JP5400065B2 - 改良されたプロモーターおよびこれを用いたl−リシンの生産方法 - Google Patents
改良されたプロモーターおよびこれを用いたl−リシンの生産方法 Download PDFInfo
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- JP5400065B2 JP5400065B2 JP2010544884A JP2010544884A JP5400065B2 JP 5400065 B2 JP5400065 B2 JP 5400065B2 JP 2010544884 A JP2010544884 A JP 2010544884A JP 2010544884 A JP2010544884 A JP 2010544884A JP 5400065 B2 JP5400065 B2 JP 5400065B2
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- lysine
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Description
本発明の他の目的は、前記プロモータの活性を有する核酸分子を含むベクターを提供することにある。
本発明の別の目的は、前記ベクターで形質転換された形質転換体を提供することにある。
本発明の別の目的は、前記形質転換体を培養する段階を含む、リシンの生産方法を提供することにある。
本発明のプロモーター活性を有するコリネバクテリウム・グルタミカム核酸分子は、ジアミノピメリン酸デヒドロゲナーゼをコードする遺伝子と作動可能に連結される。ジアミノピメリン酸デヒドロゲナーゼをコードする遺伝子は、それぞれddh遺伝子であって、コリネバクテリウム属細菌でリシンを生産する生合成経路上の主要な遺伝子である。
(1)染色体挿入用ベクター(pDZ)の製作
本実施例では、大腸菌クローニング用ベクターpACYC177(New England Biolab、GenBank accetion #X06402)を基本ベクターとして用いて、コリネバクテリウム染色体挿入用ベクターpDZを製作した。
本実施例では、リシンを生産する菌株としてのコリネバクテリウム・グルタミカム由来のddh遺伝子のプロモーターを改良するための組換えベクターを製作した。
本実施例では、コリネバクテリウムの染色体上のddh遺伝子プロモーターを改良するために、前記で製作した組換えベクターをリシン生産菌株としてのコリネバクテリウム・グルタミカムKFCC10881に形質転換させて、菌株染色体のプロモーター配列と前記ベクター上のプロモーター配列を相同組換えによって置換させることにより、染色体内に前記改良プロモーター配列を挿入させた。
母菌株であるコリネバクテリウム・グルタミカムKFCC10881菌株および実施例2で最終的に製作されたL−リシン生産菌株であるコリネバクテリウム・グルタミカムKFCC10881−ddhP1菌株を培養し、培養液からタンパク質を分離して、ジアミノピメリン酸デヒドロゲナーゼ活性を測定した。
コリネバクテリウム・グルタミカムKFCC10881−ddhP1菌株は、母菌株KFCC10881に比べて23.2倍増加したジアミノピメリン酸デヒドロゲナーゼ活性を示すことを確認した(表2)。
原糖20g、ポリペプトン10g、酵母抽出物5g、(NH4)2SO4 5g、尿素1.5g、KH2PO4 4g、K2HPO4 8g、MgSO4・7H2O 0.5g、ビオチン150μg、チアミンHCl 1500μg、カルシウム−パントテン酸3000μg、ニコチンアミド3000μg(蒸留水1L基準)
母菌株として用いたコリネバクテリウム・グルタミカムKFCC10881菌株、および実施例2で最終的に製作されたL−リシン生産菌株としてのコリネバクテリウム・グルタミカムKFCC10881−ddhP1菌株を、L−リシン生産のために下記の方法で培養した。
原糖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、カルシウム−パントテン酸2000μg、ニコチンアミド2000μg(蒸留水1L基準)
ブドウ糖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、カルシウム−パントテン酸2000μg、ニコチンアミド3000μg、CaCO3 30g(蒸留水1L基準)
表3に示すように、ジアミノピメリン酸デヒドロゲナーゼ活性が23倍以上強化されたKFCC10881−ddhP1菌株は、母菌株KFCC10881に比べてリシン生産効率が約3%以上増加したことを確認することができた。
Claims (13)
- 配列番号2の1〜354番目の残基のヌクレオチド配列を含む、核酸分子。
- 配列番号2のヌクレオチド配列を有する、請求項1に記載の核酸分子。
- 請求項1または2のいずれかの核酸分子を含む、ベクター。
- 前記核酸分子が、作動可能なように対象となる遺伝子に連結されている、請求項3に記載のベクター。
- 前記対象となる遺伝子が、ジアミノピメリン酸デヒドロゲナーゼ(ddh)をコードする遺伝子である、請求項5に記載のベクター。
- リシンを生産するのに適している、請求項1または2のいずれかの核酸分子および前記核酸分子に作動可能なように連結されたジアミノピメリン酸デヒドロゲナーゼ(ddh)をコードする遺伝子を含むベクターで形質転換された、形質転換体。
- 前記形質転換体が、コリネバクテリウム属またはブレビバクテリウム属である、請求項7に記載の形質転換体。
- 受託番号KCCM10920Pであって、CA01−0136と命名された、請求項7に記載の形質転換体。
- 請求項1または2のいずれかの核酸分子が、形質転換体の染色体内に相同組換えで挿入されることを特徴とする、請求項7に記載の形質転換体。
- 請求項1または2のいずれかの核酸分子を、プラスミド形態で保有していることを特徴とする、請求項7に記載の形質転換体。
- 請求項1または2のいずれかの核酸分子および前記核酸分子に作動可能なように連結されたジアミノピメリン酸デヒドロゲナーゼ(ddh)をコードする遺伝子を含むベクターで形質転換された、リシン生産能を有する原核生物の形質転換体を培養する工程を含む、リシンの生産方法。
- 該形質転換体が、コリネバクテリウム属またはブレビバクテリウム属に属する、請求項12に記載の方法。
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PCT/KR2009/000382 WO2009096690A2 (ko) | 2008-01-31 | 2009-01-23 | 개량된 프로모터 및 이를 이용한 l-라이신의 생산 방법 |
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JP4075087B2 (ja) | 1996-12-05 | 2008-04-16 | 味の素株式会社 | L−リジンの製造法 |
US20020086371A1 (en) | 1999-07-07 | 2002-07-04 | Degussa-Huls Aktiengesellschaft | L-lysine-producing corynebacteria and process for the preparation of L-lysine |
WO2001009306A2 (en) * | 1999-08-02 | 2001-02-08 | Archer-Daniels-Midland Company | Mutant bacterial strains l-lysine production |
JP4623825B2 (ja) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | 新規ポリヌクレオチド |
US6927046B1 (en) * | 1999-12-30 | 2005-08-09 | Archer-Daniels-Midland Company | Increased lysine production by gene amplification using coryneform bacteria |
BRPI0017148B1 (pt) * | 2000-03-09 | 2016-03-01 | Basf Aktiengesellschfat | vetor, célula hospedeira recombinante, e, método para produzir um produto químico fino |
WO2002040679A2 (en) | 2000-11-15 | 2002-05-23 | Archer-Daniels-Midland Company | Corynebacterium glutamicum promoters |
KR100397322B1 (ko) | 2000-12-30 | 2003-09-06 | 씨제이 주식회사 | 엘-라이신의 제조방법 |
AU2002325923A1 (en) * | 2001-08-06 | 2003-05-19 | Degussa Ag | Production of l-lysine by genetically modified corynebacterium glutamicum strains |
KR100620092B1 (ko) | 2004-12-16 | 2006-09-08 | 씨제이 주식회사 | 코리네박테리움 속 세포로부터 유래된 신규한 프로모터서열, 그를 포함하는 발현 카세트 및 벡터, 상기 벡터를포함하는 숙주 세포 및 그를 이용하여 유전자를 발현하는방법 |
KR100653742B1 (ko) | 2004-12-30 | 2006-12-05 | 씨제이 주식회사 | 신규한 l-라이신-유도성 프로모터 |
EP2066782A4 (en) * | 2006-09-15 | 2010-02-03 | Cj Cheiljedang Corp | CORYNEBACTERIA WITH IMPROVED L-LYSINE PRODUCTIVITY AND METHOD FOR THE PREPARATION OF L-LYSINE THEREWITH |
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2008
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- 2009-01-23 CN CN201210322618.0A patent/CN103555721A/zh active Pending
- 2009-01-23 ES ES09705987.7T patent/ES2597877T3/es active Active
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Also Published As
Publication number | Publication date |
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PL2236610T3 (pl) | 2017-01-31 |
EP2990488A1 (en) | 2016-03-02 |
CN101939432B (zh) | 2013-02-13 |
CN101939432A (zh) | 2011-01-05 |
EP2993233A1 (en) | 2016-03-09 |
DK2993233T3 (da) | 2019-08-19 |
WO2009096690A3 (ko) | 2009-10-29 |
US20110076731A1 (en) | 2011-03-31 |
PL2993233T3 (pl) | 2019-11-29 |
BRPI0906584A2 (pt) | 2019-10-01 |
CN103555721A (zh) | 2014-02-05 |
HUE030771T2 (en) | 2017-05-29 |
HRP20161224T1 (hr) | 2016-12-02 |
KR20090084099A (ko) | 2009-08-05 |
EP2236610A2 (en) | 2010-10-06 |
EP2236610B1 (en) | 2016-07-20 |
ES2743422T3 (es) | 2020-02-19 |
EP2993233B1 (en) | 2019-06-19 |
KR100987281B1 (ko) | 2010-10-12 |
EP2236610A4 (en) | 2011-03-16 |
HUE044749T2 (hu) | 2019-11-28 |
DK2236610T3 (en) | 2016-11-14 |
ES2597877T3 (es) | 2017-01-23 |
WO2009096690A2 (ko) | 2009-08-06 |
US8535915B2 (en) | 2013-09-17 |
BRPI0906584B1 (pt) | 2020-12-01 |
JP2011510651A (ja) | 2011-04-07 |
LT2236610T (lt) | 2016-10-25 |
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