JP2021511009A - L−アミノ酸を生産するコリネバクテリウム属微生物及びそれを用いたl−アミノ酸の生産方法 - Google Patents
L−アミノ酸を生産するコリネバクテリウム属微生物及びそれを用いたl−アミノ酸の生産方法 Download PDFInfo
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- 229930182816 L-glutamine Natural products 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
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- 229930182821 L-proline Natural products 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
トランスポゾンを用いたランダム突然変異ライブラリーの作製
リシン生産能が向上した菌株を得るために、次の方法でベクターライブラリーを作製した。
<複合平板培地(pH7.0)>
グルコース10g,ペプトン10g,牛肉抽出物5g,酵母抽出物5g,ブレインハートインフュージョン(Brain Heart Infusion)18.5g,NaCl 2.5g,尿素2g,ソルビトール91g,寒天20g(蒸留水1リットル中)
トランスポゾンを用いたランダム突然変異ライブラリーのスクリーニング
実施例1で得た約20,000個のコロニーをそれぞれ300μlの次の選択培地に接種し、96ディープウェルプレート(96-deep well plate)にて32℃、1000rpmで約24時間培養した。
<選択培地(pH8.0)>
グルコース10g,硫酸アンモニウム(ammonium sulfate)5.5g,MgSO4・7H2O 1.2g,KH2PO4 0.8g,K2HPO4 16.4g,ビオチン100μg,チアミンHCl 1mg,パントテン酸カルシウム2mg,ニコチンアミド2mg(蒸留水1リットル中)
選択したランダム突然変異株におけるL−リシン生産能の分析
実施例2で選択した10種の突然変異株を対象に、再現性のあるL−リシン生産能向上を示す菌株を最終選択するために、次の方法で培養を行った。種培地25mlを含有する250mlのコーナーバッフルフラスコに各菌株を接種し、30℃、200rpmで20時間振盪培養した。次に、生産培地24mlを含有する250mlのコーナーバッフルフラスコに1mlの種培養液を接種し、32℃、200rpmで72時間振盪培養した。前記種培地及び生産培地の組成は、それぞれ次の通りである。培養終了後に、HPLC(Waters社, 2478)を用いて培養液中のL−リシン濃度を分析した。各突然変異株のL−リシン生産濃度を表1に示す。
<種培地(pH7.0)>
グルコース20g,ペプトン10g,酵母抽出物5g,尿素1.5g,KH2PO4 4g,K2HPO4 8g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミンHCl 1mg,パントテン酸カルシウム2mg,ニコチンアミド2mg(蒸留水1リットル中)
<生産培地(pH7.0)>
グルコース100g,(NH4)2SO4 40g,大豆タンパク質2.5g,コーンスティープソリッド(Corn Steep Solids)5g,尿素3g,KH2PO4 1g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミン塩酸塩1mg,パントテン酸カルシウム2mg,ニコチンアミド3mg,CaCO3 30g(蒸留水1リットル中)
最終選択株におけるL−リシン生産能向上の原因解明
本実施例においては、実施例3で最終選択した突然変異株を対象に、トランスポゾンのランダムな挿入により欠損した遺伝子の同定を試みた。
プライマー1(配列番号3):ACCTACAACAAAGCTCTCATCAACC
プライマー2(配列番号4):CTACCCTGTGGAACACCTACATCT
遺伝子を欠損させる組換えベクターの作製
本実施例においては、配列番号1のアミノ酸配列からなるタンパク質の不活性化によりL−リシン生産に影響があるか否かを確認するために、コリネバクテリウム属のL−リシンを生産する微生物の染色体上で実施例4において選択した遺伝子を欠損させるための組換えプラスミドを作製した。そのために、表2のプライマー3〜6を合成した。
コリネバクテリウム・グルタミカムKCCM11016PにおいてNCgl0275遺伝子が欠損した菌株の作製及びそのL−リシン生産能の評価
代表的なL−リシン生産コリネバクテリウム属菌株であるKCCM11016P菌株に基づいて、前述したように選択したNCgl0275遺伝子が欠損した菌株を作製し、そのL−リシン生産能を評価した。
<種培地(pH7.0)>
グルコース20g,ペプトン10g,酵母抽出物5g,尿素1.5g,KH2PO4 4g,K2HPO4 8g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミンHCl 1mg,パントテン酸カルシウム2mg,ニコチンアミド2mg(蒸留水1リットル中)
<生産培地(pH7.0)>
グルコース100g,(NH4)2SO4 40g,大豆タンパク質2.5g,コーンスティープソリッド(Corn Steep Solids)5g,尿素3g,KH2PO4 1g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミン塩酸塩1mg,パントテン酸カルシウム2mg,ニコチンアミド3mg,CaCO3 30g(蒸留水1リットル中)
コリネバクテリウム・グルタミカムKCCM11347PにおいてNCgl0275遺伝子が欠損した菌株の作製及びL−リシン生産能の評価
L−リシンを生産する他のコリネバクテリウム・グルタミカムに属する菌株においても前記と同じ効果があるか否かを確認するために、実施例6と同様に、L−リシン生産菌株であるコリネバクテリウム・グルタミカムKCCM11347P(特許文献4;前記微生物は、KFCC10750として公開され、ブダペスト条約上の国際寄託機関に寄託番号KCCM11347Pとして再寄託された)を対象に、NCgl0275遺伝子が欠損した菌株を作製し、KCCM11347P−NCgl0275と命名した。
コリネバクテリウム・グルタミカムKCCM10770PにおいてNCgl0275遺伝子が欠損した菌株の作製及びL−リシン生産能の評価
L−リシンを生産する他のコリネバクテリウム・グルタミカムに属する菌株においても前記と同じ効果があるか否かを確認するために、実施例6と同様に、L−リシン生産菌株であるコリネバクテリウム・グルタミカムKCCM10770P(特許文献1)を対象に、NCgl0275遺伝子が欠損した菌株を作製し、KCCM10770P−NCgl0275と命名した。
コリネバクテリウム・グルタミカムCJ3PにおいてNCgl0275遺伝子が欠損した菌株の作製及びL−リシン生産能の評価
L−リシンを生産する他のコリネバクテリウム・グルタミカムに属する菌株においても前記と同じ効果があるか否かを確認するために、実施例6と同様に、コリネバクテリウム・グルタミカムCJ3P(非特許文献10)を対象に、NCgl0275遺伝子が欠損した菌株を作製し、CJ3P−NCgl0275と命名した。
コリネバクテリウム・グルタミカムKCCM11201PにおいてNCgl0275遺伝子が欠損した菌株の作製及びL−バリン生産能の評価
前記L−リシン以外に、L−バリン生産能を有するコリネバクテリウム・グルタミカムにおいてもNCgl0275遺伝子の欠損によりバリン生産能が向上するか否かを評価した。
<生産培地(pH7.0)>
グルコース100g,硫酸アンモニウム40g,大豆タンパク質2.5g,コーンスティープソリッド(Corn Steep Solids)5g,尿素3g,リン酸水素二カリウム1g,硫酸マグネシウム7水塩0.5g,ビオチン100μg,チアミンHCl 1mg,パントテン酸カルシウム2mg,ニコチンアミド3mg,炭酸カルシウム30g(蒸留水1リットル中)
実施例11
L−バリンを生産する他のコリネバクテリウム・グルタミカムに属する菌株においても前記と同じ効果があるか否かを確認するために、野生株コリネバクテリウム・グルタミカムATCC14067に1種の変異[ilvN(A42V);非特許文献11]を導入してL−バリン生産能が向上した菌株を作製した。
コリネバクテリウム・グルタミカムCJ8Vを用いた、NCgl0275遺伝子が欠損した菌株の作製及びL−バリン生産能の評価
L−バリンを生産する他のコリネバクテリウム・グルタミカムに属する菌株においても前記と同じ効果があるか否かを確認するために、実施例10と同様に、野生株コリネバクテリウム・グルタミカムATCC13869に1種の変異[ilvN(A42V)]を導入してL−バリン生産能を有するようになった変異株を作製した。前記組換え菌株をコリネバクテリウム・グルタミカムCJ8Vと命名した。
Claims (7)
- 配列番号1のアミノ酸配列からなるタンパク質の活性が不活性化された、L−アミノ酸を生産するコリネバクテリウム属微生物。
- 前記L−アミノ酸は、塩基性アミノ酸、脂肪族アミノ酸又は分枝鎖アミノ酸である、請求項1に記載のコリネバクテリウム属微生物。
- 前記L−アミノ酸は、L−リシン(L-lysine)又はL−バリン(L-valine)である、請求項1に記載のコリネバクテリウム属微生物。
- 前記コリネバクテリウム属微生物は、コリネバクテリウム・グルタミカム(Corynebacterium glutamicum)である、請求項1に記載のコリネバクテリウム属微生物。
- 請求項1〜4のいずれか一項に記載の微生物を培地で培養するステップと、
前記微生物又は培地からL−アミノ酸を回収するステップとを含む、L−アミノ酸の生産方法。 - 前記L−アミノ酸は、塩基性アミノ酸、脂肪族アミノ酸又は分枝鎖アミノ酸である、請求項5に記載のL−アミノ酸の生産方法。
- 前記L−アミノ酸は、L−リシン(L-lysine)又はL−バリン(L-valine)である、請求項5に記載のL−アミノ酸の生産方法。
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PCT/KR2019/001067 WO2019147059A1 (ko) | 2018-01-25 | 2019-01-25 | L-아미노산을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아미노산의 생산방법 |
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KR102311391B1 (ko) * | 2020-05-21 | 2021-10-12 | 씨제이제일제당 주식회사 | L- 분지쇄 아미노산 생산능이 강화된 미생물 및 이를 이용하여 l-분지쇄 아미노산을 생산하는 방법 |
KR102281361B1 (ko) * | 2021-01-26 | 2021-07-22 | 씨제이제일제당 (주) | 신규한 아스파라긴 신타제 변이체 및 이를 이용한 l-발린 생산 방법 |
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