JP2021513341A - 変異型ホモセリンデヒドロゲナーゼ、及びそれを用いたホモセリン又はホモセリン由来l−アミノ酸の生産方法 - Google Patents
変異型ホモセリンデヒドロゲナーゼ、及びそれを用いたホモセリン又はホモセリン由来l−アミノ酸の生産方法 Download PDFInfo
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Abstract
Description
本実施例においては、コリネバクテリウム・グルタミカムKFCC10881(特許文献1)を親株として、ホモセリンデヒドロゲナーゼ(homoserine dehydrogenase,以下、Hom,EC:1.1.1.3)のL−トレオニンによるフィードバック阻害を解除するために、L−トレオニンアナログである2−amino−3−hydroxy−valerate(以下、AHV)に対する耐性を付与する実験を行った。
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,MgSO47H2O 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(蒸留水1リットル中)
最小培地(pH7.2)
グルコース5g,KH2PO4 1g,(NH4)2SO4 5g,MgSO47H2O 0.4g,NaCl 0.5g,ビオチン200μg,チアミンHCl 100μg,パントテン酸カルシウム100μg,ニコチンアミド0.03g,尿素2g,Na2B4O710H2O 0.09mg,(NH4)6Mo7O274H2O 0.04mg,ZnSO47H2O 0.01mg,CuSO45H2O,MnCl24H2O 0.01mg,FeCl36H2O 1mg,CaCl2 0.01mg(蒸留水1リットル中)
実施例1で得た155株のAHV耐性菌株に対してL−トレオニン生産能試験を行った。種培地25mlを含有する250mlのコーナーバッフルフラスコに実施例1で得た155株の菌株を接種し、その後30℃、200rpmで20時間振盪培養した。次のL−トレオニン生産培地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、30℃、200rpmで48時間振盪培養した。
L−トレオニン生産培地(pH7.2)
グルコース30g,KH2PO4 2g,Urea 3g,(NH4)2SO4 40g,Peptone 2.5g,CSL(Sigma) 5g(10ml),MgSO4・7H2O 0.5g,Leucine 400mg,CaCO3 20g(蒸留水1リットル中)
実施例1で選択した菌株におけるL−トレオニン生合成酵素の塩基配列の分析を次のように行った。KEGG(Kyoto Encyclopedia of Genes and Genomes)が提供する遺伝子情報に基づいて、コリネバクテリウム・グルタミカムATCC13032のホモセリンデヒドロゲナーゼをコードするhomの塩基配列(配列番号1,NCgl1136)、ホモセリンキナーゼをコードするthrBの塩基配列(配列番号2,遺伝子番号NCgl1137)をそれぞれ確保した。homとthrBは、オペロン構造をとることが知られている(非特許文献7)。
実施例2で確認した変異体T285I、R398Q、G378W、G378Sが野生型菌株に導入された菌株を作製するために、配列番号14及び15のプライマーを作製した。
前述したように作製した菌株を対象にHom酵素活性を測定した。実施例4で作製したCTR−T285I、CTR−R398Q、CTR−G378W、CTR−G378S、及び対照群としての野生型菌株(ATCC13032)を下記種培地25mlに接種し、その後対数期の後半まで培養した。遠心分離により菌体を回収し、0.1Mのpotassium phosphate(pH7.6)緩衝液で2回洗浄し、その後30%の濃度のグリセリンを含有する同じ緩衝液2mlに最終懸濁した。菌体懸濁液を一般的なガラスビーズボルテックス法により10分間物理的に破砕し、その後2回の遠心分離(13,000rpm,4℃,30分)により上清を回収し、Hom酵素活性の測定のための粗酵素液(crude extract)として用いた。Hom酵素活性の測定のために、酵素活性測定用反応液(potassium phosphate(pH7.0)緩衝液,25mM NADPH,5mM aspartate semi−aldehyde)0.9mlに粗酵素液0.1mlを添加し、30℃で反応させた。Hom酵素活性(U)は、L−トレオニンの有無(0mM,10mM)による1分当たり消費したNADPH umol数により決定した。酵素活性の測定結果を表2に示す。
野生種コリネバクテリウム・グルタミカムATCC13032からL−トレオニン生産菌株を作製した。具体的には、トレオニン生合成経路において最初の重要な酵素として作用するaspartate kinase(lysC)のフィードバック阻害(feedback inhibition)を解除するために、lysCの377番目のアミノ酸であるロイシン(Leucine)をリシン(Lysine)に置換した(配列番号22)。
イソロイシン生産菌株を作製するために、実施例6で作製した菌株において、公知のL−トレオニンデヒドラターゼ(L-threonine dehydratase, isoleucine生合成経路の最初の酵素)をコードする変異遺伝子ilvA(V323A)(非特許文献6)の発現を強化するためのベクターを作製した。
8−1:変異型Homに置換されたATCC13032菌株の作製
実施例7と同様に、ATCC13032菌株にT285I及びR398Qの2種の変異を導入し、その菌株をATCC13032::HomFBRと命名した。
本実施例においては、コリネバクテリウム・グルタミカムATCC13032の染色体DNAを鋳型としたPCRにより、O−アセチル−ホモセリン分解経路のシスタチオニンγ−シンターゼ(cystathionine gamma-synthase)をコードするmetB遺伝子を確保した。米国国立衛生研究所の遺伝子バンク(NIH GenBank)からmetB遺伝子の塩基配列情報(NCBI登録番号Ncgl2360,配列番号31)を確保し、それに基づいてmetB遺伝子のN−terminal部分とlinker sequenceを含有するプライマー(配列番号32,33)、C−terminal部分とlinker部分を含有するプライマー(配列番号34,35)を合成した。コリネバクテリウム・グルタミカムATCC13032の染色体DNAを鋳型とし、配列番号32及び33と配列番号34及び35のオリゴヌクレオチドをプライマーとしてPCRを行った。重合酵素としてはPfuUltraTM高信頼DNAポリメラーゼ(Stratagene)を用いた。PCR条件は、96℃で30秒間の変性、53℃で30秒間のアニーリング、72℃で1分間の重合反応を30サイクル行うものとした。その結果、metB遺伝子のN−terminal部分とlinkerを含有する500bpの増幅された遺伝子と、metB遺伝子のC−terminal部分とlinkerを含有する500bpの増幅された遺伝子が得られた。
本実施例においては、コリネバクテリウム・グルタミカムATCC13032の染色体DNAを鋳型としたPCRにより、O−アセチル−ホモセリン分解経路のO−アセチル−ホモセリンチオールリアーゼ(O-acetylhomoserine(thiol)-lyase)をコードするmetY遺伝子を確保した。米国国立衛生研究所の遺伝子バンク(NIH GenBank)からmetY遺伝子の塩基配列情報(NCBI登録番号Ncgl0625,配列番号36)を確保し、それに基づいてmetY遺伝子のN−terminal部分とlinker sequenceを含有するプライマー(配列番号37,38)、C−terminal部分とlinker部分を含有するプライマー(配列番号39,40)を合成した。
実施例8−1〜8−3で作製した、metB、metY、metBY遺伝子が欠損して変異型hom遺伝子に置換されたATCC13032、ATCC13032ΔmetB、ATCC13032ΔmetY、ATCC13032ΔmetBΔmetY、ATCC13032::HomFBR、ATCC13032::HomFBRΔmetB、ATCC13032::HomFBRΔmetY、ATCC13032::HomFBRΔmetBΔmetY菌株のO−アセチル−ホモセリン生産能を比較した。
L−O−アセチルホモセリン生産培地(pH7.2)
グルコース30g,KH2PO4 2g,Urea 3g,(NH4)2SO4 40g,Peptone 2.5g,CSL(Sigma) 5g(10ml),MgSO4・7H2O 0.5g,Methionine 400mg,Leucine 400mg,CaCO3 20g(蒸留水1リットル中)
9−1:mcbR遺伝子の欠損のための組換えベクターの作製
本実施例においては、メチオニン生産菌株を作製するために、実施例6で作製した菌株において公知のメチオニンシステイン転写調節因子タンパク質をコードするmcbR(非特許文献9)を不活性化するベクターを作製した。
実施例9で作製したpDZ−△mcbRベクターを、染色体上での相同組換えにより実施例6で作製したCJP1−G378W、CJP1−T285I,R398Q、CJP1−G378W,R398Q、CJP1−T285I,G378W、及びCJP1菌株に、エレクトロポレーションにより形質転換した(非特許文献10)。その後、X−galを含む固体培地において2次組換えを行った。2次組換えが終了した前記コリネバクテリウム・グルタミカム形質転換株を対象に、プライマー46とプライマー47を用いたPCRにより、mcbR遺伝子が欠損した菌株を確認した。その組換え菌株をそれぞれコリネバクテリウム・グルタミカムCJP1−G378W/ΔmcbR、CJP1−T285I,R398Q/ΔmcbR、CJP1−G378W,R398Q/ΔmcbR、CJP1−T285I,G378W/ΔmcbR、CJP1/ΔmcbRと命名した。
<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 1000μg,パントテン酸カルシウム2000μg,ニコチンアミド2000μg(蒸留水1リットル中)
<生産培地(pH8.0)>
グルコース50g,(NH4)2S2O3 12g,Yeast extract 5g,KH2PO4 1g,MgSO4・7H2O 1.2g,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,ニコチンアミド3000μg,CaCO3 30g(蒸留水1リットル中)
Claims (15)
- 配列番号1のアミノ酸配列において、285番目のアミノ酸がイソロイシンに置換されるか、398番目のアミノ酸がグルタミンに置換されるか、又はそれらの組み合わせにより置換された変異型ホモセリンデヒドロゲナーゼ。
- 前記配列番号1のアミノ酸配列において、さらに378番目のアミノ酸がトリプトファンに置換された、請求項1に記載の変異型ホモセリンデヒドロゲナーゼ。
- 請求項1または2に記載の変異型ホモセリンデヒドロゲナーゼをコードするポリヌクレオチド。
- 請求項1または2に記載の変異型ホモセリンデヒドロゲナーゼを含む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、ホモセリン又はホモセリン由来L−アミノ酸を生産する、請求項4に記載のコリネバクテリウム属微生物。
- 前記ホモセリン由来L−アミノ酸は、L−トレオニン、L−イソロイシン、O−アセチルホモセリン及びL−メチオニンからなる群から選択される少なくとも1種である、請求項5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L−アラニンを生産する、請求項4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・グルタミカムである、請求項4に記載のコリネバクテリウム属微生物。
- 請求項4に記載の微生物を培地で培養する工程と、前記培養した微生物又は培養した培地からホモセリン又はホモセリン由来L−アミノ酸を回収する工程とを含む、ホモセリン又はホモセリン由来L−アミノ酸の生産方法。
- 前記ホモセリン由来L−アミノ酸は、L−トレオニン、L−イソロイシン、O−アセチル−L−ホモセリン及びL−メチオニンからなる群から選択される少なくとも1種である、請求項9に記載のホモセリン又はホモセリン由来L−アミノ酸の生産方法。
- 請求項1に記載の変異型ホモセリンデヒドロゲナーゼを含む、ホモセリン又はホモセリン由来L−アミノ酸生産用組成物。
- 請求項1に記載の変異型ホモセリンデヒドロゲナーゼをコリネバクテリウム属微生物から培養するステップを含む、ホモセリン又はホモセリン由来L−アミノ酸の排出増加方法。
- ホモセリン又はホモセリン由来L−アミノ酸の生産用組成物の製造のための、請求項1に記載の変異型ホモセリンデヒドロゲナーゼの使用。
- ホモセリン又はホモセリン由来L−アミノ酸の生産用組成物の製造のための、請求項3に記載のポリヌクレオチドの使用。
- ホモセリン又はホモセリン由来L−アミノ酸の生産用組成物の製造のための、請求項4に記載のコリネバクテリウム属微生物の使用。
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KR101947959B1 (ko) * | 2018-05-28 | 2019-02-13 | 씨제이제일제당 (주) | 변이형 호모세린 디하이드로게나제 및 이를 이용한 호모세린 또는 호모세린 유래 l-아미노산의 생산 방법 |
KR102175112B1 (ko) | 2019-04-22 | 2020-11-06 | 씨제이제일제당 주식회사 | L-쓰레오닌 생산능이 강화된 미생물 및 이를 이용한 쓰레오닌 생산방법 |
KR102207867B1 (ko) | 2020-01-21 | 2021-01-26 | 씨제이제일제당 주식회사 | Nadp 의존적 글리세르알데하이드-3-포스페이트 디하이드로지나제를 포함하는 미생물을 이용하여 l-아미노산을 생산하는 방법 |
KR102363913B1 (ko) * | 2020-06-26 | 2022-02-18 | 씨제이제일제당 (주) | L-쓰레오닌 디하이드라타아제의 신규 변이체 및 이를 이용한 l-이소류신 생산 방법 |
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KR102273639B1 (ko) * | 2021-04-20 | 2021-07-06 | 씨제이제일제당 주식회사 | 신규한 이중기능성 메틸렌테트라히드로폴레이트 탈수소효소/메테닐테트라하이드로폴레이트 사이클로하이드롤라아제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 |
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KR20220157144A (ko) | 2021-05-20 | 2022-11-29 | 씨제이제일제당 (주) | 신규 프로모터 및 이의 용도 |
KR102421911B1 (ko) * | 2022-02-16 | 2022-07-21 | 대상 주식회사 | 징크 바인딩 디하이드로게나제 신규 변이체 및 이를 이용한 l-방향족 아미노산 생산 방법 |
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US11555213B2 (en) | 2023-01-17 |
HUE062601T2 (hu) | 2023-11-28 |
ES2944588T3 (es) | 2023-06-22 |
AU2019279282A1 (en) | 2020-07-16 |
PH12020551031A1 (en) | 2021-08-16 |
TWI740150B (zh) | 2021-09-21 |
CN110945121A (zh) | 2020-03-31 |
SG11202006718YA (en) | 2020-08-28 |
BR112019020697B1 (pt) | 2022-04-26 |
AR115415A1 (es) | 2021-01-13 |
CA3091741A1 (en) | 2019-12-05 |
AU2019279282B2 (en) | 2022-03-03 |
CN110945121B (zh) | 2021-09-28 |
RU2733426C1 (ru) | 2020-10-01 |
US20210002682A1 (en) | 2021-01-07 |
CA3091741C (en) | 2023-10-31 |
CN112143719B (zh) | 2023-11-24 |
EP3597738B1 (en) | 2023-04-05 |
WO2019231159A1 (ko) | 2019-12-05 |
US11236374B2 (en) | 2022-02-01 |
US20210403962A1 (en) | 2021-12-30 |
MX2020007591A (es) | 2021-01-15 |
TW202000914A (zh) | 2020-01-01 |
EP3597738A4 (en) | 2021-01-27 |
EP3597738A1 (en) | 2020-01-22 |
CN112143719A (zh) | 2020-12-29 |
JP6938795B2 (ja) | 2021-09-22 |
KR101947959B1 (ko) | 2019-02-13 |
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