに電気穿孔法(文献[Tauch et al.、FEMS Microbiology letters 123(1994)343-347]参照)を用いて1次組換えを誘導した。この時、電気穿孔した菌株をカナマイシンが50μg/μl含まれる寒天培地に塗抹してコロニーを分離した後、ゲノム上の誘導した位置に適切に挿入されたかを、PCRおよび塩基配列分析により確認した。このように分離された菌株を再度2次組換えを誘導するために液体培地に接種し、一晩以上培養して10%sucroseを含有した寒天培地に塗抹してコロニーを分離した。最終分離したコロニー中でカナマイシンに対する耐性の有無を確認した後、抗生剤耐性のない菌株のうちアセチルグルタメートキナーゼに変異が導入されたかを、塩基配列分析により確認した(文献[Schafer et al.、Gene 145(1994)69-73]参照)。最終的に、アセチルグルタメートキナーゼのアミノ酸配列(配列番号2)のうち258番目に位置したグルタミンがロイシンに置換された、L-アルギニンの生産が可能なコリネバクテリウムグルタミカム変異株AB1を作製した。
argB遺伝子の258番目のアミノ酸をグルタミンからアラニンに置換した組換えベクター(pk19msb+argB-E258A)を用いたことを除けば、実施例1と同様の方法でアセチルグルタメートキナーゼの活性が強化されたコリネバクテリウムグルタミカム変異株を作製した。
argB遺伝子の245番目のアミノ酸をヒスチジンからアスパラギン酸に置換した組換えベクター(pk19msb+argB-H245D)を用いたことを除けば、実施例1と同様の方法でアセチルグルタメートキナーゼの活性が強化されたコリネバクテリウムグルタミカム変異株を作製した。
親菌株と比較して、実施例1~3で作製されたコリネバクテリウムグルタミカム変異株AB1~AB3のL-アルギニン生産能を評価した。
実施例1-1のpk19msb+argB-E258Lクローニングベクターを、コリネバクテリウムグルタミカム14GRの代わりに、コリネバクテリウムグルタミカム15GD(KCCM13220P)に導入したことを除けば、実施例1と同様の方法でアセチルグルタメートキナーゼのアミノ酸配列(配列番号2)のうち258番目に位置したグルタミンがロイシンに置換された、L-シトルリンの生産が可能なコリネバクテリウムグルタミカム変異株CB1を作製した。
実施例2のpk19msb+argB-E258Aクローニングベクターを、コリネバクテリウムグルタミカム14GRの代わりに、コリネバクテリウムグルタミカム15GDに導入したことを除けば、実施例1と同様の方法でアセチルグルタメートキナーゼのアミノ酸配列(配列番号2)のうち258番目に位置したグルタミンがアラニンに置換された、L-シトルリンの生産が可能なコリネバクテリウムグルタミカム変異株CB2を作製した。
実施例3のpk19msb+argB-H245Dクローニングベクターを、コリネバクテリウムグルタミカム14GRの代わりに、コリネバクテリウムグルタミカム15GDに導入したことを除けば、実施例1と同様の方法でアセチルグルタメートキナーゼのアミノ酸配列(配列番号2)のうち245番目に位置したヒスチジンがアスパラギン酸に置換された、L-シトルリンの生産が可能なコリネバクテリウムグルタミカム変異株CB3を作製した。
親菌株と比較して、実施例4~6で作製されたコリネバクテリウムグルタミカム変異株CB1~CB3のL-シトルリン生産能を評価した。
寄託機関名:韓国微生物保存センター(KCCM)
受託番号:KCCM13219P
受託日付:20220629
寄託機関名:韓国微生物保存センター(KCCM)
受託番号:KCCM13220P
受託日付:20220629
Claims (3)
- 配列番号2のアミノ酸配列のうち、258番目のアミノ酸においてグルタミンから非極性アミノ酸への置換又は245番目のアミノ酸においてヒスチジンからアスパラギン酸への置換を有するアセチルグルタメートキナーゼを発現することで、親株と比べてL-アルギニンまたはL-シトルリンの生産能が向上したコリネバクテリウム グルタミカム(Corynebacterium glutamicum)変異株。
- 前記非極性アミノ酸は、グリシン、アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニンおよびトリプトファンからなる群より選択された1種である、請求項1に記載のコリネバクテリウム グルタミカム変異株。
- 請求項1に記載のコリネバクテリウム グルタミカム変異株を培地で培養するステップと、
前記培養された変異株または変異株が培養された培地からL-アルギニンまたはL-シトルリンを回収するステップとを含むL-アルギニンまたはL-シトルリンの生産方法。
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KR1020220090642A KR20240013961A (ko) | 2022-07-21 | 2022-07-21 | L-아르기닌 또는 l-시트룰린 생산능이 향상된 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌 또는 l-시트룰린의 생산 방법 |
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WO2006035831A1 (ja) | 2004-09-28 | 2006-04-06 | Kyowa Hakko Kogyo Co., Ltd. | L-アルギニン、l-オルニチンまたはl-シトルリンの製造法 |
JP2017079705A (ja) | 2015-10-30 | 2017-05-18 | 味の素株式会社 | グルタミン酸系l−アミノ酸の製造法 |
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KR100854234B1 (ko) * | 2006-07-13 | 2008-08-25 | 씨제이제일제당 (주) | 역가가 향상된 유전자 argF 염기서열 및 그를 포함하는형질전환 균주를 이용한 L―알지닌의 생산방법 |
KR100830289B1 (ko) * | 2007-01-18 | 2008-05-16 | 씨제이제일제당 (주) | L-아르기닌 생산 변이주 및 이의 제조방법 |
CN106434594B (zh) * | 2016-11-08 | 2019-09-10 | 江南大学 | 一种n-乙酰谷氨酸激酶突变体及其应用 |
KR102269637B1 (ko) | 2018-12-26 | 2021-06-28 | 대상 주식회사 | L-시트룰린 또는 l-아르기닌 생산능이 향상된 변이 균주 및 이를 이용한 l-시트룰린 또는 l-아르기닌의 제조 방법 |
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WO2006035831A1 (ja) | 2004-09-28 | 2006-04-06 | Kyowa Hakko Kogyo Co., Ltd. | L-アルギニン、l-オルニチンまたはl-シトルリンの製造法 |
JP2017079705A (ja) | 2015-10-30 | 2017-05-18 | 味の素株式会社 | グルタミン酸系l−アミノ酸の製造法 |
Non-Patent Citations (1)
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IKEDA M et al.,"Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer.",Applied and environmental microbiology,2009年,Vol. 75, No. 6,p.1635-1641 |
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WO2024019216A1 (ko) | 2024-01-25 |
JP2024014657A (ja) | 2024-02-01 |
US20240026282A1 (en) | 2024-01-25 |
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