JP2015506686A - プトレシン生産能が向上した組換え微生物及びそれを用いてプトレシンを生産する方法 - Google Patents
プトレシン生産能が向上した組換え微生物及びそれを用いてプトレシンを生産する方法 Download PDFInfo
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- JP2015506686A JP2015506686A JP2014553263A JP2014553263A JP2015506686A JP 2015506686 A JP2015506686 A JP 2015506686A JP 2014553263 A JP2014553263 A JP 2014553263A JP 2014553263 A JP2014553263 A JP 2014553263A JP 2015506686 A JP2015506686 A JP 2015506686A
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Abstract
Description
野生型コリネバクテリウム菌株の染色体からプトレシン生合成に有効な遺伝子をスクリーニングするために、野生型コリネバクテリウム菌株の染色体ライブラリーを作製した。具体的には、野生型コリネバクテリウムグルタミカムATCC13032菌株から抽出された染色体を制限酵素Sau3AIで処理してランダムに切断し、そこから5〜8kbの断片を選択し、大腸菌とコリネバクテリウムのシャトルベクターであるpECCG122(特許文献2)にクローニングすることにより染色体ライブラリーを作製した。
実施例2−1.A15クローンに含まれる5つの遺伝子のクローニング及び形質転換体の作製
実施例1で得たA15クローンの塩基配列は既に公開されている。ATCC13032菌株の公知の塩基配列に基づいて、NCgl0100遺伝子を増幅するためのプライマーとして配列番号1及び2のNCgl0100−F、NCgl0100−Rを作製し、N末端の436個のアミノ酸が除去された形態のtNCgl0100遺伝子を増幅するためのプライマーとして配列番号2及び3のNCgl0100−R、tNCgl0100−Fを作製し、NCgl0101遺伝子を増幅するためのプライマーとして配列番号4及び5のNCgl0101−F、NCgl0101−Rを作製し、NCgl0102とNCgl0103遺伝子を同時に増幅するためのプライマーとして配列番号6及び7のNCgl0102−F、NCgl0103−Rを作製し、NCgl0104遺伝子を増幅するためのプライマーとして配列番号8及び9のNCgl0104−F、NCgl0104−Rを作製した。また、発現プロモーターであるP(CJ7)(又はpcj7)(特許文献4参照)を増幅するためのプライマーとして配列番号10及び11のP(CJ7)−F、P(CJ7)−Rを作製した(表1)。
実施例2−1で得た全6種の形質転換体を対象に、実施例1と同様の方法で高濃度プトレシンを含む培地で成長のよかった形質転換体を選択した(図2)。図2は本発明で作製した各形質転換菌株の成長の程度を比較した結果を示す写真であり、1、2、3、4、5及び6は、それぞれ前記6種の発現ベクターであるpHC139T、pHC139T−P(CJ7)−NCgl0100、pHC139T−P(CJ7)−tNCgl0100、pHC139T−P(CJ7)−NCgl0101、pHC139T−P(CJ7)−NCgl0102−NCgl0103、及びpHC139T−P(CJ7)−NCgl0104が導入された菌株を示す。図2に示すように、pHC139T−P(CJ7)−NCgl0101が導入された形質転換体(4番)のみ高濃度プトレシンを含む培地で成長がよかったことが確認されたので、前記NCgl0101をプトレシン有効遺伝子として選択した。
実施例2で有効遺伝子として確認されたNCgl0101遺伝子を過剰発現する菌株のプトレシン生産能を評価した。プトレシン生産菌株KCCM11138PにpHC139T−P(CJ7)−NCgl0101が導入された菌株を用いた。
実施例4−1.ATCC13032ベースのプトレシン生産菌株におけるNCgl0101欠損菌株の作製
NCgl0101を過剰発現する場合は高濃度プトレシン培地で細胞成長性が増加するが、実施例3によれば、NCgl0101を過剰発現する場合にプトレシン生産量が減少することが確認された。このような結果に基づいて、NCgl0101欠損時にプトレシン生産性に及ぼす影響を確認した。
コリネバクテリウムグルタミカムATCC13032ベースのプトレシン生産菌株であるKCCM11138Pと同じ遺伝型を有するコリネバクテリウムグルタミカムATCC13869ベースのプトレシン生産菌株DAB12−a(ArgF欠損、NCgl1221欠損、大腸菌speC導入、Argオペロン−ArgCJBDプロモーター置換により強化)を対象に、NCgl0101欠損菌株を作製した。
プトレシン生産菌株においてNCgl0101欠損がプトレシン生産に及ぼす効果を確認するために、前記実施例4−1及び4−2で作製されたコリネバクテリウムグルタミカム変異株を対象に、プトレシン生産能を比較した。
Claims (11)
- プトレシンを生産するように変異させたコリネバクテリウム(Corynebacterium)属微生物において、配列番号17又は19で表されるアミノ酸配列を有するタンパク質の活性が内在的活性より低下又は欠失した、プトレシン生産能が向上したコリネバクテリウム属微生物。
- オルニチンデカルボキシラーゼ(speC)の活性がさらに導入された、請求項1に記載の微生物。
- 前記オルニチンデカルボキシラーゼが配列番号22のアミノ酸配列を有する、請求項2に記載の微生物。
- オルニチンカルバモイルトランスフェラーゼ(ArgF)及びグルタミン酸エクスポーター(NCgl1221)からなる群から選択される少なくとも1種の活性が内在的活性よりさらに低下した、請求項1に記載の微生物。
- ArgFが配列番号20のアミノ酸配列を有し、NCgl1221が配列番号21のアミノ酸配列を有する、請求項4に記載の微生物。
- アセチルガンマグルタミルホスフェートレダクターゼ(ArgC)、アセチルグルタミン酸シンターゼ又はオルニチンアセチルトランスフェラーゼ(ArgJ)、アセチルグルタミン酸キナーゼ(ArgB)、及びアセチルオルニチンアミノトランスフェラーゼ(ArgD)からなる群から選択される少なくとも1種の活性がさらに向上した、請求項1に記載の微生物。
- 前記ArgC、ArgJ、ArgB及びArgDが、それぞれ配列番号23、24、25及び26のアミノ酸配列を有する、請求項6に記載の微生物。
- 活性低下が、1)前記タンパク質をコードする遺伝子の一部又は全部の欠失、2)前記遺伝子発現の減少、3)前記タンパク質の活性が低下するように染色体上の前記遺伝子配列の変異、又は4)それらの組み合わせにより行われる、請求項1に記載の微生物。
- コリネバクテリウムグルタミカム(Corynebacterium glutamicum)である、請求項1に記載の微生物。
- 配列番号17又は19で表されるアミノ酸配列を有するタンパク質の活性が内在的活性より低下又は欠失した、向上したプトレシン生産能を有するコリネバクテリウム属微生物を培養する工程と、
前記工程で得られた培養物からプトレシンを分離する工程とを含むプトレシンの生産方法。 - コリネバクテリウム属微生物がコリネバクテリウムグルタミカムである、請求項10に記載のプトレシンの生産方法。
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