JP5816258B2 - 5’−キサンチル酸及び5’−グアニル酸の生産能が向上した微生物、及びこれを用いた5’−キサンチル酸及び5’−グアニル酸の生産方法 - Google Patents
5’−キサンチル酸及び5’−グアニル酸の生産能が向上した微生物、及びこれを用いた5’−キサンチル酸及び5’−グアニル酸の生産方法 Download PDFInfo
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Description
XMP+ATP+NH3 → GMP+AMP+PPi
XMPアミナーゼ
本実施例では、韓国公開特許第10−2007−94433号に開示されたベクターpDZを用いて遺伝子の染色体挿入を行った。pDZベクターを用いてコリネバクテリウムの染色体ヌクレオチドを挿入するためには、染色体上の挿入される部位と相同性を有する配列が、挿入されるpDZベクター内に含まれなければならないので、挿入しようとする配列を両隣に含んでいるpDZベクターを作製した。
配列番号2:CAGCCAATCTTGCAGCCAA
配列番号3:TGAGCGTCGGTCACTCAACTA
配列番号4:CGGGATCCCAGCCAATCTTGCAGTCCAA
作製されたpDZ−putAベクターをKCCM10530菌株に形質転換し、実施例1で記述したように2次組み換え過程を経て染色体上でputA遺伝子の直ぐ隣に1コピーのputA遺伝子をさらに挿入してコピー数を2個に増加させたXMP生産菌株コリネバクテリウムアンモニアゲネスKCJ−1346を得た。前記KCJ−1346と命名した新規な微生物を、2010年2月24日に国際寄託機関としての韓国微生物保存センター(Korean Culture Center of Microorganisms:KCCM、韓国ソウル市西大門区弘済1洞361−221ユリムビル)に受託番号KCCM11068Pで寄託した。連続的に挿入されたputA遺伝子は2コピーのputAの上流(upstream)と下流(downstream)の塩基配列部位を増幅することが可能なプライマー(配列番号5及び配列番号6)を用いたPCRで最終確認した。
配列番号6:AGCAGGCCATTAAAACGACC
実施例2で最終的に作製されたXMP生産菌株としてのコリネバクテリウムアンモニアゲネスKCJ−1346をXMP生産のために下記の方法で培養した。下記種培地3mLを含有する14mLのチューブにコリネバクテリウムアンモニアゲンス母菌株KCCM10530とKCJ−1346を接種し、30℃で20時間200rpmにて振盪培養した。下記の生産培地32mL(本培地24mL+別殺培地8mL)を含んでいる250mLのコーナーバッフルフラスコに0.4mLの種培養液を接種し、30℃で96時間230rpmにて振盪培養した。培養終了の後、HPLCを用いた方法によって5’−キサンチル酸の生産量を測定した。コリネバクテリウムアンモニアゲネスKCCM10530とKCJ−1346に対する培養液中のXMPは、下記表1のとおりである。
実施例2で最終的に作製されたXMP生産菌株としてのコリネバクテリウムアンモニアゲネスKCJ−1346のプロリンデヒドロゲナーゼ活性を確認するために、下記の方法で酵素活性を測定した。バクトペプトン10g/L、バクトビーフ抽出物5g/L、バクト酵母抽出物5g/L、NaCl2.5g/L、アデニン50mg/L、グアニン50mg/Lの培地に菌株を接種し、30℃で12時間培養してOD10まで培養させた。培養の後、10mLの細胞を培養液から回収して50mM HEPES、10mM 酢酸カリウム、10mM CaCl2、10mM MgCl2バッファで2回洗浄して100mM Tris−HClバッファ(pH7.5)1mLに浮遊させた。
実施例2で最終的に作製されたXMP生産菌株としてのコリネバクテリウムアンモニアゲネスKCJ−1346の細胞内ATP濃度測定のために、次の方法で培養した。
実施例2で最終的に作製されたXMP生産菌株としてのコリネバクテリウムアンモニアゲネスKCJ−1346をGMP生産のために、次の方法で培養した。
転換反応添加物:フィチン酸(phytic acid)1.8g/L、MgSO44.8g/L、ナイミーン(nymeen)3mL/L、キシレン(xylene)2%、アデニン100mg/L、Na2HPO47.7g/L、グルコース46g/L
Claims (9)
- プロリンデヒドロゲナーゼ活性が内在的活性より増加した、5’−キサンチル酸又は5’−グアニル酸生産コリネバクテリウム属微生物であり、前記プロリンデヒドロゲナーゼがコリネ型バクテリアのputA遺伝子からコードされるコリネバクテリウム属微生物であって、前記プロリンデヒドロゲナーゼ活性の増加が、コリネ型バクテリアのputA遺伝子のコピー数の増加又はプロモーターの取替えによりもたらされることを特徴とする、コリネバクテリウム属微生物。
- 前記微生物はコリネ型バクテリアのputA遺伝子の発現量の増加によりプロリンデヒドロゲナーゼ活性が増加したことを特徴とする、請求項1に記載のコリネバクテリウム属微生物。
- 前記putA遺伝子は配列番号7の塩基配列を有するものである、請求項2に記載のコリネバクテリウム属微生物。
- putA遺伝子を含むベクターが導入されて形質転換された、請求項1に記載のコリネバクテリウム属微生物。
- 前記微生物はATP生産能が増加した微生物である、請求項1に記載のコリネバクテリウム属微生物。
- 前記微生物はコリネバクテリウムアンモニアゲネス(Corynebacterium ammoniagenes)である、請求項1に記載のコリネバクテリウム属微生物。
- 前記微生物は受託番号がKCCM11068Pであることを特徴とする、請求項1に記載のコリネバクテリウム属微生物。
- 請求項1〜7のいずれか1項に記載のコリネバクテリウム属微生物を培養し、培養液から5’−キサンチル酸又は5’−グアニル酸を生産する方法。
- (a)コリネ型バクテリアのputA遺伝子を含むベクターで形質転換されたコリネバクテリウム属微生物を培養する段階と、
(b)前記(a)段階で培養されたコリネバクテリウム属微生物に5’−キサンチル酸アミナーゼを添加する段階と、
(c)前記(b)段階の培養液から5’−グアニル酸(GMP)を収得する段階とを含んでなる、5’−グアニル酸の生産方法。
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PCT/KR2011/001866 WO2011115439A2 (ko) | 2010-03-19 | 2011-03-17 | 5'-크산틸산 및 5'-구아닐산 생산능이 향상된 미생물 및 이를 이용한 5'-크산틸산 또는 5'-구아닐산의 생산방법 |
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KR102013873B1 (ko) | 2018-01-25 | 2019-08-23 | 씨제이제일제당 주식회사 | 퓨린 뉴클레오티드를 생산하는 코리네박테리움 속 미생물 및 이를 이용한 퓨린 뉴클레오티드의 생산방법 |
KR101929158B1 (ko) * | 2018-06-07 | 2018-12-13 | 씨제이제일제당 (주) | 5'-크산틸산을 생산하는 미생물 및 이를 이용한 5'-크산틸산의 제조방법 |
KR102006976B1 (ko) * | 2019-02-26 | 2019-08-06 | 씨제이제일제당 주식회사 | 신규 프로모터 및 이를 이용한 퓨린 뉴클레오티드 제조방법 |
KR102185850B1 (ko) * | 2020-02-21 | 2020-12-02 | 씨제이제일제당 주식회사 | 퓨린 뉴클레오티드를 생산하는 미생물 및 이를 이용한 퓨린 뉴클레오티드의 생산방법 |
KR102281365B1 (ko) * | 2021-01-26 | 2021-07-22 | 씨제이제일제당 (주) | 신규한 프롤린 탈수소효소 변이체 및 이를 이용한 l-발린 생산 방법 |
KR102419166B1 (ko) | 2021-09-23 | 2022-07-08 | 씨제이제일제당 주식회사 | 신규한 글루타민 가수분해 gmp 합성효소 변이체 및 이를 이용한 퓨린 뉴클레오티드의 생산방법 |
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