JP2011512155A - トランスポゾンを利用した形質転換用ベクター、前記ベクターで形質転換された微生物及びこれを利用したl−リジン生産方法 - Google Patents
トランスポゾンを利用した形質転換用ベクター、前記ベクターで形質転換された微生物及びこれを利用したl−リジン生産方法 Download PDFInfo
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- JP2011512155A JP2011512155A JP2010547576A JP2010547576A JP2011512155A JP 2011512155 A JP2011512155 A JP 2011512155A JP 2010547576 A JP2010547576 A JP 2010547576A JP 2010547576 A JP2010547576 A JP 2010547576A JP 2011512155 A JP2011512155 A JP 2011512155A
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- corynebacterium glutamicum
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
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Abstract
Description
実施例1:トランスポゾン遺伝子が挿入されたベクター(pDZTn)の製作及びこれを利用した遺伝子の挿入方法
本実施例では、コリネバクテリウム属微生物の染色体挿入用ベクターpDZを基本ベクターとして使用し、コリネバクテリウム属微生物におけるトランスポゾン遺伝子が挿入されたベクターpDZTnを製作した。製作過程は、以下の通りである。
コリネバクテリウムグルタミクムKCCM10770P由来のlysC/asd遺伝子を確保するために、NIH GenBankを根拠にしてlysC/asd遺伝子の塩基配列情報(NCBI登録番号NC_003450, Ncgl0247〜0248)を確保して、これに基づいて1対のプライマー(表2、配列番号7〜8)を合成した。
実施例2と同様な方法により、コリネバクテリウムグルタミクムKCCM10770P由来のdapA/dapB遺伝子を確保するために、NIH GenBankを根拠にしてdapA/dapB遺伝子の塩基配列情報(NCBI登録番号NC_003450, Ncgl1896〜1898)を確保した。その結果、dapA遺伝子は、dapB遺伝子とオペロンを構成しており、その間に、機能が明かされていないORF(Ncg11987)が存在していたため、dapB遺伝子のプロモーター部位を含んだdapB−ORF(Ncgl1897)-dapA遺伝子全体を増幅するために、2対のプライマー(表4、配列番号11〜12)を合成した。
クロストリジウムアセトブチリクム(Clostridium acetobutylicum)ATCC824由来のフルクトキナーゼ遺伝子の塩基配列は、既に明白に明かされて公開されている。米国国立保健院ジェンバンク(NIH GenBank)からクロストリジウムアセトブチリクムATCC824のフルクトキナーゼ遺伝子情報(登録番号NP_347064)を確保した。報告された塩基配列に基づいて、一対のプライマー(表6、配列番号14及び15)を合成して、クロストリジウムアセトブチリクムATCC 824菌株の染色体DNAを鋳型としたPCR法を通じて前記遺伝子を増幅した。 PCR条件は、変性94℃、20秒間;アニーリング52℃、20秒間;及び重合72℃、1分10秒間;を30回繰り返した。
L−リジン生産菌株であるコリネバクテリウムグルタミクムKCCM10770P−CJ2のアスパラギン酸キナーゼ活性をアスパルチルヒドロキサメートを利用した測定法(Pecher J-F, Capony J-P (1968) On the colorimetric determination of acyl phosphates.Anal Biochem 22: 536〜539)で測定した結果、コリネバクテリウムグルタミクムKCCM10770P−CJ2菌株が、母菌株であるコリネバクテリウムグルタミクムKCCM10770Pより2.1倍高い活性を有することを確認した。
フルクトキナーゼ発現ベクターから細胞内にフルクトキナーゼが発現されて、活性があるかを、公知文献に記載された方法により測定した(Andreas Pikisなど、Microbiology, 148, 843-852(2002))。コリネバクテリウムグルタミクムKCCM10770P−CJ3をLB培地で一日間培養した後、遠心分離して、細胞のみを獲得した。得られた細胞を適切なバッファに懸濁した後、超音波破砕法により細胞を破壊して、再び高速遠心分離して上澄み液を得た。上記の方法により得られた上澄み液をフルクトース、ホスホグルコースイソメラーゼ、グルコース−6−ホスフェートジヒドロゲナーゼ、ATP及びNADP+が入っている反応液と反応して生成されるNADPHの量を、波長340nmで吸光光度計を利用して間接的にフルクトキナーゼの活性を計算した結果を表9に示した。表11に示したように、コリネバクテリウムグルタミクムKCCM10770P−CJ3は、母菌株であるコリネバクテリウムグルタミクムKCCM10770P菌株より約2倍以上の活性を示すことから、フルクトキナーゼ遺伝子が発現されていることを確認した。
実施例3で最終的に製作されたL−リジン生産菌株であるコリネバクテリウムグルタミクムKCCM10770P−CJ2を、L−リジン生産のために、下記のような方法で培養した。
原糖20g、ペプトン10g、酵母抽出物5g、尿素1.5g、KH2PO4 4g、K2HPO4 8g、MgSO47H2O 0.5g、バイオチン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)
生産培地(pH7.0)
ブドウ糖100g、(NH4)2SO4 40g、大豆蛋白質2.5g、玉蜀黍浸漬固形分(Corn Steep Solids)5g、尿素3g、KH2PO4 1g、MgSO47H2O 0.5g、バイオチン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)
上記の表10で示したように、二つのリジン生合成遺伝子が挿入されたコリネバクテリウムグルタミクムKCCM10770P−CJ2は、コリネバクテリウムグルタミクム母菌株KCCM10770Pに比べ、リジン生産が10%増加されたことを確認することができた。
Claims (10)
- コリネバクテリウム属微生物由来のトランスポゾン遺伝子とマルチクローニングサイトを含むことを特徴とする形質転換用ベクター。
- 前記トランスポゾン遺伝子が配列番号1及び配列番号2のヌクレオチド配列を有することを特徴とする、請求項1に記載の形質転換用ベクター。
- 前記マルチクローニングサイトに、コリネバクテリウム属微生物由来のアスパラギン酸キナーゼを暗号化するlysC遺伝子、アスパラギン酸セミアルデヒドジヒドロゲナーゼを暗号化するasd遺伝子、ジヒドロジピコリネートシンターゼを暗号化するdapA遺伝子及びジヒドロジピコリネートリダクターゼを暗号化するdapB遺伝子からなる群から選択された一つまたはそれ以上の遺伝子が挿入されていることを特徴とする、請求項1に記載の形質転換用ベクター。
- 前記マルチクローニングサイトに外来のフルクトキナーゼを暗号化するsrk遺伝子が挿入されていることを特徴とする、請求項1に記載の形質転換用ベクター。
- 前記srk遺伝子は、クロストリジウムアセトブチリクム(Clostridium acetobutylicum)ATCC824由来のものであることを特徴とする、請求項4に記載の形質転換用ベクター。
- 請求項1〜5のいずれかに記載の形質転換用ベクターを使用して形質転換されたことを特徴とする、L−リジン生産能を有するコリネバクテリウムグルタミクム。
- 形質転換される微生物は、コリネバクテリウムグルタミクム(Corynebacterium glutamicum)KCCM10770Pであることを特徴とする、請求項6に記載のL−リジン生産能を有するコリネバクテリウムグルタミクム。
- 前記コリネバクテリウムグルタミクムは、コリネバクテリウムグルタミクム(Corynebacterium glutamicum)KCCM10770P−CJ2(受託番号:KCCM10939P)であることを特徴とする、請求項6に記載のL−リジン生産能を有するコリネバクテリウムグルタミクム。
- 前記形質転換用ベクターのマルチクローニングサイトに挿入された遺伝子は、2次クロスオーバーにより染色体に統合されることを特徴とする、請求項6に記載のL−リジン生産能を有するコリネバクテリウムグルタミクム。
- 請求項6〜9のいずれかに記載の微生物を培養して、細胞または培養物中にリジンを生産する段階と、
前記細胞または培養物からリジンを回収する段階と、を含むことを特徴とする、L−リジンを生産する方法。
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CN102027119B (zh) | 2014-03-05 |
KR20090107665A (ko) | 2009-10-14 |
US20100330624A1 (en) | 2010-12-30 |
KR101126041B1 (ko) | 2012-03-19 |
WO2009125992A2 (ko) | 2009-10-15 |
DE112009000453T5 (de) | 2010-12-30 |
US8323933B2 (en) | 2012-12-04 |
CN102027119A (zh) | 2011-04-20 |
JP5412446B2 (ja) | 2014-02-12 |
WO2009125992A3 (ko) | 2010-01-21 |
BRPI0908450A2 (pt) | 2015-08-18 |
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