JP2021510298A - クエン酸シンターゼの活性が弱化された変異型ポリペプチド及びそれを用いたl−アミノ酸生産方法 - Google Patents
クエン酸シンターゼの活性が弱化された変異型ポリペプチド及びそれを用いたl−アミノ酸生産方法 Download PDFInfo
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Abstract
Description
コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)のgltA遺伝子の発現量又はその活性が減衰された変異体を発掘することを目的として、以下の方法でライブラリを製作した。
野生型のコリネバクテリウム・グルタミクムATCC13032においてgltA遺伝子が欠損した菌株を製作するために、以下のようにgltA遺伝子が欠損したベクターpDZ-ΔgltAを製造した。具体的には、gltA遺伝子の5'及び3'末端に位置するDNA断片が(各600bp)pDZベクター(大韓民国登録特許第10-0924065号(特許文献2))に連結された形態で製作された。報告されたgltA遺伝子の塩基配列(配列番号2)に基づいて5'断片及び3'断片に制限酵素XbaI認識部位を挿入したプライマー配列番号7及び8と、これらからそれぞれ600 bp離れた位置でプライマー配列番号9及び10を合成した(表2)。コリネバクテリウム・グルタミクムATCC13032の染色体を鋳型とし、5'末端遺伝子断片は、プライマー配列番号7及び9を用いてPCRを通じて製作した。同様な方法で、gltA遺伝子の3'末端に位置する遺伝子断片は、配列番号8及び10を用いてPCRを通じて製作した。PCR条件は、94℃で2分間の変性後、94℃で1分間の変性、56℃で1分間のアニーリング、72℃で40秒間の重合を30回繰り返した後、72℃で10分間の重合反応を行った。
グルコース10g、ペプトン10g、Beef extract 5g、酵母エキス5g、Brain Heart Infusion 18.5g、NaCl 2.5g、尿素2g、Sorbitol 91g、寒天20g(蒸留水1リットル基準)
グルコース20g、ペプトン10g、酵母エキス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、ニコチン酸アミド2000μg(蒸留水1リットル基準)
3種の選別菌株WT ::gltA(mt)-1〜3のgltA遺伝子塩基配列を確認するために、実施例1に明示されたプライマーを(配列番号5及び6)用いて染色体内のgltA遺伝子を含むDNA断片をPCR増幅した。PCR条件は、94℃で2分間の変性後、94℃で1分間の変性、56℃で1分間のアニーリング、72℃で40秒間の重合を30回繰り返した後、72℃で10分間の重合反応を行った。
上記アミノ酸配列1において241番目のアミノ酸の位置に、野生型が有するアスパラギンを除く他のproteogenicアミノ酸への置換を試みた。
既存に報告された方法(Ooyen et al., Biotechnol. Bioeng., 109(8):2070-2081, 2012(非特許文献1))を通じて前記選別された菌株を対象に、クエン酸シンターゼ(CS)活性を測定した。実施例1で使用した方法でKCCM11016Pの菌株にgltA遺伝子を欠損し、その菌株をKCCM11016P ::ΔgltAと命名した。KCCM11016P、KCCM11016P ::ΔgltA菌株を対照群として使用し、選別菌株19種を下記のような方法で培養し、糖消費速度、リジン生産収率、培養培地内の代表副産物であるグルタミン酸(Glutamic acid; GA)の濃度、及びCS酵素活性を測定した。
グルコース20g、ペプトン10g、酵母エキス5g、尿素1.5g、KH2PO44g、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、ニコチン酸アミド2000μg(蒸留水1リットル基準)
グルコース100g、(NH4)2SO4 40g、大豆タンパク質2.5g、トウモロコシ浸漬固形分(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1000μg、パントテン酸カルシウム2000μg、ニコチン酸アミド3000μg、CaCO3 30g(蒸留水1リットル基準)。
L-リジンを生産するコリネバクテリウム・グルタミクムKCCM10770P(大韓民国登録特許第10-0924065号(特許文献2))及びKCCM11347P(大韓民国登録特許第10-0073610号(特許文献5))に、実施例5で選別されたgltA遺伝子変異3種を導入した。上記3種は、CS活性が減少してリジン収率が増加し、糖消費速度が親株比同様のものを選別した。実施例4のpDZ-gltA(N241S)、pDZ-gltA(N241Y)、pDZ-gltA(N241T)のベクター3種を電気パルス法でコリネバクテリウム・グルタミクムKCCM10770P及びKCCM11347P菌株2種に導入し、KCCM10770P::gltA(N241S)、KCCM10770P ::gltA(N241Y)、KCCM10770P ::gltA(N241T)、KCCM11347P ::gltA(N241S)、KCCM11347P ::gltA(N241Y)、KCCM11347P ::gltA(N241T)の菌株6種を製作した。対照群として使用されたKCCM10770P及びKCCM11347Pの菌株とgltA遺伝子の塩基置換変異導入菌株6種の上記菌株を実施例5と同様の方法で培養し、リジンの生産能、糖消費速度及び培養液の成分を分析した。
L-リジンを生産する他のコリネバクテリウム・グルタミクムに属する菌株でも、上記と同様の効果があるかを確認するために、野生株に3種の変異[pyc(P458S)、hom(V59A)、lysC(T311I)]を導入し、L-リジン生産能を有するようになったコリネバクテリウム・グルタミクムCJ3P(Binder et al. Genome Biology, 2012, 13:R40(非特許文献16))を対象に、実施例6と同様の方法でgltA(N241T)変異が導入された菌株を作製した。上記製作された菌株は、CJ3 ::gltA(N241T)と命名した。対照群であるCJ3P菌株とCJ3 ::gltA(N241T)を前記実施例5と同様の方法で培養し、リジンの生産能、糖消費速度及び培養液の成分を分析して下記表6に示した。
gltA(N241T)変異導入によるL-トレオニン生産能の変化を明確に確認するために、L-トレオニン、L-イソロイシン生合成経路の共通中間体であるホモセリン(homoserine)を生産するホモセリンデヒドロゲナーゼ(homoserin dehydrogenase)をコードする遺伝子に変異を導入して強化した。具体的には、実施例7で使用されたCJ3P ::gltA(N241T)菌株に既に公知となったhom(G378E)変異(R. Winkels, S. et al., Appl. Microbiol. Biotechnol. 45, 612-620, 1996(非特許文献17))が導入された菌株を作製した。また、その対照群としてCJ3Pにhom(G378E)変異のみが導入された菌株も製作した。変異導入のための組換えベクターは、以下のような方法で製作された。
gltA(N241T)変異導入によるL-イソロイシン生産能に及ぼす効果を確認するために、既に公知となったトレオニンデヒドラターゼ(L-threonine dehydratase)をコードする遺伝子に変異を導入し強化した。具体的には、実施例7で使用されたCJ3P ::gltA(N241T)-hom(G378E)菌株に既に公知となったilvA(V323A)変異(S. Morbach et al., Appl. Enviro. Microbiol., 62(12):4345-4351, 1996(非特許文献18))が導入された菌株を作製した。また、その対照としてCJ3P ::hom(G378E)にilvA(V323A)変異のみが導入された菌株も製作した。変異導入のための組換えベクターは、以下のような方法で製作された。
Claims (13)
- 配列番号1のアミノ酸配列において241番目のアスパラギン(asparagine)が他のアミノ酸で置換されたクエン酸シンターゼ(Citrate synthase)活性を有する、変異型ポリペプチド。
- 前記241番目のアスパラギンは、リジン以外の他のアミノ酸で置換される、請求項1に記載の変異型ポリペプチド。
- 前記他のアミノ酸は、芳香族アミノ酸または親水性アミノ酸である、請求項1に記載の変異型ポリペプチド。
- 前記241番目のアスパラギンは、トレオニン(Threonine)、セリン(Serine)またはチロシン(Tyrosine)で置換される、請求項1に記載の変異型ポリペプチド。
- 請求項1に記載の変異型ポリペプチドをコードするポリヌクレオチド。
- 請求項1に記載の変異型ポリペプチドを含む、コリネバクテリウム属(Corynebacterium sp.)微生物。
- 前記コリネバクテリウム属微生物は、L-アミノ酸を生産する、請求項6に記載のコリネバクテリウム属(Corynebacterium sp.)微生物。
- 前記コリネバクテリウム属微生物は、アスパラギン酸由来のL-アミノ酸を生産する、請求項6に記載のコリネバクテリウム属(Corynebacterium sp.)微生物。
- 前記コリネバクテリウム属微生物は、リジン、トレオニン、メチオニン、ホモセリンまたはその誘導体、及びイソロイシンからなる群から選択される1種以上のL-アミノ酸を生産する、請求項6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・グルタミクム(Corynebacterium glutamicum)である、請求項6に記載のコリネバクテリウム属微生物。
- 請求項6に記載の微生物を培地で培養する段階; 及び
前記培養された微生物または培地からL-アミノ酸を回収する段階を含む、L-アミノ酸の生産方法。 - 前記L-アミノ酸は、アスパラギン酸由来のL-アミノ酸である、請求項11に記載のL-アミノ酸の生産方法。
- 前記L-アミノ酸は、リジン、トレオニン、メチオニン、ホモセリンまたはその誘導体、及びイソロイシンからなる群から選択される1種以上である、請求項11に記載のL-アミノ酸の生産方法。
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KR102104658B1 (ko) | 2013-05-13 | 2020-04-27 | 우정케미칼주식회사 | 고기능성 폴리아미드 중합체, 이를 포함하는 방사 도프 조성물 및 그의 성형물 |
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KR101825777B1 (ko) * | 2014-06-05 | 2018-02-07 | 씨제이제일제당 (주) | O-아세틸-호모세린을 생산하는 미생물 및 이를 이용하여 o-아세틸-호모세린을 생산하는 방법 |
KR101641770B1 (ko) | 2014-06-23 | 2016-07-22 | 씨제이제일제당 (주) | O-아세틸 호모세린을 생산하는 미생물 및 상기 미생물을 이용하여 o-아세틸 호모세린을 생산하는 방법 |
KR101835935B1 (ko) * | 2014-10-13 | 2018-03-12 | 씨제이제일제당 (주) | L-아르기닌을 생산하는 코리네박테리움 속 미생물 및 이를 이용한 l-아르기닌의 제조 방법 |
KR101915433B1 (ko) * | 2018-02-13 | 2018-11-05 | 씨제이제일제당 (주) | 시트레이트 신타아제 (Citrate synthase)의 활성이 약화된 변이형 폴리펩타이드 및 이를 이용한 L-아미노산 생산방법 |
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EP3561055A1 (en) | 2019-10-30 |
KR101915433B1 (ko) | 2018-11-05 |
RU2732815C1 (ru) | 2020-09-22 |
PH12020550844A1 (en) | 2021-05-17 |
CN112175895A (zh) | 2021-01-05 |
BR112019016462A2 (pt) | 2020-04-07 |
BR122020018773B1 (pt) | 2021-06-22 |
US20230046561A1 (en) | 2023-02-16 |
CN112175895B (zh) | 2024-05-03 |
EP3561055A4 (en) | 2020-10-14 |
AU2019221267B2 (en) | 2022-03-31 |
CN110546254B (zh) | 2020-11-24 |
US11499173B2 (en) | 2022-11-15 |
ZA202003471B (en) | 2021-06-30 |
MX2020006106A (es) | 2020-08-24 |
US20210355514A1 (en) | 2021-11-18 |
JP6998466B2 (ja) | 2022-02-04 |
AU2019221267A1 (en) | 2020-06-25 |
US11667936B2 (en) | 2023-06-06 |
CN110546254A (zh) | 2019-12-06 |
WO2019160301A1 (ko) | 2019-08-22 |
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