JP2012511925A - 5’−キサントシン一リン酸生産能が向上されたコリネバクテリア菌株およびそれを用いた5’−キサントシン一リン酸の生産方法 - Google Patents
5’−キサントシン一リン酸生産能が向上されたコリネバクテリア菌株およびそれを用いた5’−キサントシン一リン酸の生産方法 Download PDFInfo
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Abstract
【解決手段】また、図1の切断地図に示された構造を有する組換えベクター、前記ベクターで形質転換されたコリネバクテリア菌株、および前記形質転換された菌株を培養することによって、5’−キサントシン一リン酸を生産する方法に関する。
【選択図】図1
Description
mqo遺伝子のヌクレオチド配列(NCBI ID_3345228)をNIHジェンバンク(GenBank)のデータから得た。前記配列に基づいて、2対のプライマー(配列番号1〜4)を合成した。
コリネバクテリウムKCCM−10530を鋳型としながら、高信頼DNAポリメラーゼ PfuΜltraTM(ストラタジーン(Stratagene))の存在下で前記配列番号1〜4のプライマーを使用して95℃で30秒間変性させ、55℃で30秒間アニーリングさせ;68℃で2分間伸長させることを25回繰り返した。こうして得たPCR生成物は、それぞれ2.1kbのmqo遺伝子2つのコピー(mqo−A、mqo−B)であり、これを配列番号1と2、および配列番号3と4の2つのセットをそれぞれ使用して増幅させた。
配列番号1:gctctagaATCGGTCATTCCATGAACCC
配列番号2:cgcggatccCATCGATATCGCCAACTCCA
配列番号3:cgcggatccATCGGTCATTCCATGAACCC
配列番号4:gctctagaCATCGATATCGCCAACTCCA
適合した制限酵素で処理した後に(mqo−A:XbaI+BamHI、mqo−B:BamHI+XbaI)、PCR生成物であるmqo−Aおよびmqo−Bを3片接合を介してXbaIとエビアルカリ性リン酸加水分解酵素で以前に処理したpDZベクターへ挿入した(大韓民国特許出願第10−2007−94433号参考)。最終的に、mqo遺伝子2つのコピーが連続的にクローン化された組換えpDZ−mqoベクターを得た。図1は、コリネバクテリウムゲノムに統合させるための組換えpDZ−mqoベクターの構造を示す模式図である。
前記pDZ−mqoベクターをKCCM−10530菌株に形質転換し、ゲノム上のmqo遺伝子に隣接した位置で一つのmqo遺伝子を挿入するためにゲノムと相同組換えを行った。こうして、そのゲノム上に2つのコピーのmqo遺伝子を有する、コリネバクテリウム・アンモニアゲネスKCJ−1304と命名した新規XMP生産菌株を得た。2つコピーのmqo遺伝子の連続的挿入は、2つコピーのmqo遺伝子のヌクレオチド配列の上流と下流を標的とするプライマーセット(配列5および6)を用いたPCRで確認した。
配列番号5 CTTTTCGATGACGCCCAA
配列番号6 CCACTTTATCGGGTGAGACCA
実施例2で製造されたXMP生産コリネバクテリウム・アンモニアゲネスKCJ−1304に対して下記のようにリンゴ酸デヒドロゲナーゼ活性を分析した。前記菌株をバクトペプトン10g/l、バクト牛肉抽出物5g/l、バクト酵母抽出物5g/l、NaCl2.5g/l、アデニン50mg/l、およびグアニン50mg/lを含有した培地に接種し、30℃で12時間OD10が得られるまでインキュベーションさせた。細胞培養液10mlを回収して50mM HEPES、10mM 酢酸カリウム、10mM CaCl2および10mM MgCl2を含む緩衝液で2回洗浄し、同じ緩衝液1mlに懸濁させた。超音波粉砕機(sonicator)を用いて中断させた後、細胞溶解物を遠心分離させた。上澄液を再び遠心分離してペレットを得た後、これを緩衝液100μlに懸濁させた。この懸濁液のうち、10μlを酵素液として使用した。50mM HEPES、10mM酢酸カリウムおよび50μM2,6−ジクロロインドフェノール(Cl2Ind)を混合して反応緩衝液を製造した。Cl2Indは、解凍させて反応直前に混合した。前記反応混合物980μlに基質として100mMリンゴ酸10μlおよび酵素液10μlを添加した後、30℃で15分間振盪しながらインキュベーションさせた。酵素活性は、還元されたCl2Indの濃度を測定することによって決定した。Cl2Indは、600nmで22cm−1mM−1の吸収係数を有した。
XMPを生産するために、実施例2で製造されたXMP生産菌株であるコリネバクテリウム・アンモニアゲネスKCJ−1304を下記のように培養した。母菌株コリネバクテリウム・アンモニアゲネスKCCM−10530および変異株KCJ−1304をそれぞれ下記の種培地3mlを含有する14mlチューブにそれぞれ接種し、30℃で20時間200rpmで振盪しながらインキュベーションさせた。その後、種培養液を0.4mlの量でそれぞれ250mlコーナーバッフル(corner-baffle)フラスコに含まれた32mlの下記の生産培地(24ml本培地+8ml培地A)に添加した後、続いて30℃で230rpmで96時間振盪培養した。以後、HPLCを用いて5’−XMPの生産を定量的に測定した。コリネバクテリウム・アンモニアゲネズKCJ−1304から生産されたXMPの量を下記の表2に示した。
生産培地(本培地):グルコース80g/l、硫酸マグネシウム10g/l、硫酸鉄20mg/l、硫酸亜鉛10mg/l、硫酸マンガン10mg/l、アデニン30mg/l、グアニン30mg/l、ビオチン100μg/l、硫酸銅1mg/l、塩化チアミン5mg/l、塩化カルシウム10mg/l、pH7.2
生産培地(培地A):リン酸一水素カリウム10g/l、リン酸二水素カリウム10g/l、尿素7g/l、硫酸アンモニウム5g/l
Claims (8)
- 野生型より高いリンゴ酸デヒドロゲナーゼ活性を有する、5’−キサントシン一リン酸を生産するためのコリネバクテリア菌株。
- リンゴ酸デヒドロゲナーゼの活性がmqo発現増加によって増進された請求項1に記載のコリネバクテリア菌株。
- 図1の切断地図に示された構造を有する組換えベクター。
- 請求項3の組換えベクターで形質転換されたコリネバクテリア菌株。
- 向上したリンゴ酸デヒドロゲナーゼの活性を示す請求項4に記載のコリネバクテリア菌株。
- 菌株が向上した5’−キサントシン一リン酸生産能を有するコリネバクテリウム・アンモニアゲネスである請求項4に記載のコリネバクテリア菌株。
- 菌株がコリネバクテリウム・アンモニアゲネスKCCM10972Pである請求項6に記載のコリネバクテリア菌株。
- 請求項4によるコリネバクテリア菌株を培養する段階;および培養液から5’−キサントシン一リン酸を回収する段階を含む、5’−キサントシン一リン酸の生産方法。
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KR1020080128847A KR101072708B1 (ko) | 2008-12-17 | 2008-12-17 | 5'-크산틸산 생산능이 향상된 미생물 및 이를 이용한 5'-크산틸산의 생산방법 |
KR10-2008-0128847 | 2008-12-17 | ||
PCT/KR2009/007559 WO2010071367A2 (en) | 2008-12-17 | 2009-12-17 | A corynebacteria strain having enhanced 5'-xanthosine monophosphate productivity and a method of producing 5'-xanthosine monophosphate using the same |
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KR101072720B1 (ko) * | 2008-12-17 | 2011-10-11 | 씨제이제일제당 (주) | 5'-구아닐산 생산능이 향상된 미생물 및 이를 이용한 5'-구아닐산의 생산방법 |
KR101929158B1 (ko) * | 2018-06-07 | 2018-12-13 | 씨제이제일제당 (주) | 5'-크산틸산을 생산하는 미생물 및 이를 이용한 5'-크산틸산의 제조방법 |
KR102288398B1 (ko) * | 2021-01-29 | 2021-08-10 | 씨제이제일제당 주식회사 | 신규한 nad(p)-의존성 산화환원효소 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 |
KR102273637B1 (ko) * | 2021-01-29 | 2021-07-06 | 씨제이제일제당 주식회사 | 신규한 펩티딜-디펩티다제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 |
KR102258159B1 (ko) * | 2021-01-29 | 2021-05-27 | 씨제이제일제당 (주) | 신규한 말레이트 디하이드로게나제 변이체 및 이를 이용한 l-라이신 생산 방법 |
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KR101283119B1 (ko) | 2005-01-12 | 2013-07-05 | 유노보, 인크. | 용기를 배기하고 밀봉하기 위한 방법 및 장치 |
KR100957690B1 (ko) | 2008-01-22 | 2010-05-12 | 씨제이제일제당 (주) | 5'-크산틸산 생산능이 향상된 코리네박테리움 속 미생물및 이를 이용한 5'-크산틸산의 생산 방법 |
KR101072720B1 (ko) * | 2008-12-17 | 2011-10-11 | 씨제이제일제당 (주) | 5'-구아닐산 생산능이 향상된 미생물 및 이를 이용한 5'-구아닐산의 생산방법 |
KR101210704B1 (ko) * | 2010-03-19 | 2012-12-10 | 씨제이제일제당 (주) | 5'-크산틸산 및 5'-구아닐산 생산능이 향상된 미생물 및 이를 이용한 5'-크산틸산 또는 5'-구아닐산의 생산방법 |
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KR20100070219A (ko) | 2010-06-25 |
US8815545B2 (en) | 2014-08-26 |
EP2358866A4 (en) | 2012-08-22 |
CN102317433A (zh) | 2012-01-11 |
DK2358866T3 (en) | 2015-11-23 |
WO2010071367A2 (en) | 2010-06-24 |
WO2010071367A3 (en) | 2010-11-04 |
CN102317433B (zh) | 2016-09-07 |
EP2358866B1 (en) | 2015-10-07 |
EP2358866A2 (en) | 2011-08-24 |
JP5553842B2 (ja) | 2014-07-16 |
ES2553571T3 (es) | 2015-12-10 |
KR101072708B1 (ko) | 2011-10-11 |
CN104130966A (zh) | 2014-11-05 |
US20110300582A1 (en) | 2011-12-08 |
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