JP2023518743A - プレフェナートデヒドラターゼ活性強化によるl-トリプトファンを生産する方法 - Google Patents
プレフェナートデヒドラターゼ活性強化によるl-トリプトファンを生産する方法 Download PDFInfo
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Abstract
Description
1)上記タンパク質をコードする遺伝子の細胞内コピー数の増加、
2)上記タンパク質をコードする染色体上の遺伝子発現調節配列を活性が強力な配列で交換する方法、
3)上記タンパク質活性が増加するように上記タンパク質の開始コドンまたは5’-UTR領域の塩基配列を変形させる方法、
4)上記タンパク質活性が増加するように染色体上のポリヌクレオチド配列を変形させる方法、
5)上記タンパク質の活性を示す外来ポリヌクレオチドまたは上記ポリヌクレオチドのコドン最適化された変異型ポリヌクレオチドの導入、または
6)上記方法の組み合わせなどにより行われ得るが、これに制限されない。
上記プレフェナートデヒドラターゼ、活性強化及びL-トリプトファンを生産する微生物については前述した通りである。
コリネバクテリウム染色体内の遺伝子の挿入及び交換のためのプラスミド(pDCM2、図1、配列番号3)をデザインし、バイオニクス(株)の遺伝子合成(Gene-synthesis)サービスを用いてプラスミドを合成した。一般に知られているsacBシステム関連の論文[Gene, 145 (1994) 69-73]を参考にしてクローニングに活用するのに容易な制限酵素(restriction enzyme)を含むようにプラスミドを設計した。このように合成されたpDCM2プラスミドは次のような特性を有する。
2)選別マーカーとしてカナマイシン耐性遺伝子を有する。
3)2次陽性選別(positive-selection)マーカーとしてレバンスクラーゼ(Levan sucrose)遺伝子(sacB)を有する。
4)最終製作された菌株にはpDCM2プラスミドに由来した、如何なる遺伝子情報も残らない。
プレフェナートデヒドラターゼ(prephenate dehydratase、以下「pheA」)の活性を強化させるために、強いプロモーターとして知られた変異型lysCプロモーター(米国登録公報US 8426577 B2)を基盤に一部の配列を変更して配列番号4としてデザインし、バイオニクス(株)の遺伝子合成サービスを用いて合成し、これをPlysCm1プロモーターと命名した。上記PlysCm1プロモーターを利用し、pheA遺伝子を追加挿入したり、pheA遺伝子の野生型プロモーターをPlysCm1で交換することにより、プレフェナートデヒドラターゼ活性を強化するプラスミドを製作した。
上記PlysCm1プロモーターを利用し、pheA遺伝子を追加挿入するために、野生種のコリネバクテリウム・グルタミカムATCC13869染色体DNAを鋳型とし、配列番号5及び配列番号6のプライマー対を用いて染色体上の相同組換え(Homologous recombiantion)が発生するアップストリーム(Upstream)領域を、配列番号7及び配列番号8のプライマー対を用いてダウンストリーム(Downsteam)領域を増幅した後、それぞれの遺伝子断片を得た。ここで使用されたプライマー配列は、下記表1の通りである。
pheA遺伝子の野生型プロモーターをPlysCm1で交換することにより、プレフェナートデヒドラターゼ活性を強化するプラスミドを製作しようとした。具体的には、野生種のコリネバクテリウム・グルタミカム(ATCC13869)染色体DNAを鋳型として配列番号13及び配列番号14のプライマー対を用いて染色体上の相同組換え(Homologous recombiantion)が発生するpheA遺伝子の野生型プロモーターアップストリーム(Upstream)領域の遺伝子断片を得た。そして先に製作したpDCM2-Tn::PlysCm1_pheAプラスミドを鋳型としてプライマー配列番号15及び配列番号16を用いてPlysCm1プロモーター及びそのダウンストリームを共に含む遺伝子断片を得た。ここで使用されたプライマー配列は、下記表3の通りである。
ハーバスピリラム・リゾスファエラエ(Herbaspirillum rhizosphaerae)由来の膜タンパク質をコードする遺伝子(登録番号NZ_LFLU01000012.1)をトリプトファン生産菌株であるCA04-8352菌株(韓国登録特許第10-1968317号)に導入して製作されたCA04-8405の菌株(KCCM12099P、米国公開公報US 2020-0063219 A1)に上記実施例2-1で製作したpDCM2-Tn::PlysCm1_pheAを電気穿孔法(Appl. Microbiol.Biotechnol. (1999) 52:541-545)を用いて形質転換した後、2次交差過程を経て、PlysCm1_pheA遺伝子が追加で挿入された菌株を得た。当該相同組換えアップストリーム領域及びダウンストリーム領域の外部部位をそれぞれ増幅できる配列番号17及び配列番号18のプライマー対を用いてPCR増幅及びゲノムシーケンスを通じてその遺伝子が挿入されたことを確認した。上記遺伝子が挿入された菌株をCM05-9157と命名した。ここで使用されたプライマー配列は、下記表4の通りである。
ブドウ糖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 1000μg、パントテン酸カルシウム2000μg、ニコチンアミド2000μg(蒸溜水1リットルを基準)
ブドウ糖30g、(NH4)2SO4 15g、MgSO4 7H2O 1.2g、KH2PO4 1g、酵母抽出物5g、ビオチン900μg、チアミン塩酸塩4500μg、パントテン酸カルシウム4500μg、CaCO3 30g(蒸溜水1リットルを基準)。
Claims (8)
- プレフェナートデヒドラターゼ(prephenate dehydratase)活性が強化された、L-トリプトファンを生産する微生物。
- 上記プレフェナートデヒドラターゼは、配列番号1またはこれと90%以上の配列同一性を有するアミノ酸配列を含む、請求項1に記載の微生物。
- 上記活性強化は、上記プレフェナートデヒドラターゼをコードする遺伝子のコピー数の増加または上記遺伝子プロモーターの強いプロモーターへの交換によるものである、請求項1に記載の微生物。
- 上記微生物は、コリネバクテリウム属(Corynebacterium sp.)である、請求項1に記載の微生物。
- 上記微生物は、コリネバクテリウム・グルタミカム(Corynebacterium glutamicum)である、請求項4に記載の微生物。
- プレフェナートデヒドラターゼ活性が強化された、L-トリプトファンを生産する微生物を培地で培養する段階を含む、L-トリプトファンの生産方法。
- 上記方法は、培養された培地または微生物からL-トリプトファンを回収する段階をさらに含む、請求項6に記載のL-トリプトファンの生産方法。
- プレフェナートデヒドラターゼ活性が強化された、L-トリプトファンを生産する微生物を含む、L-トリプトファン生産用組成物。
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PCT/KR2021/002763 WO2021187781A1 (ko) | 2020-03-17 | 2021-03-05 | 프리페네이트 디하이드라타아제 활성 강화를 통한 l-트립토판을 생산하는 방법 |
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KR102278000B1 (ko) * | 2020-03-17 | 2021-07-15 | 씨제이제일제당 주식회사 | 프리페네이트 디하이드라타아제 활성 강화를 통한 l-트립토판을 생산하는 방법 |
KR102464883B1 (ko) | 2020-12-11 | 2022-11-09 | 씨제이제일제당 주식회사 | 신규한 감마-아미노부티르산 퍼미에이즈 변이체 및 이를 이용한 이소류신 생산 방법 |
KR102470602B1 (ko) | 2020-12-11 | 2022-11-25 | 씨제이제일제당 주식회사 | 신규한 분지 연쇄 아미노산 아미노트렌스퍼라아제 변이체 및 이를 이용한 이소류신 생산 방법 |
KR102284726B1 (ko) * | 2021-01-25 | 2021-08-02 | 씨제이제일제당 주식회사 | 신규한 타우토머레이즈 pptA 변이체 및 이를 이용한 L-트립토판 생산 방법 |
KR102284725B1 (ko) * | 2021-01-25 | 2021-08-02 | 씨제이제일제당 주식회사 | 신규한 페로체라테이즈 변이체 및 이를 이용한 l-트립토판 생산 방법 |
KR102495918B1 (ko) | 2021-01-26 | 2023-02-06 | 씨제이제일제당 주식회사 | aroG 알돌라아제 (Phospho-2-dehydro-3-deoxyheptonate aldolase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
KR102271333B1 (ko) | 2021-01-29 | 2021-06-30 | 씨제이제일제당 (주) | 신규한 미코티온 리덕타제 변이체 및 이를 이용한 l-라이신 생산 방법 |
KR102527096B1 (ko) * | 2021-02-01 | 2023-04-28 | 씨제이제일제당 주식회사 | 프리페네이트 탈수 효소 (Prephenate dehydratase) 변이체 및 이를 이용한 분지쇄 아미노산 생산 방법 |
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KR20230094761A (ko) | 2021-12-21 | 2023-06-28 | 씨제이제일제당 (주) | L-이소루신 생산 미생물 및 이를 이용한 l-이소루신 생산 방법 |
KR20230131654A (ko) | 2022-03-07 | 2023-09-14 | 씨제이제일제당 (주) | 변이형 l-쓰레오닌 배출 단백질 및 이를 이용한 l-쓰레오닌 생산 방법 |
KR20230131653A (ko) | 2022-03-07 | 2023-09-14 | 씨제이제일제당 (주) | 변이형 l-쓰레오닌 배출 단백질 및 이를 이용한 l-쓰레오닌 생산 방법 |
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