JP2004513603A - Corynebacterium glutamicum gene encoding a protein involved in membrane synthesis and transport - Google Patents

Abstract

An isolated nucleic acid molecule, the indicated MCT nucleic acid, which encodes a novel MCT protein from Corynebacterium glutamicum is described. The present invention also provides antisense nucleic acid molecules, recombinant expression vectors containing MCT nucleic acid molecules, and host cells into which the expression vectors are introduced. The present invention further relates to isolated MCT proteins, mutated MCT proteins, fusion proteins, antigenic peptides, and C.I. A method is provided for improving the production of a desired compound from Glutamicum based on genetic manipulation of the MCT gene of this organism.

Description

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【０１３２】
［実施例］
実施例１：コリネバクテリウム−グルタミカム ＡＴＣＣ１３０３２の全ゲノムＤＮＡの製造
コリネバクテリウム−グルタミカム （ＡＴＣＣ１３０３２）の培養は、激しく攪拌したＢＨＩ媒体（Ｄｉｆｃｏ）中で、３０℃で終夜行なわれた。細胞は遠心分離により回収され、上澄みを処分し、細胞は、５ｍｌの緩衝液Ｉ（最初の培養容積の５％、すべての示された容積は、１００ｍｌの培養容積のために計算されたものである。）中再懸濁された。緩衝液Ｉの組成は、１４０．３４ｇ／ｌのショ糖、２．４６ｇ／ｌのＭｇＳＯ_４ｘ７Ｈ_２Ｏ、１０ｍｌ／ｌのＫＨ_２ＰＯ_４溶液（１００ｇ／ｌ、ＫＯＨでｐＨ６．７に調整）、５０ｍｌ／ｌのＭ１２濃縮物（１０ｇ／ｌの（ＮＨ_４）_２ＳＯ_４、１ｇ／ｌのＮａＣｌ、２ｇ／ｌのＭｇＳＯ_４ｘ７Ｈ_２Ｏ、０．２ｇ／ｌのＣａＣｌ_２、０．５ｇ／ｌの酵母抽出物（Ｄｉｆｃｏ）、１０ｍｌ／ｌのトレース−エレメンツ−ミックス（２００ｍｇ／ｌのＦｅＳＯ_４ｘＨ_２Ｏ、１０ｍｇ／ｌのＺｎＳＯ_４ｘ７Ｈ_２Ｏ、３ｍｇ／ｌのＭｎＣｌ_２ｘ４Ｈ_２Ｏ、３０ｍｇ／ｌのＨ_３ＢＯ_３、２０ｍｇ／ｌのＣｏＣｌ_２ｘ６Ｈ_２Ｏ、１ｍｇ／ｌのＮｉＣｌ_２ｘ６Ｈ_２Ｏ、３ｍｇ／ｌのＮａ_２ＭｏＯ_４ｘ２Ｈ_２Ｏ、５００ｍｇ／ｌの錯化剤（ＥＤＴＡまたはクエン酸）、１００ｍｌ／ｌのビタミン−ミックス（０．２ｍｇ／ｌのビオチン、０．２ｍｇ／ｌの葉酸、２０ｍｇ／ｌのｐ−アミノ安息香酸、２０ｍｇ／ｌのリボフラビン、４０ｍｇ／ｌのパントテン酸カルシウム、１４０ｍｇ／ｌのニコチン酸、４０ｍｇ／ｌの塩酸ピリドキソール、２００ｍｇ／ｌのミオ−イノシトール）である。リゾチームを最終濃度が２．５ｍｇ／ｍｌになるまで懸濁液に加えた。約４時間、３７℃での培養の後、細胞壁を劣化させ得られたプロトプラストを遠心分離によって回収した。ペレットを５ｍｌの緩衝液Ｉで一度、５ｍｌのＴＥ緩衝液（１０ｍＭのトリスＨＣｌ、１ｍＭのＥＤＴＡ、ｐＨ８）で一度洗浄した。ペレットを４ｍｌのＴＥ緩衝液に再懸濁し、０．５ｍｌのＳＤＳ溶液（１０％）と０．５ｍｌのＮａＣｌ溶液（５Ｍ）を加えた。プロテイナーゼＫを最終濃度２００μｇ／ｍｌになるまで加えた後、懸濁液を約１８時間、３７℃で培養した。ＤＮＡを標準的な操作で、フェノール、フェノール−クロロホルム−イソアミルアルコールおよびクロロホルム−イソアミルアルコールによる抽出により精製した。それから、ＤＮＡを１／５０容積の３Ｍ酢酸ナトリウムおよび２容積のエタノールを加えて析出させ、次いで３０分間、−２０℃でインキュベートし、ＳＳ３４ローター（Ｓｏｒｖａｌｌ）を使用した高速遠心分離機で１２０００ｒｐｍで遠心分離を３０分間行なった。ＤＮＡを２０μｇ／ｍｌのＲＮアーゼＡを含む１ｍｌのＴＥ緩衝液に溶解し、４℃で、少なくとも３時間１０００ｍｌのＴＥ緩衝液に対して透析した。この間、緩衝液を３度交換した。透析したＤＮＡ溶液の０．４ｍｌの部分に、０．４ｍｌの２ＭＬｉＣｌと０．８ｍｌのエタノールを加えた。３０分間、−２０℃でインキュベートした後、遠心分離（１３０００ｒｐｍ、Ｂｉｏｆｕｇｅ　Ｆｒｅｓｃｏ，Ｈｅｒａｅｕｓ，Ｈａｎａｕ，Ｇｅｒｍａｎｙ）によりＤＮＡを集めた。ＤＮＡペレットをＴＥ緩衝液に溶解した。この操作により製造されたＤＮＡは、すべての目的、サザンブロット法またはゲノムライブラリーの構築に使用することができた。
【０１３３】
実施例２：コリネバクテリウム−グルタミカム ＡＴＣＣ１３０３２のＥｓｃｈｅｒｉｃｈｉａ Ｃｏｌｉにおけるゲノムライブラリーの構築
実施例１に記載したように製造されたＤＮＡを用いて、コスミッドおよびプラスミドライブラリーが、公知で、よく確立された方法に従って構築された（例えば、Ｓａｍｂｒｏｏｋ，Ｊら、（１９８９）’Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ’， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ ＰｒｅｓｓまたはＡｕｓｕｂｅｌ，Ｆ．Ｍら（１９９４） ’Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ’， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ．を参照）。
【０１３４】
どんなプラスミドまたはコスミッドでも使用することができた。特別に、プラスミドｐＢＲ３２２（Ｓｕｔｃｌｉｆｆｅ， Ｊ．Ｇ．（１９７９） Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，７５：３７３７−３７４１）；ｐＡＣＹＣ１７７ （Ｃｈａｎｇｅ とＣｏｈｅｎ（１９７８） Ｊ．Ｂａｃｔｅｒｉｏｌ １３４：１１４１−１１５６），ｐＢＳの系列のプラスミド（ｐＢＳＳＫ＋，ｐＢＳＳＫ−およびその他；Ｓｔｒａｔａｇｅｎｅ ＬａＪｏｌｌａ，ＵＳＡ），またはＳｕｐｅｒＣｏｓ１（Ｓｔｒａｔａｇｅｎｅ ＬａＪｏｌｌａ，ＵＳＡ）、Ｌｏｒｉｓｔ６（Ｇｉｂｓｏｎ，Ｔ．Ｊ．，Ｒｏｓｅｎｔｈａｌ Ａ．およびＷａｔｅｒｓｏｎ，Ｒ．Ｈ．（１９８７） Ｇｅｎｅ ５３：２８３−２８６）のコスミッドが使用された。特にＣ．ｇｌｕｔａｍｉｃｕｍにおいて使用するためのジーンライブラリーは、プラスミドｐＳＬ１０９（ Ｌｅｅ，Ｈ．Ｓ．およびＡ．Ｊ．Ｓｉｎｓｋｅｙ（１９９４） Ｊ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．４：２５６−２６３）を用いて構築することができる。
【０１３５】
実施例３：ＤＮＡシーケンスとコンピューターによる機能解析
実施例２に記載されたゲノムライブラリーを標準的な方法、特にＡＢＩ３７７シーケンスマシーンを用いた鎖成長停止反応法（例えばＦｌｅｉｓｃｈｍａｎ，Ｒ．Ｄ．ら（１９９５）’Ｗｈｏｌｅ−ｇｅｎｏｍｅ Ｒａｎｄｏｍ Ｓｅｑｕｅｎｃｉｎｇ ａｎｄ Ａｓｓｅｍｂｌｙ ｏｆ Ｈａｅｍｏｐｈｉｌｕｓ Ｉｎｆｌｕｅｎｚａｅ Ｒｄ．’，Ｓｃｉｅｎｃｅ， ２６９：４９６−５１２参照）に従ったＤＮＡシーケンスに使用した。以下のヌクレオチド配列のシーケンスプライマーが使用された：５’−ＧＧＡＡＡＣＡＧＴＡＴＧＡＣＣＡＴＧ−３’または５’−ＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ−３’。
【０１３６】
実施例４：生体内突然変異誘発
コリネバクテリウム−グルタミカムの生体内突然変異誘発が、Ｅ．ｃｏｌｉまたは他の微生物（例えばＢａｃｉｌｌｕｓ ｓｐｐ．またはＳａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅなどの酵母）であってその遺伝子情報の完全性を保つための能力が減じられているものを通して、プラスミド（または他のベクター）の通過により行なうことができる。典型的なミューテーター株は、ＤＮＡ修復システム（例えばｍｕｔＨＬＳ，ｍｕｔＤ，ｍｕｔＴその他；Ｒｕｐｐ，Ｗ．Ｄ．（１９９６） ＤＮＡ ｒｅｐａｉｒ ｍｅｃｈａｎｉｓｍｓ，Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ａｎｄ Ｓａｌｍｏｎｅｌｌａ，ｐ２２７７−２２９４，ＡＳＭ：Ｗａｓｈｉｎｇｔｏｎ参照）の遺伝子に変異を持っている。このような株は当業者によく知られている。このような株の使用は、例えばＧｒｅｅｎｅｒ，Ａ．およびＣａｌｌａｈａｎ，Ｍ．（１９９４） Ｓｔｒａｔｅｇｉｅｓ ７：３２−３４に記載されている。
【０１３７】
実施例５：Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ および コリネバクテリウム−グルタミカムの間のＤＮＡ転換
いくつかのコリネバクテリウムとブレビバクテリウム種は自律的に複製する（例えば、Ｍａｒｔｉｎ， Ｊ．Ｆ．ら（１９８７） Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ５：１３７−１４６参照）内因性プラスミド（例えばｐＨＭ１５１９またはｐＢＬ１）を含む。Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉとＣｏｒｙｎｅｂａｃｕｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍのシャトルベクターは、Ｅ．ｃｏｌｉの標準ベクター（Ｓａｍｂｒｏｏｋ，Ｊ．ｅｔ ａｌ．（１９８９），’Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ：Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ’，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ ＰｒｅｓｓまたはＡｕｓｕｂｅｌ，Ｆ．Ｍ．ｅｔ ａｌ．（１９９４）’Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ’， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ）を用いて、これに、開始または複製のおよび適当なコリネバクテリウム−グルタミカム由来のマーカーを加えることで容易に構築することができる。このような複製の開始点は、好ましくはコリネバクテリウムとブレビバクテリウム種から単離された内因性プラスミドからもたらされる。これらの種の形質転換マーカーとして、カナマイシン耐性の遺伝子（Ｔｎ５またはＴｎ９０３トランスポゾンから誘導されたもの）またはクロラムフェニコール耐性遺伝子（Ｗｉｎｎａｃｋｅｒ，Ｅ．Ｌ．（１９８７）’Ｆｒｏｍ Ｇｅｎｅｓ ｔｏ Ｃｌｏｎｅｓ−Ｉｎｔｒｏｄｕｃｔｉｏｎ ｔｏ Ｇｅｎｅ Ｔｅｃｈｎｏｌｏｇｙ，ＶＣＨ，Ｗｅｉｎｈｅｉｍ）が特に用いられる。Ｅ．ｃｏｌｉ とＣ．ｇｌｕｔａｍｉｃｕｍの双方でおいて複製するシャトルベクターの広範囲の構築の文献は数多く例があり、遺伝子過剰発現を含むいくつかの目的に使用することができる（例えば、Ｙｏｓｈｉｈａｍａ，Ｍ．ｅｔ ａｌ．（１９８５） Ｊ．Ｂａｃｔｅｒｉｏｌ． １６２：５９１−５９７， Ｍａｒｔｉｎ，Ｊ．Ｆ．ｅｔ ａｌ．（１９８７） Ｂｉｏｔｅｃｈｎｏｌｏｇｙ，５：１３７−１４６ およびＥｉｋｍａｎｎｓ，Ｂ．Ｊ．ｅｔ ａｌ．（１９９１） Ｇｅｎｅ， １０２：９３−９８参照）。
【０１３８】
標準的な方法を用いて、上述のように注目の遺伝子をシャトルベクターの一つヘクローンすることが可能であり、またこのようなハイブリッドベクターをコリネバクテリウム−グルタミカム株に導入することが可能である。Ｃ．ｇｌｕｔａｍｉｃｕｍの形質転換を、プロトプラスト形質転換（Ｋａｓｔｓｕｍａｔａ，Ｒ．ｅｔ ａｌ．（１９８４） Ｊ．Ｂａｃｔｅｒｉｏｌ． １５９３０６−３１１），エレクトロポレーション（Ｌｉｅｂｌ，Ｅ．ｅｔ ａｌ．（１９８９）ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌ．Ｌｅｔｔｅｒｓ，５３：３９９−３０３）そして特別のベクターが用いられた場合には、接合（例えば Ｓｃｈａｅｆｅｒ，Ａ ｅｔ ａｌ．（１９９０）Ｊ．Ｂａｃｔｅｒｉｏｌ．１７２：１６６３−１６６６に記載）によって達成することができる。Ｃ．ｇｌｕｔａｍｉｃｕｍからのプラスミドＤＮＡの製造（周知の標準的な方法を用いて）およびそのＥ．Ｃｏｌｉへの形質転換によるＣ．ｇｌｕｔａｍｉｃｕｍからＥ．ｃｏｌｉへのシャトルベクターの転換も可能である。この形質転換の段階は、標準的な方法を用いて行なうことができるが、ＭＣＴ−不足Ｅ．ｃｏｌｉ株、例えばＮＭ５２２（Ｇｏｕｇｈ ａｎｄ Ｍｕｒｒａｙ（１９８３）Ｊ．Ｍｏｌ．Ｂｉｏｌ．１６６：１−１９）を使用することが有利である。
【０１３９】
遺伝子は、ｐＣＧ１（Ｕ．Ｓ．ｐａｔｅｎｔ Ｎｏ．４６１７２６７）またはその断片、任意でＴＮ９０３（Ｇｒｉｎｄｌｅｙ，Ｎ．Ｄ．ａｎｄ Ｊｏｙｃｅ，Ｃ．Ｍ．（１９８０）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ７７（１２）：７１７６−７１８０）からのカナマイシン耐性遺伝子を含むプラスミドを用いたＣ．ｇｌｕｔａｍｉｃｕｍ 株中に過剰発現させることができる。加えて、遺伝子は、プラスミドｐＳＬ１０９（Ｌｅｅ，Ｈ．Ｓ ａｎｄ Ａ．Ｊ．Ｓｉｎｓｋｅｙ（１９９４） Ｊ．Ｍｉｃｒｏｂｉｏｌ Ｂｉｏｔｅｃｈｎｏｌ．４：２５６−２６３）を用いてＣ．Ｇｌｕｔａｍｉｃｕｍ株中に過剰発現することができる。
【０１４０】
複製プラスミドに加えて、遺伝子の過剰発現は、ゲノムへの組み込みによって達成することができる。Ｃ．ｇｌｕｔａｍｉｃｕｍ または他のコリネバクテリウムまたはブレビバクテリウム種における遺伝子組み込みは、周知の方法、例えばゲノム領域での相同性組換え、制限エンドヌクレアーゼ仲介の組み込み（ＲＥＭＩ）（例えばＤＥ ｐａｔｅｎｔ １９８２３８３４）またはトランスポゾンの使用などによって達成することができる。調節領域（例えば、プロモーター、レプレッサー、および／またはエンハンサー）を配列の修飾、挿入、サイト−ダイレクテド法（例えば相同性組換え）による削除、またはランダムイベントに基づく方法（例えばトランスポゾン突然変異誘発またはＲＥＭＩ）により変形することにより、注目遺伝子の活性を変化させることもできる。転写ターミネーターとして機能する核酸配列は、本発明の一以上の遺伝子のコード領域へ３’挿入されることもできる。このようなターミネーターは、周知であり、例えばＷｉｎｎａｃｋｅｒ， Ｅ．Ｌ． （１９８７）Ｆｒｏｍ Ｇｅｎｅｓ ｔｏ Ｃｌｏｎｅｓ−Ｉｎｔｒｏｄｕｃｔｉｏｎ ｔｏ Ｇｅｎｅ Ｔｅｃｈｎｏｌｏｇｙ． ＶＣＨ： Ｗｅｉｎｈｅｉｍ中に記載されている。
【０１４１】
実施例６：変異タンパク質の発現の評価
形質転換された宿主細胞中の変異タンパク質の活性の観察は、変異タンパク質が、野生種のタンパク質と類似した形態と類似した量で発現されるという事実に基づいている。変異遺伝子（遺伝子産物に対する転写に用いられるｍＲＮＡの量のインジケーター）の転写のレベルを確認するために有用な方法は、ノーザン・ブロット法（Ａｕｓｕｂｅｌ ｅｔ ａｌ．（１９８８） Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｗｉｌｅｙ： Ｎｅｗ Ｙｏｒｋ参照）であり、注目遺伝子に結合するように設計されたプライマーが、検出タグ（通常放射性または化学ルミネセンス）により、生物の培養の総ＲＮＡを抽出し、ゲルに流し、安定マトリックスに移動させ、このプローブでインキュベートする場合に、プローブの結合および結合の量が、この遺伝子のｍＲＮＡの存在と量を示すようにラベルされている。この情報は、変異遺伝子の転写の程度の証拠である。全部の細胞のＲＮＡは、いくつかの周知の方法、例えば、 Ｂｏｒｍａｎｎ， Ｅ．Ｒ． ｅｔ ａｌ． （１９９２） Ｍｏｌ． Ｍｉｃｒｏｂｉｏｌ． ６：３１７−３２６に記載された方法により、コリネバクテリウム−グルタミカムから製造することができる。
【０１４２】
このｍＲＮＡから翻訳されたタンパク質の存在と比較量の評価のために、ウェスタン・ブロッド法などの標準的な技術が使用され得る（例えばＡｕｓｕｂｅｌ ｅｔ ａｌ． （１９８８） Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｗｉｌｅｙ： Ｎｅｗ Ｙｏｒｋ参照）。この方法では、全部の細胞のタンパク質は、抽出され、ゲル電気泳動により分離され、ニトロセルロースのようなマトリックスに移され、設計されたタンパク質に特異的に結合する抗体などのプローブとともにインキュベートされる。このプローブは、一般に容易に検出可能な化学ルミネセンスまたは比色のラベルでタグがつけられている。観察されたラベルの存在と量は、細胞中の所望の変異タンパク質の存在と量を示す。
【０１４３】
実施例７：遺伝子的に修飾されたＣｏｒｙｎｅｂａｃｔｅｒｉａの成長−媒体および培養条件
遺伝子的に修飾されたコリネバクテリウム−グルタミカムは、合成または天然成長媒体中で培養される。Ｃｏｒｙｎｅｂａｃｔｅｒｉａの異なった成長媒体の多くはどちらも知られており、容易に入手可能である（Ｌｉｅｂ ｅｔ ａｌ． （１９８９） Ａｐｐｌ． Ｍｉｃｒｏｂｉｏｌ． Ｂｉｏｔｅｃｈｎｏｌ．， ３２：２０５−２１０； ｖｏｎ ｄｅｒ Ｏｓｔｅｎ ｅｔ ａｌ． （１９９８） Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｌｅｔｔｅｒｓ， １１：１１−１６； Ｐａｔｅｎｔ ＤＥ ４，１２０，８６７； Ｌｉｅｂｌ （１９９２）
’Ｔｈｅ Ｇｅｎｕｓ Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ， ｉｎ： Ｔｈｅ Ｐｒｏｃａｒｙｏｔｅｓ， Ｖｏｌｕｍｅ ＩＩ， Ｂａｌｏｗｓ， Ａ． ｅｔ ａｌ．， ｅｄｓ． Ｓｐｒｉｎｇ−Ｖｅｒｌａｇ）。これらの媒体は、一種以上の炭素源、窒素源、無機塩、ビタミンおよび痕跡の要素からなる。好ましい炭素源は、モノ−、ジ−またはポリサッカライドなどの砂糖である。例えば、グルコース、フルクトース、マンノース、ガラクトース、リボース、ソルボース、リブロース、ラクトース、マルトース、スクロース、ラフィノース、デンプンまたはセルロースが非常によい炭素源として働く。糖蜜または他の砂糖精製の副生成物のような複合化合物によって媒体に砂糖を供給することも可能である。異なった炭素源の混合物を供給することも有利である。他の可能な炭素源は、メタノール、エタノール、酢酸、乳酸などのアルコールおよび有機酸である。窒素源は通常有機または無機の窒素化合物か、またはこれらの化合物を含む材料である。窒素源として例えば、アンモニアガス、アンモニウム塩（例えばＮＨ_４Ｃｌまたは（ＮＨ_４）_２ＳＯ_４）、ＮＨ_４ＯＨ、硝酸塩、尿素、アミノ酸、コーンスティープリカー、大豆粉、大豆タンパク質、酵母抽出物、肉抽出物などの複合窒素源が含まれる。
【０１４４】
媒体に含まれる無機塩化合物は、クロリド−、リン酸、硫酸のカルシウム塩、マグネシウム塩、ナトリウム塩、コバルト塩、モリブデン塩、カリウム塩、マグネシウム塩、亜鉛塩、銅塩、鉄塩を含む。金属イオンを溶液中に保持するために媒体中にキレート化合物を加えることができる。特に有用なキレート化合物は、カテコールやプロトカテチュエートなどのジヒドロキシフェノールまたはクエン酸などの有機酸を含む。ビタミンなどの成長因子または成長促進剤、例えばビオチン、リボフラビン、チアミン、葉酸、ニコチン酸、パントテン酸塩、ピリドキシンなどを含む媒体が典型的である。成長因子および塩はしばしば酵母抽出物、糖蜜、コーンスティープリカーなどの複合媒体成分に由来する。正確な媒体化合物の組成は、直接の実験に強く依存し、各々の特定の場合に個々に決定される。媒体の最適化の情報は、テキストブックＡｐｐｌｉｅｄ Ｍｉｃｒｏｂｉｏｌ． Ｐｈｙｓｉｏｌｏｇｙ， Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ （ｅｄｓ． Ｐ．Ｍ． Ｒｈｏｄｅｓ， Ｐ．Ｆ． Ｓｔａｎｂｕｒｙ， ＩＲＬ Ｐｒｅｓｓ （１９９７） ｐｐ．５３−７３， ＩＳＢＮ ０１９ ９６３５７７ ３） から入手可能である。成長媒体を市販品、例えば標準１（Ｍｅｒｃｋ）またはＢＨＩ（ｇｒａｉｎ ｈｅａｒｔ ｉｎｆｕｓｉｏｎ， ＤＩＦＣＯ）などから選定することも可能である。
【０１４５】
すべての媒体成分は、熱（１．５×１０^５Ｐａ、１２１℃で２０分間）またはろ過殺菌法のいずれかにより殺菌される。成分は、一緒に、または所望により分割して殺菌することができる。すべての媒体成分は、成長の初期に存在させるか、または任意に連続的にまたは回分式に加えることができる。
【０１４６】
培養条件は各々の実験のために分けて定義される。温度は、１５℃から４５℃の範囲であるべきである。温度を一定に保つか、実験中に変えることができる。媒体のｐＨは、５から８．５、好ましくは７．０前後の範囲であるべきであり、媒体に緩衝液を添加することで一定に保つことができる。この目的のための緩衝液の例は、リン酸カリウム緩衝液である。ＭＯＰＳ， ＨＥＰＥＳ， ＡＣＥＳその他の合成緩衝液を代わりに、または同時に使用することができる。成長の間にＮａＯＨまたはＮＨ_４ＯＨを添加することより一定の培養ｐＨを保つことも可能である。酵母抽出物などの複合媒体成分が使用される場合、多くの複合成分は高い緩衝能力があるという事実によって、追加の緩衝液が必要ないかもしれない。微生物の培養に発酵槽を使用する場合、ｐＨはアンモニアガスにより制御することもできる。
【０１４７】
インキュベート時間は通常数時間から数日の範囲である。この時間は、ブロスの中に蓄積される生成物を最大にするように選択される。開示された成長実験は、種々の容器、例えばミクロタイター板、ガラス管、ガラスフラスコ、または種々のサイズのガラスまたは金属発酵槽等の中で行なうことができる。多数のクローンのスクリーニングのために、微生物をミクロタイター板、ガラス管、振とうフラスコ中、バッフル付きで、またはなしで培養すべきである。好ましくは１００ｍｌの振とうフラスコを使用し、所望の成長媒体の１０容積％で満たす。フラスコを１００から３００ｒｐｍの速さの範囲を使用して、ロータリーシェーカー（ふり幅２５ｍｍ）で振とうすべきである。蒸発ロスは湿潤雰囲気中に保つことによって少なくすることができる。代わりに、蒸発ロスの数学的補正が行なわれるべきである。
【０１４８】
遺伝子的に修飾されたクローンを試験した場合、修飾されていないコントロールのクローンまたはいかなる挿入もなされていない基本的なプラスミドを含むコントロールのクローンも試験するべきである。媒体を寒天平板上、例えばＣＭプレート（１０ｇ／ｌのグルコース、２．５ｇ／ｌのＮａＣｌ、２ｇ／ｌの尿素、１０ｇ／ｌのポリペプトン、５ｇ／ｌの酵母抽出物、５ｇ／ｌの肉抽出物、２２ｇ／ｌの寒天、２Ｍ ＮａＯＨによるｐＨ６．８としたもの）を３０℃でインキュベートしたもので成長させた細胞を用いて０．５−１．５のＯＤ_６００まで接種する。媒体の接種は、ＣＭプレートからのＣ．ｇｌｕｔａｍｉｃｕｍ細胞のサリーン懸濁液の導入、またはこのバクテリアの前培養液の添加のいずれかによりなされる。
【０１４９】
実施例８：変異タンパク質の機能のインビトロ解析
酵素の活性と速度論的パラメーターの決定は、先行技術でよく確立されている。得られた改変された酵素の活性決定のための実験は、当業者の能力の範囲内で、野生種酵素の比活性に対して行なわれなければならない。一般に酵素についての概要は、構造、速度論的、原理、方法、適用および多数の酵素の活性の決定例に関する特定の詳細と同様に、例えば以下の参照に見出される：Ｄｉｘｏｎ， Ｍ．， ａｎｄ Ｗｅｂｂ， Ｅ．Ｃ．， （１９７９） Ｅｎｚｙｍｅｓ． Ｌｏｎｇｍａｎｓ： Ｌｏｎｄｏｎ； Ｆｅｒｓｈｔ， （１９８５） Ｅｎｚｙｍｅ Ｓｔｒｕｃｔｕｒｅ ａｎｄ Ｍｅｃｈａｎｉｓｍ．　Ｆｒｅｅｍａｎ： Ｎｅｗ Ｙｏｒｋ； Ｗａｌｓｈ， （１９７９） Ｅｎｚｙｍａｔｉｃ Ｒｅａｃｔｉｏｎ Ｍｅｃｈａｎｉｓｍｓ． Ｆｒｅｅｍａｎ： Ｓａｎ Ｆｒａｎｃｉｓｃｏ； Ｐｒｉｃｅ， Ｎ．Ｃ．， Ｓｔｅｖｅｎｓ， Ｌ． （１９８２） Ｆｕｎｄａｍｅｎｔａｌｓ ｏｆ Ｅｎｚｙｍｏｌｏｇｙ． Ｏｘｆｏｒｄ Ｕｎｉｖ． Ｐｒｅｓｓ：Ｏｘｆｏｒｄ； Ｂｏｙｅｒ， Ｐ．Ｄ．， ｅｄ（１９８３） Ｔｈｅ Ｅｎｚｙｍｅｓ， ３ｒｄ ｅｄ． Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ： Ｎｅｗ Ｙｏｒｋ； Ｂｉｓｓｗａｎｇｅｒ， Ｈ．， （１９９４） Ｅｎｚｙｍｋｉｎｅｔｉｃ， ２ｎｄ ｅｄ． ＶＣＨ： Ｗｅｉｎｈｅｉｍ （ＩＳＢＮ ３５２７３００３２５）， Ｂｅｒｇｍｅｙｅｒ， Ｈ．Ｕ．， Ｂｅｒｇｍｅｙｅｒ， Ｊ．， Ｇｒａｓｓｌ， Ｍ．， ｅｄｓ． （１９８３−１９８６） Ｍｅｔｈｏｄｓ ｏｆ Ｅｎｚｙｍａｔｉｃ Ａｎａｌｙｓｉｓ， ３ｒｄ ｅｄ．， ｖｏｌ． Ｉ−ＸＩＩ， Ｖｅｒｌａｇ Ｃｈｅｍｉｅ： Ｗｅｉｎｈｅｉｍ； ａｎｄ Ｕｌｌｍａｎｎ’ｓ Ｅｎｃｙｃｌｏｐｅｄｉａ ｏｆ Ｉｎｄｕｓｔｒｉａｌ Ｃｈｅｍｉｓｔｒｙ （１９８７） ｖｏｌ． Ａ９， ’Ｅｎｚｙｍｅｓ’． ＶＣＨ： Ｗｅｉｎｈｅｉｍ， ｐ． ３５２−３６３．。
【０１５０】
ＤＮＡと結合するタンパク質の活性は、数種のよく確立された方法、例えばＤＮＡバンド−シフトアッセイ（ゲル遅延アッセイとも呼ばれる）により測定することができる。他の分子の発現におけるこのようなタンパク質の効果は、レポーター遺伝子アッセイ（例えばＫｏｌｍａｒ， Ｈ． ｅｔ ａｌ． （１９９５） ＥＭＢＯ Ｊ． １４： ３８９５−３９０４および引用文献に記載されたような） を用いて測定される。レポーター遺伝子試験系は、周知であり、ベータ−ガラクトシダーゼ、緑蛍光性タンパク質およびその他数種の酵素を用いて、原核および真核細胞のどちらにでも適用が確立されている。
【０１５１】
膜輸送タンパク質の活性の決定は、例えばＧｅｎｎｉｓ， Ｒ．Ｂ． （１９８９） ’Ｐｏｒｅｓ， Ｃｈａｎｎｅｌｓ ａｎｄ Ｔｒａｎｓｐｏｒｔｅｒｓ’， ｉｎ Ｂｉｏｍｅｍｂｒａｎｅｓ， Ｍｏｌｅｃｕｌａｒ Ｓｔｒｕｃｔｕｒｅ ａｎｄ Ｆｕｎｃｔｉｏｎ， Ｓｐｒｉｎｇｅｒ； Ｈｅｉｄｅｌｂｅｒｇ， ｐ． ８５−１３７； １９９−２３４； ａｎｄ ２７０−３２２に記載されたような技術に従って行なわれる。
【０１５２】
実施例９：所望の製造物の製造における変異タンパク質の感化の解析
所望の化合物（例えばアミノ酸）の製造におけるＣ．Ｇｌｕｔａｍｉｃｕｍの遺伝子修飾の効果は、適当な条件（例えば上述の）下の改変された微生物成長および媒体及び／または細胞成分の増加した所望の製造物（即ちアミノ酸）の生成の解析によって評価することができる。このような解析法は当業者に周知であり分光法、薄層クロマトグラフィー、種々の染色法、酵素的および微生物学的方法、高速液体クロマトグラフィーのような解析クロマトグラフィー（例えばＵｌｌｍａｎ， Ｅｎｃｙｃｌｏｐｅｄｉａ ｏｆ Ｉｎｄｕｓｔｒｉａｌ Ｃｈｅｍｉｓｔｒｙ， ｖｏｌ． Ａ２， ｐ． ８９−９０ ａｎｄ ｐ． ４４３−６１３， ＶＣＨ： Ｗｅｉｎｈｅｉｍ （１９８５）； Ｆａｌｌｏｎ， Ａ． ｅｔ ａｌ．， （１９８７） ’Ａｐｐｌｉｃａｔｉｏｎｓ ｏｆ ＨＰＬＣ ｉｎ Ｂｉｏｃｈｅｍｉｓｔｒｙ ’ｉｎ： Ｌａｂｏｒａｔｏｒｙ Ｔｅｃｈｎｉｑｕｅｓ ｉｎ Ｂｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ｖｏｌ． １７； Ｒｅｈｍ ｅｔ ａｌ． （１９９３） Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ｖｏｌ． ３， Ｃｈａｐｔｅｒ ＩＩＩ： ’Ｐｒｏｄｕｃｔ ｒｅｃｏｖｅｒｙ ａｎｄ ｐｕｒｉｆｉｃａｔｉｏｎ’， ｐａｇｅ ４６９−７１４， ＶＣＨ： Ｗｅｉｎｈｅｉｍ； Ｂｅｌｔｅｒ， Ｐ．Ａ． ｅｔ ａｌ． （１９８８） Ｂｉｏｓｅｐａｒａｔｉｏｎｓ： ｄｏｗｎｓｔｒｅａｍ ｐｒｏｃｅｓｓｉｎｇ ｆｏｒ ｂｉｏｔｅｃｈｎｏｌｏｇｙ， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ； Ｋｅｎｎｅｄｙ， Ｊ．Ｆ． ａｎｄ Ｃａｂｒａｌ， Ｊ．Ｍ．Ｓ． （１９９２） Ｒｅｃｏｖｅｒｙ ｐｒｏｃｅｓｓｅｓ ｆｏｒ ｂｉｏｌｏｇｉｃａｌ ｍａｔｅｒｉａｌｓ， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ； Ｓｈａｅｉｗｉｔｚ， Ｊ．Ａ． ａｎｄ Ｈｅｎｒｙ， Ｊ．Ｄ．（１９８８） Ｂｉｏｃｈｅｍｉｃａｌ ｓｅｐａｒａｔｉｏｎｓ， ｉｎ： Ｕｌｌｍａｎｎ’ｓ Ｅｎｃｙｃｌｏｐｅｄｉａ ｏｆ Ｉｎｄｕｓｔｒｉａｌ Ｃｈｅｍｉｓｔｒｙ， ｖｏｌ． Ｂ３， Ｃｈａｐｔｅｒ １１， ｐａｇｅ １−２７， ＶＣＨ： Ｗｅｉｎｈｅｉｍ； ａｎｄ Ｄｅｃｈｏｗ， Ｆ．Ｊ． （１９８９） Ｓｅｐａｒａｔｉｏｎ ａｎｄ ｐｕｒｉｆｉｃａｔｉｏｎ ｔｅｃｈｎｉｑｕｅｓ ｉｎ ｂｉｏｔｅｃｈｎｏｌｏｇｙ， Ｎｏｙｅｓ Ｐｕｂｌｉｃａｔｉｏｎｓ参照）を含む。
【０１５３】
最終発酵生成物の測定に加えて、所望の化合物の生産に使用された代謝経路の他の成分、例えば中間体、副産物を、化合物の製造の効率を決定するために解析することもできる。解析法は、媒体中の栄養剤（例えば砂糖、炭化水素、窒素源、ホスファートおよび他のイオン）のレベルの測定、バイオマス組成物と成長の測定、通常の生合成経路の代謝物の製造の解析、発酵の間に生成したガスの測定を含む。これらの測定のための標準的な方法は、Ａｐｐｌｉｅｄ Ｍｉｃｒｏ Ｐｈｙｓｉｏｌｏｇｙ， Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ， Ｐ．Ｍ． Ｒｈｏｄｅｓ ａｎｄ Ｐ．Ｆ． Ｓｔａｎｂｕｒｙ， ｅｄｓ．， ＩＲＬ Ｐｒｅｓｓ， ｐ． １０３−１２９； １３１−１６３； ａｎｄ １６５−１９２ （ＩＳＢＮ： ０１９９６３５７７３） および引用文献に概説してある。
【０１５４】
実施例１０：Ｃ．ｇｌｕｔａｍｉｃｕｍ培養からの所望の製造物の精製
Ｃ．ｇｌｕｔａｍｉｃｕｍ細胞からまたは上述の培養物の上澄みからの所望の製造物の回収は、周知の種々の方法により行なうことができる。所望の製造物が細胞から隠されていない場合、細胞を培養物から低速遠心分離により回収することができ、細胞を機械的力または超音波のような標準的な方法で溶解することができる。細胞の破片を遠心分離で取り除き、可溶性タンパク質を含む上澄み画分を所望の化合物のさらなる精製のために保持する。製造物がＣ．ｇｌｕｔａｍｉｃｕｍ細胞から隠されている場合、細胞を培養物から低速遠心分離により取り除き、上澄み画分をさらなる精製のために保持する。
【０１５５】
双方の精製法からの上澄み画分を、適当なレジンであって、所望の分子がクロマトグラフィーレジンに保持され、サンプル中の多くの不純物が保持されないか、または不純物がレジンにより保持され、サンプルが保持されないようなレジンを用いたクロマトグラフィーで処理する。このようなクロマトグラフィーの工程は、必要に応じて、同じまたは異なるクロマトグラフィーレジンを用いて繰り返すことができる。当業者は、適当なクロマトグラフィーレジンの選択と、精製される特別の分子への最も効率的な適用に精通しているであろう。精製された製造物はろ過または限外ろ過により濃縮し、製造物の安定性が最も大きくなる温度で保存することができる。
【０１５６】
広い範囲の精製法が周知であり、前述した精製法は範囲の減縮を意味しない。このような精製法が、例えば Ｂａｉｌｅｙ， Ｊ．Ｅ． ＆ Ｏｌｌｉｓ， Ｄ．Ｆ． Ｂｉｏｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ Ｆｕｎｄａｍｅｎｔａｌｓ， ＭｃＧｒａｗ−Ｈｉｌｌ： Ｎｅｗ Ｙｏｒｋ （１９８６）に記載されている。
【０１５７】
単離した化合物の同定と純度が、先行技術で標準的な方法で評価される。これらは、高速液体クロマトグラフィー（ＨＰＬＣ）、分光学的方法、染色法、薄層クロマトグラフィー、ＮＩＲＳ、酵素的または微生物学的アッセイを含む。このような解析方法は、Ｐｅｔｅｋ ｅｔ ａｌ． （１９９４） Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ． ６０： １３３−１４０； Ｍａｌａｋｈｏｖａ ｅｔ ａｌ．（１９９６） Ｂｉｏｔｅｋｈｎｏｌｏｇｉｙａ １１： ２７−３２； ａｎｄ Ｓｃｈｍｉｄｔ ｅｔ ａｌ． （１９８８） Ｂｉｏｐｒｏｃｅｓｓ Ｅｎｇｉｎｅｅｒ． １９： ６７−７０． Ｕｌｍａｎｎ’ｓ Ｅｎｃｙｃｌｏｐｅｄｉａ ｏｆ Ｉｎｄｕｓｔｒｉａｌ Ｃｈｅｍｉｓｔｒｙ， （１９９６） ｖｏｌ． Ａ２７， ＶＣＨ： Ｗｅｉｎｈｅｉｍ， ｐ． ８９−９０， ｐ． ５２１−５４０， ｐ．５４０−５４７， ｐ． ５５９−５６６， ５７５−５８１ ａｎｄ ｐ． ５８１−５８７； Ｍｉｃｈａｌ， Ｇ． （１９９９） Ｂｉｏｃｈｅｍｉｃａｌ Ｐａｔｈｗａｙｓ： Ａｎ Ａｔｌａｓ ｏｆ Ｂｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ； Ｆａｌｌｏｎ， Ａ． ｅｔ ａｌ． （１９８７） Ａｐｐｌｉｃａｔｉｏｎｓ ｏｆ ＨＰＬＣ ｉｎ Ｂｉｏｃｈｅｍｉｓｔｒｙ ｉｎ： Ｌａｂｏｒａｔｏｒｙ Ｔｅｃｈｎｉｑｕｅｓ ｉｎ Ｂｉｏｃｈｅｍｉｓｔｒｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ｖｏｌ． １７に記載されている。
【０１５８】
実施例１１：本発明の遺伝子配列の解析
配列の比較と、２個の配列の間の相同性の百分率の決定は、公知の技術であり、数学的アルゴリズム、例えばＫａｒｌｉｎ ａｎｄ Ａｌｔｓｃｈｕｌ （１９９０） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ． ８７： ２２６４−６８のアルゴリズムで、Ｋａｒｌｉｎ ａｎｄ Ａｌｔｓｃｈｕｌ （１９９３） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９０： ５８７３−７７に記載のように変形されたものなどを使用して行なうことができる。このようなアルゴリズムは、Ａｌｔｓｃｈｕｌ， ｅｔ ａｌ． （１９９０） Ｊ． Ｍｏｌ． Ｂｉｏｌ． ２１５： ４０３−１０のＮＢＬＡＳＴおよびＸＢＬＡＳＴプログラム（バージョン２．０）に組み入れられている。ＢＬＡＳＴヌクレオチド探索は、ＮＢＬＡＳＴプログラム、スコア＝１００、語長＝１２によって、本発明のＭＣＴ核酸分子に対するヌクレオチド配列の相同性を得るために行なうことができる。ＢＬＡＳＴタンパク質探索は、ＸＢＬＡＳＴプログラム、スコア＝５０、語長＝３によって、本発明のＭＣＴタンパク質分子に対するアミノ酸配列の相同性を得るために行なうことができる。比較の目的で、間隙のあけられたアライメントを得るために、ギャップドＢＬＡＳＴをＡｌｔｓｃｈｕｌ ｅｔ ａｌ．， （１９９７） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２５（１７）：３３８９−３４０２に記載のように使用することができる。ＢＬＡＳＴおよびギャップドＢＬＡＳＴプログラムの使用に際して、当業者はプログラム（例えばＸＢＬＡＳＴおよびＮＢＬＡＳＴ）のパラメーターの、解析される特定の配列のための最適化の方法についてよく知っている。
【０１５９】
配列の比較に使用される数学的アルゴリズムの他の例は、Ｍｅｙｅｒｓ とＭｉｌｌｅｒ （（１９８８） Ｃｏｍｐｕｔ． Ａｐｐｌｎ． Ｂｉｏｓｃｉ． ４： １１−１７）のアルゴリズムである。このようなアルゴリズムは、ＧＣＧシーケンスアライメントソフトウェアパッケージの一部であるＡＬＩＧＮプログラム（バージョン２．０）に組み入れられている。ＡＬＩＧＮプログラムをアミノ酸配列の比較のために使用する場合、ＰＡＭ１２０重量残渣表、１２のギャップ長さペナルティー、４のギャップペナルティーを使用することができる。さらに配列解析に使用されるアルゴリズムが公知であり、ＡＤＶＡＮＣＥおよびＡＤＡＭ（Ｔｏｒｅｌｌｉ ａｎｄ Ｒｏｂｏｔｔｉ （１９９４） Ｃｏｍｐｕｔ． Ａｐｐｌ． Ｂｉｏｓｃｉ． １０：３−５に記載）および ＦＡＳＴＡ（ Ｐｅａｒｓｏｎ ａｎｄ Ｌｉｐｍａｎ （１９８８） Ｐ．Ｎ．Ａ．Ｓ． ８５：２４４４−８に記載）を含む。
【０１６０】
２本のアミノ酸配列の間の相同性の百分率は、ＧＣＧソフトウェアパッケージ中のＧＡＰプログラム（ｈｔｔｐ：　／／ｗｗｗ．ｇｃｇ．ｃｏｍで入手可能）を用い、Ｂｌｏｓｕｍ６２マトリックスまたはＰＡＭ２５０マトリックスのいずれか、１２、１０、８、６、または４の間隙重さ、２、３または４の長さ重さを用いて行なうことができる。２本の核酸配列の間の相同性の百分率は、標準的なパラメーター、例えば間隙重さが５０、および長さ重さが３などを用いて、ＧＣＧソフトウェアパッケージ中のＧＡＰプログラムを用いて決定することができる。
【０１６１】
本発明の遺伝子配列と、遺伝子バンクに存在する配列の比較解析が先行技術において公知の技術を用いて行なわれた（例えばＢｅｘｅｖａｎｉｓ ａｎｄ Ｏｕｅｌｌｅｔｔｅ， ｅｄｓ． （１９９８） Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ： Ａ Ｐｒａｃｔｉｃａｌ Ｇｕｉｄｅ ｔｏ ｔｈｅ Ａｎａｌｙｓｉｓ ｏｆ Ｇｅｎｅｓ ａｎｄ Ｐｒｏｔｅｉｎｓ． Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ： Ｎｅｗ Ｙｏｒｋ参照）。本発明の遺伝子配列が、遺伝子バンクに存在する遺伝子と、３段階の工程で比較された。第一段階で、遺伝子バンクに存在するヌクレオチド配列に対する本発明の配列の各々について、ＢＬＡＳＴＮ解析（例えばローカルアライメント解析）が行なわれた。最初にヒットした５００個についてさらに解析が続けられた。次いでＦＡＳＴＡ探索（例えば、結合されたローカルおよびグローバルアライメント解析で、配列の限定された領域を整列させる）が、これらの５００個について行われた。各々の本発明の遺伝子配列は次いで全体的に、ＦＡＳＴＡの頭の３個のヒットの各々について、ＧＣＧソフトウェアパッケージ中のＧＡＰプログラムを用いて（標準的なパラメーターを使用して）整列させられる。正しい結果を得るために、遺伝子バンクから抽出された配列の長さは、周知の方法により質問の配列の長さに調整された。この解析の結果は、表４にまとめられている。得られた結果は、遺伝子バンクの参照の各々と比較した本発明の遺伝子の各々においてＧＡＰ（グローバル）解析を単独で行なったときに得られるであろう結果と一致したが、このようなデータベースを広げて行なったＧＡＰ（グローバル）解析と比較して明らかに計算時間が短縮された。切り捨て値を上回るアライメントが得られなかった本発明の配列を、アライメントの情報なく表４に示した。表４に、’％相同性（ＧＡＰ）’の見出しで記載されたＧＡＰアライメント相同性百分率は、’，’は小数点を表すヨーロッパの数の形式で、並べられているということは、当業者によってさらに理解されるであろう。例えば、このカラム中の’４０，３４５’の値は、４０．３４５％を表す。
【０１６２】
実施例１２：ＤＮＡマイクロアレイの構築と操作
本発明の配列を追加で、ＤＮＡマイクロアレイ（デザイン、方法論、ＤＮＡアレイの使用は周知であり、例えばＳｃｈｅｎａ， Ｍ． ｅｔ ａｌ． （１９９５） Ｓｃｉｅｎｃｅ ２７０： ４６７−４７０； Ｗｏｄｉｃｋａ， Ｌ． ｅｔ ａｌ． （１９９７） Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ １５：１３５９−１３６７； ＤｅＳａｉｚｉｅｕ， Ａ． ｅｔ ａｌ． （１９９８） Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ １６： ４５−４８； ａｎｄ ＤｅＲｉｓｉ， Ｊ．Ｌ． ｅｔ ａｌ． （１９９７） Ｓｃｉｅｎｃｅ ２７８： ６８０−６８６に記載されている。）の構築と適用に使用することができる。
【０１６３】
ＤＮＡマイクロアレイは、ニトロセルロース、ナイロン、ガラス、シリコーンまたは他の材料からなる固体または柔軟性のある支持体である。核酸分子を、注文された方法で表面に取り付けることができる。適当なラベリングのあと、他の核酸または核酸混合物を固定された核酸分子にハイブリダイズすることができ、限定された領域でハイブリダイズされた分子の個々の信号強度を検知し、測定するためにラベルを使用することができる。この方法論は、適用された核酸サンプルまたは混合物中のすべてあるいは選択された核酸の相対量または絶対量を同時に定量化することを可能にする。このためＤＮＡマイクロアレイは、並行して多数の（６８００以上）核酸の発現を解析することを可能とする（Ｓｃｈｅｎａ， Ｍ． （１９９６） ＢｉｏＥｓｓａｙｓ １８（５）： ４２７−４３１参照）。
【０１６４】
本発明の配列は、ポリメラーゼ連鎖反応のような核酸増幅反応によって、１種以上のＣ．ｇｌｕｔａｍｉｃｕｍ遺伝子の限定された領域を増幅することができるオリゴヌクレオチドプライマーの設計に使用することができる。５’または３’オリゴヌクレオチドプライマーあるいは適当なリンカーの選択と設計は、上述の支持媒体（例えばＳｃｈｅｎａ， Ｍ． ｅｔ ａｌ． （１９９５） Ｓｃｉｅｎｃｅ ２７０： ４６７−４７０にも記載されている。）の表面に対する、得られたＰＣＲ産物の共有結合を可能とする。
【０１６５】
核酸マイクロアレイは、Ｗｏｄｉｃｋａ，Ｌ．ｅｔ ａｌ．（１９９７） Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ １５：１３５９−１３６７に記載されたようにその場のオリゴヌクレオチド合成によっても構築することができる。写真平板法により、マトリックスの正確に限定された領域が露光される。光不安定な保護基はこれによって活性化され、ヌクレオチド付加が進行し、一方光からマスクされた領域はいかなる修飾も進行しない。続く保護と光活性化のサイクルは、限定された位置における異なったオリゴヌクレオチドの合成を可能とする。小さい、限定された本発明の遺伝子の領域は、マイクロアレイ上で、固相オリゴヌクレオチド合成により合成することができる。
【０１６６】
ヌクレオチドのサンプルまたは混合物の中に存在する本発明の核酸分子は、マイクロアレイに対してハイブリダイズすることができる。これらの核酸分子は標準的な方法でラベルすることができる。要するに、核酸分子（例えば、ｍＲＮＡ分子またはＤＮＡ分子）は、例えば、逆転写またはＤＮＡ合成の間、放射性同位元素によりまたは蛍光によりラベルされたヌクレオチドの組み込みによりラベルされる。ラベルされた核酸のマイクロアレイに対するハイブリダイゼーションは、例えば Ｓｃｈｅｎａ， Ｍ． ｅｔ ａｌ． （１９９５） ｓｕｐｒａ； Ｗｏｄｉｃｋａ， Ｌ． ｅｔ ａｌ． （１９９７）， ｓｕｐｒａ； ａｎｄ ＤｅＳａｉｚｉｅｕ Ａ． ｅｔ ａｌ （１９９８）， ｓｕｐｒａに記載されている。ハイブリダイズされた分子の検出と定量化は、特定の組み込まれたラベルにより行なわれる。放射性のラベルは例えばＳｃｈｅｎａ， Ｍ． ｅｔ ａｌ． （１９９５） ｓｕｐｒａに記載されたように検出することができ、蛍光ラベルは例えばＳｈａｌｏｎ ｅｔ ａｌ． （１９９６） Ｇｅｎｏｍｅ Ｒｅｓｅａｒｃｈ ６： ６３９−６４５の方法により検出することができる。
【０１６７】
本発明の配列のＤＮＡマイクロアレイ技術に対する適用は、上述したように、Ｃ．ｇｌｕｔａｍｉｃｕｍまたは他のＣｏｒｙｎｅｂａｃｔｅｒｉａの異なった株の比較解析を可能とする。例えば、個々の転写の特徴に基づく内部株の変種の研究および特定するために重要な遺伝子の同定および／または所望の株の特性、例えば病原性、生産性、およびストレス寛容などの同定は、核酸アレイ方法論により促進される。発酵反応の進行の間の、本発明の遺伝子の発現の特徴の比較は、核酸アレイ技術を用いることで可能である。
【０１６８】
実施例１３：細胞タンパク質ポピュレーションの動力学的解析（プロテオミクス）
本発明の遺伝子、組成、方法は、タンパク質のポピュレーションの相互作用および動力学（プロテオミクスと定義する）の研究に適用することができる。注目のタンパク質ポピュレーションは、Ｃ．ｇｌｕｔａｍｉｃｕｍ（例えば他の生物のタンパク質ポピュレーションと比較して）、特定の環境または代謝条件下（例えば高いまたは低い温度で、または高いまたは低いｐＨでの発酵の間）で活性なタンパク質または特定の成長および発達の相の間に活性なタンパク質の総タンパク質ポピュレーションを含むが、これに限定されない。
【０１６９】
タンパク質ポピュレーションは、種々の周知の技術、例えばゲル電気泳動などにより解析することができる。細胞タンパク質は、例えば細胞溶解または抽出により得られ、種々の電気泳動技術を用いて互いに分離することができる。ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）は、タンパク質をその分子量に基づいて大部分分離する。等電点のポリアクリルアミドゲル電気泳動（ＩＥＦ−ＰＡＧＥ）は、タンパク質をその等電点（アミノ酸配列を反映するのみならずタンパク質の後翻訳修飾も反映する）により分離する。他の、さらに好ましいタンパク質解析の方法は、２−Ｄ−ゲル電気泳動（例えばＨｅｒｍａｎｎ ｅｔ ａｌ． （１９９８） Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １９： ３２１７−３２２１； Ｆｏｕｎｔｏｕｌａｋｉｓ ｅｔ ａｌ． （１９９８） Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １９： １１９３−１２０２； Ｌａｎｇｅｎ ｅｔ ａｌ． （１９９７） Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １８： １１８４−１１９２； Ａｎｔｅｌｍａｎｎ ｅｔ ａｌ． （１９９７） Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １８： １４５１−１４６３に記載されている）として知られるＩＥＦ−ＰＡＧＥとＳＤＳ−ＰＡＧＥの連続する組み合わせである。他の分離技術、例えばキャピラリーゲル電気泳動などもタンパク質の分離に利用することができ、このような技術は、周知である。
【０１７０】
これらの方法論により分離されたタンパク質は、標準的な技術、例えば染色またはラベリングにより可視化することができる。適当な染色は公知であり、Ｃｏｏｍａｓｓｉｅ ブリリアントブルー、銀染色、蛍光染料、例えばＳｙｐｒｏ　Ｒｕｂｙ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ）を含む。Ｃ．ｇｌｕｔａｍｉｃｕｍの媒体中にある、放射性物質によりラベルされたアミノ酸またはその他のタンパク質前駆体（例えば^３５Ｓ−メチオニン、^３５Ｓ−システイン、^１４Ｃ−ラベルされたアミノ酸、^１５Ｎ−アミノ酸、^１５ＮＯ_３または^１５ＮＨ_４ ^＋またはＣ_１３ラベルされたアミノ酸）の含有物は、分離に先立ってこれらの細胞からタンパク質をラベルすることを可能とする。同様に、蛍光ラベルを使用することができる。これらのラベルされたタンパク質は、抽出され、単離され、前述した技術に従って分離される。
【０１７１】
これらの技術により可視化されたタンパク質はさらに、使用された染料またはラベルの量を測定することにより解析することができる。得られたタンパク質の量は、定量的に、例えば光学的方法を用いて決定することができ、同様のゲルまたは他のゲル中の他のタンパク質の量と比較することができる。ゲル上のタンパク質の比較は、例えば光学的比較、分光学的、ゲルのイメージスキャンおよび解析、または写真フィルムおよびスクリーンの使用を通じて行なうことができる。このような技術は周知である。
【０１７２】
得られたタンパク質を同定するために、直接シーケンスまたは他の標準的な技術を使用することができる。例えば、Ｎ−および／またはＣ末端アミノ酸シーケンス（例えばエドマン分解）を、マススペクトロメトリー（特にＭＡＬＤＩまたはＥＳＩ技術（例えばＬａｎｇｅｎ ｅｔ ａｌ． （１９９７） Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ １８： １１８４−１１９２参照））として使用することができる。ここで提供されたタンパク質配列を、これらの技術によりＣ．ｇｌｕｔａｍｉｃｕｍタンパク質の同定のために使用することができる。
【０１７３】
これらの方法により得られた情報は、タンパク質の存在、活性、種々の生物学的条件（例えば、異なった生物、発酵の時点、媒体条件、または異なったビオトープ、その他）からの異なったサンプルの間の修飾の様式の比較に使用することができる。このような実験から単独で、または他の技術との組み合わせにより得られたデータは、様々な適用、例えば与えられた（例えば代謝的）状況にある種々の生物の挙動の比較、ファインケミカルを産出する株の生産性の上昇、またはファインケミカルの製造の効率の上昇などに使用することができる。
【０１７４】
同等物
当業者は、わずかの型にはまった実験を用いて、ここに記載された発明の特定の態様に対する多くの同等物を認識するか、確認することができるであろう。このような同等物は、請求の範囲に含まれる。[0001]
[Related application]
This application is based on priority of US provisional application Ser. No. 60 / 141,031 filed on Jun. 25, 1999. Further, the present application relates to German Patent Application No. 19931454.3 filed on Jul. 8, 1999, German Patent Application No. 19931478.0 filed on Jul. 8, 1999, and German Patent Application No. filed on Jul. 8, 1999. German Patent Application No. 19932122.1, filed on Jul. 9, 1999 German Patent Application No. 19932124.8, filed on Jul. 9, 1999, filed on Jul. 9, 1999 German Patent Application No. 199 32125.6, filed Jul. 9, 1999, German Patent Application No. German Patent Application No. 19933218.0, filed on July 9, 1999, German Patent Application No. 199322180.9, filed on July 9, 1999, German Patent Application No. 199,312,82.5, filed July 9, 1999 German Patent Application No. 199332190.6, filed on Jul. 9, 1999 German Patent Application No. 19932191.4, filed on Jul. 9, 1999 German Patent Application No. 19932209.0, Jul. 1999 German Patent Application No. 19933222.0 filed on May 9, German Patent Application No. 1993227.9 filed on Jul. 9, 1999, German Patent Application No. 1993228.7 filed on Jul. 9, 1999 German Patent Application No. 1993229.5, filed on Jul. 9, 1999, German Patent Application No. Patent Application No. 19932230.9, filed on July 14, 1999 German Patent Application No. 19932927.3, filed on July 14, 1999 German Patent Application No. 19933005.0, July 1999 German Patent Application No. 19933006.9 filed on the 14th, German Patent Application No. 199407764.9 filed on August 27, 1999, German Patent Application No. 199407765.7 filed on August 27, 1999, German Patent Application No. 199 40 766.5 filed on Aug. 27, 1999, German Patent Application No. 199 40830.0 filed on Aug. 27, 1999, German Patent Application No. 199 40831.9 filed on Aug. 27, 1999 Patent Specification, filed Aug. 27, 1999, German Patent Application No. 19940832.7 German Patent Application No. 199 40833.5 filed on Aug. 27, 1999, German Patent Application No. 1941378.9 filed on Aug. 31, 1999, German Patent Application No. 19941379.7 filed on Aug. 31, 1999 German Patent Application No. 19941395.9, filed on Sep. 3, 1999 German Patent Application No. 19942077.7, filed on Aug. 31, 1999, German Patent Application No. No. 19942078.5, German Patent Application No. 19942079.3 filed on Sep. 3, 1999 and German Patent Application No. 19942088.2 filed on Sep. 3, 1999 are the priority basis. The entire contents of the above application are incorporated herein by reference in their entirety.
[0002]
[Background of the Invention]
Products and by-products from natural metabolic processes in cells are used in a variety of industries, including the food, feed, cosmetics and pharmaceutical industries. Collectively called "fine chemicals," these molecules include organic acids, amino acids of protein and non-protein sources, nucleic acids, nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors (cofactors). ) And enzymes. These productions are most conveniently performed by large-scale cultivation of bacteria developed to produce and secrete large quantities of the desired molecule. One particularly useful organism for this purpose is the gram-positive, non-pathogenic bacterium Corynebacterium glutamicum (Corynebacterium glutamicum). By selecting strains (species), a number of mutant strains have been developed which produce a range of desired compounds. However, selecting an improved strain for the production of a particular molecule is a time-consuming and difficult task.
[0003]
[Summary of the Invention]
According to the present invention, a novel bacterial nucleic acid molecule is provided and used for various purposes. Examples of applications include identification of microorganisms used to produce fine chemicals, C.I. B. Fine chemical production regulation in G. glutamicum or related bacteria; C. glutamicum or related bacteria classification or identification; Use as a reference point for generating a glutamicum genome map, and as a marker for transformation. These novel nucleic acid molecules encode proteins referred to herein as membrane constituent (synthetic) and membrane transport (MCT) proteins.
[0004]
C. Glutamicum is a gram-positive aerobic bacterium and is commonly used in the industrial sector for the mass production of various fine chemicals and for the degradation of hydrocarbons (eg in petroleum) and the oxidation of terpaids. Modulation of the expression of the MCT nucleic acid molecule of the invention or modification of the sequence of the MCT nucleic acid molecule modulates the production of one or more fine chemicals obtained from the microorganism (eg, Corynebacterium or Brevibacterium). ) To enhance the termination or production of one or more fine chemicals from the seed).
[0005]
The MCT nucleic acids of the invention can be used to identify Corynebacterium glutamicum or microorganisms closely related thereto, or to C. elegans in a mixed population of microorganisms. Used to recognize the presence of glutamicum or a microorganism closely related thereto. The present invention relates to a number of C.I. The present invention provides a sequence of the nucleic acid of the G. glutamicum gene, which is capable of extracting genomic DNA extracted from a single culture of microorganisms or a mixed population culture of microorganisms under stringent conditions. The detection is performed by spanning a probe region specific to Glutamicum to evaluate whether or not the target organism exists. Corynebacterium glutamicum itself is nonpathogenic, but is associated with pathogenic species in humans, such as Corynebacterium diphtheria (causative agent of diphtheria). Therefore, detection of such microorganisms has important clinical relevance.
[0006]
The MCT nucleic acid molecule of the present invention comprises C.I. It also acts as a reference point for chromosomal mapping of the glutamicum genome or the genome of a related microorganism.
[0007]
Similarly, these molecules, variants or parts thereof, also act as genetically engineered Corynebacterium or Brevibacterium species markers.
[0008]
The MCT proteins encoded by the novel nucleic acid molecules of the present invention function, for example, with respect to the metabolism (eg, biosynthesis or degradation) of compounds required for membrane (membrane) biosynthesis, It is possible to help transport.
The effectiveness of the cloning vector used in Corynebacterium glutamicum is described, for example, in Sinskey et al., US Pat. No. 4,649,119, C.I. G. glutamicum and related Brevibacterium species (eg, lactofermentum) genetic replication techniques (eg, lactofermentum) (Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. -311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)). The nucleic acid molecules of the present invention may be used for genetic manipulation of this organism, thereby improving the production or more efficient production of one or more fine chemicals. The production of fine chemicals and the improvement of production efficiency may be a direct effect or an indirect effect by manipulating the gene of the present invention.
[0009]
When the modified protein was introduced and the MCT protein of the present invention was modified (changed), C.I. The yield, production and / or production efficiency of the fine chemical obtained from the glutamicum strain may be directly affected. The MCT protein that releases fine chemicals from such cells has been improved in number or activity, and is capable of secreting large amounts of compounds into the extracellular medium and recovering them from the culture medium more than before. Easy. Similarly, those MCT proteins involved in the uptake of nutrients required for the biosynthesis of one or more fine chemicals (eg, phosphate compounds, sulfate compounds, nitrogen compounds, etc.) have been improved in number and activity, and It is also possible to increase the intracellular concentration of precursors, cofactors or intermediate products. In addition, while fatty acids and lipids are important fine chemicals, the activity or activity of one or more of the MCT proteins of the invention involved in the biosynthesis of these compounds may be optimized or increased, or By reducing the effects of one or more MCR proteins associated with degradation, C.I. It is also possible to improve the yield, production and / or production efficiency of glutamicum-derived fatty acids and lipid molecules.
[0010]
By mutating one or more MCT genes of the present invention, C.I. It is also possible to produce MCT proteins with altered activity that indirectly affect the production of one or more desired fine chemicals derived from Glutamicum. For example, normal metabolic excretion in cells, which may increase in quantity due to overproduction of the desired fine chemicals, may adversely affect nucleotides and proteins in the cell (decrease cell viability) Books involved in the release of excreta so that they can be efficiently released from cells before disrupting the fine chemical biosynthetic pathway (which may reduce the yield, production or production efficiency of the desired fine chemical). The number or activity of the MCT proteins of the invention may be increased. Furthermore, if the intracellular mass of the desired fine chemical is relatively large, this may have a toxic effect on the cells, so that a transporter capable of releasing these compounds from the cells may be used. By increasing the activity or the number, it is possible to increase the viability of the cells in the culture and obtain a large number of cells in the culture for producing the desired fine chemical. The MCT proteins of the present invention can also be engineered to provide relative amounts of various lipid and fatty acid molecules. Since lipids have different physical properties for each type, changing the lipid composition in the membrane significantly changes the fluidity of the membrane. Changes in membrane fluidity affect the transport of molecules across the membrane and the integrity of the cell, both of which affect C. elegans in large-scale fermentation cultures. It has a significant impact on the production of fine chemicals derived from glutamicum.
[0011]
According to the present invention, C.I. A novel nucleic acid molecule encoding a protein capable of participating in metabolism of a compound necessary for the synthesis of a cell membrane in Glutamicum, that is, an MCT protein is provided. As used herein, a nucleic acid molecule encoding an MCT protein is referred to as an MCT nucleic acid molecule. In a preferred embodiment, the MCT protein is C. It is involved in the metabolism of compounds required for the synthesis of cell membranes in Glutamicum, or the transport of molecules across this membrane. Examples of such proteins include those encoded by the genes listed in Table 1.
[0012]
The present invention further relates to isolated nucleic acid molecules (eg, cDNA, DNA, RNA) comprising nucleotide sequences encoding MCT proteins or biologically active proteins thereof, and nucleic acids encoding MCTs (eg, DNA Or nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of mRNA). In a particularly preferred embodiment, the isolated nucleic acid molecule is an odd sequence of SEQ ID NO in the sequence listing (eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 5). 7), SEQ ID NO: NO, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 7 ...) or any of the nucleotide sequences represented by these coding sequences or complementary regions in such nucleotide sequences. In another particularly preferred embodiment, the isolated nucleic acid molecule of the invention comprises an odd number of SEQ ID NO in the sequence listing (eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 ...) or hybridizes to at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and more. Preferably, it comprises about 95%, 96%, 97%, 98%, or more than 99% homologous nucleotide sequences or portions thereof. In a preferred embodiment, the isolated nucleic acid molecule is represented by an even number SEQ ID NO (eg, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 ...). Encoding any of the amino acid sequences identified. This preferred MCT protein preferably has at least one of the MCT activities described herein.
[0013]
In other embodiments, the isolated nucleic acid molecule encodes a protein or a portion of a protein, wherein the protein or portion thereof has an amino acid sequence of the invention (eg, an even SEQ ID NO in the sequence listing). ID NO), for example, an amino acid sequence that is sufficiently homologous to a protein or a portion thereof to maintain MCT activity. The protein encoded by the above-described nucleic acid or a part thereof is C.I. Preferably, Glutamicum retains its ability to participate in the metabolism of compounds necessary for the formation of the cell membrane or the transport of molecules across the membrane. In a preferred embodiment, the protein encoded by the nucleic acid molecule has at least about 50% of the amino acid sequence of the invention (eg, the entire amino acid sequence selected from the even SEQ ID NO sequence in the sequence listing), Preferably, they are at least about 60%, more preferably at least about 70%, 80% or 90%, and more preferably about 95%, 96%, 97%, 98% or 99% or more homologous. In another preferred embodiment, the protein is a C. cerevisiae substantially homologous to the entire amino acid sequence of the invention. Glutamicum protein (encoded by an open reading frame corresponding to an odd number of SEQ ID NO in the sequence listing (eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 5) : 7 ...).
[0014]
In another preferred embodiment, the isolated nucleic acid molecule is C.I. A protein derived from Glutamicum and containing a biologically active domain at least about 50% homologous to any of the amino acid sequences of the present invention (eg, any of the even SEQ 数 ID NO sequences in the sequence listing) (Eg, an MCT fusion protein), and It has the ability to participate in the metabolism of compounds required for the construction of the cell membrane of Glutamicum, or the transport of molecules across this membrane, or has one or more of the activities listed in Table 1. The nucleic acid molecule in this case also includes a heterologous polypeptide or a heterologous nucleic acid sequence encoding a regulatory region.
[0015]
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length, and the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention (eg, an odd number in the sequence listing). Sequence of SEQ ID NO). Preferably, the isolated nucleic acid molecule corresponds to a naturally occurring nucleic acid molecule. The isolated nucleic acid can be a naturally occurring C. aureus. More preferably, it encodes the glutamicum MCT protein or a biologically active part thereof.
[0016]
In addition, the present invention includes vectors, such as recombinant expression vectors containing a nucleic acid molecule of the present invention, and host cells into which such vectors have been incorporated. In one embodiment, the host cells are used to produce the MCT protein by culturing such host cells in a suitable medium. The MCT protein thus produced is isolated from the medium or host cells.
[0017]
Further, the invention includes genetically altered microorganisms into which the MCT gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introducing a nucleic acid molecule of the invention encoding a wild-type or mutant MCT sequence as a transgene. In another embodiment, the endogenous MCT gene in the genome of the microorganism is modified by homologous recombination with the modified MCT gene, eg, functionally disrupted. In another embodiment, the endogenous or derived MCT gene in the microorganism encodes a functional MCT protein despite being altered by one or more point mutations, deletions, or inversions. . In yet other embodiments, one or more regions (eg, promoter, repressor, inducer) of the microbial MCT gene are altered such that expression of the MCT gene is regulated (eg, deletion, truncation, Inversion, point mutation). The microorganism preferably belongs to the genus Corynebacterium or Brevibacterium, and more preferably belongs to Corynebacterium glutamicum. In a preferred embodiment, the microorganism is preferably used for the production of a desired compound, for example an amino acid, especially lysine.
[0018]
Furthermore, the present invention provides a method for recognizing the presence or activity of Corynebacterium diphtheriae. The method includes detecting from a sample (subject / patient) one or more of the nucleic acid or amino acid sequence of the present invention (eg, the sequence shown in SEQ ID NO: 1 to 676 in the sequence listing) in the subject; Thereby, the presence or activity of Corynebacterium diphtheria in the subject is detected.
[0019]
The invention also includes an isolated MCT protein or a portion thereof, eg, a biologically active portion. In a preferred embodiment, the isolated MCT protein, or a portion thereof, is isolated from C. elegans against chemically or environmentally hazardous conditions. It has the ability to improve the resistance of glutamicum. In another preferred embodiment, the isolated MCT protein or a portion thereof is C. It is involved in the metabolism of compounds required for the formation of the cell membrane of glutamicum, or the transport of molecules across this membrane. In another preferred embodiment, the isolated MCT protein, or a portion thereof, is derived from the amino acid sequence of the invention (e.g., even SEQ ID NO: It is preferably homologous to the extent that it maintains the ability of Glutamicum to participate in the metabolism of compounds required for the formation of cell membranes or the transport of molecules across this membrane.
[0020]
Further, according to the present invention, there is provided a method for producing an isolated MCT protein. In a preferred embodiment, the MCT protein comprises an amino acid sequence of the invention (eg, an even SEQ 数 ID NO sequence in the sequence listing). In another preferred embodiment, the present invention relates to the entire amino acid sequence of the present invention (for example, the sequence of even SEQ 数 ID NO in the sequence listing) (open reading frame indicated by the odd SEQ ID NO in the sequence listing). (Encoded) substantially homologous to the isolated protein. In yet another embodiment, the protein comprises at least 50%, preferably at least 60%, more preferably at least 70%, based on the amino acid sequence of the invention (eg, the even SEQ ID NO sequence in the sequence listing). 80% or 90%, and more preferably at least about 95%, 96%, 97%, 98% or more than 99% homologous. In another preferred embodiment, the isolated MCT protein is at least about 50% or more homologous amino acid sequence to any of the amino acid sequences of the invention (eg, any of the even SEQ 数 ID NO sequences in the Sequence Listing). And C.I. It is capable of participating in the metabolism of compounds required for the construction of the cell membrane of Glutamicum, or the transport of molecules across this membrane, or having one or more of the activities listed in Table 1.
[0021]
Further, the isolated MCT protein may be encoded by, for example, a nucleotide sequence that hybridizes under stringent conditions to any of the even SEQ ID NO NO nucleotide sequences listed in the sequence listing, or at least about 50%, Amino acid sequence encoded by a nucleotide sequence that is preferably at least about 60%, more preferably at least about 70%, 80%, 90%, more preferably at least about 95%, 96%, 97%, 98% or more than 99% homologous May be included. Preferably, the MCT protein is one having one or more of the MCT biological activities described herein.
[0022]
The MCT polypeptide, or a biologically active portion of the peptide, cooperates with the non-MCT polypeptide to form a fusion protein. In a preferred embodiment, the fusion protein has a different activity than the MCT protein alone. In another preferred embodiment, the fusion protein is C. It is involved in the metabolism of compounds required for the formation of the cell membrane of glutamicum, or the transport of molecules across this membrane. In a particularly preferred embodiment, the production of the desired compound from the cell is modulated by introducing the fusion protein into a host cell.
[0023]
In addition, the present invention screens for molecules that modulate the activity of the MCT protein by interacting with the protein itself, the substrate or the binding target of the MCT protein, or by modulating the transcription or translation of the MCT nucleic acid molecule of the present invention. Provide a method.
[0024]
Further, the present invention includes a method for producing a fine chemical. In this method, cells containing a vector that controls the expression of the MCT nucleic acid molecule of the present invention are cultured, thereby producing a fine chemical. According to a preferred embodiment, the method includes the step of obtaining a cell containing such a vector, in which the vector governing MCT nucleic acid expression is transferred to the cell. In another preferred embodiment, the method further comprises recovering the fine chemical obtained from the culture. In a preferred embodiment, the cells are derived from a Corynebacterium or Brevibacterium species or are selected from the strains listed in Table 3.
[0025]
Furthermore, the method of the present invention includes a method for regulating the production of a molecule derived from a microorganism. In this method, a cell is contacted with an agent that modulates MCT protein activity or MCT nucleic acid expression to alter the activity associated with the cell relative to the activity without the agent. In this case, the cells may have more than one C.I. Preferably, the glutamicum resistance pathway is regulated or the transport of the compound across the membrane is regulated, thereby increasing the yield or production rate of the desired fine chemical by the microorganism. As an agent that modulates MCT protein activity, an agent that stimulates MCT protein activity or MCT nucleic acid expression can be used. Examples of agents that stimulate the activity of the MCT protein or the expression of the MCT nucleic acid include small molecules, active MCT protein, and nucleic acid introduced into cells encoding the MCT protein. Examples of agents that inhibit MCT activity or expression include small molecules, antisense MCT nucleic acid molecules.
[0026]
Further, the present invention provides a method for adjusting the yield of a desired compound from cells obtained by introducing a wild-type or mutant MCT gene, which is present on a separate plasmid or integrated into the genome of a host cell. including. The introduction when integrated into the genome may be random. It is also possible to perform homologous recombination such that the natural gene is replaced by the introduced copy. This produces the desired compound from the cells to be regulated. In a preferred embodiment, the yield is increased. Fine chemicals obtained in a particularly preferred embodiment are amino acids. It is particularly preferred that the amino acid is L-lysine.
[0027]
[Detailed description of the invention]
The present invention relates to C.I. MCT nucleic acid and protein molecules involved in the metabolism of cell membrane components of Glutamicum or the transport of molecules across this membrane are provided. The molecules of the present invention are C.I. It can be used to control the production of fine chemicals from microorganisms such as glutamicum. The regulation of the production of fine chemicals is directly controlled (for example, when the biosynthesis of fatty acids is overexpressed or optimized, the protein is directly linked to the yield, production and / or production efficiency of the modified C. glutamicum-derived fatty acids). Or the indirect effect that results in an increase in the yield, production and / or production efficiency of the desired compound (eg, when the metabolism of cell membrane components is regulated, the yield of cell membrane components , Manufacturing and / or manufacturing efficiency, thereby affecting the manufacture of one or more fine chemicals). The present invention will be described in more detail below.
[0028]
[1. Fine Chemical]
The term "fine chemical" refers to a molecule that, as is generally recognized, is produced from living organisms and used in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. Examples of such compounds include organic acids such as tartaric acid, itaconic acid, diaminopimelic acid, proteinogenic or non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides (Kuninaka, A., (1996) Nucleotides and related compounds, p. 561-612, (discussed in Biotechnology Volume 6, Rehm et al., VCH, Ed., Weinheim, and references cited therein), lipids, saturated and unsaturated fatty acids (eg, arachidonic acid). , Diols (eg, propane diol and butane diol), carbohydrates (eg, hyaluronic acid and trehalose), aromatic compounds (eg, aromatic amines, vanillin and indigo), vitamins and Cofactors (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references described therein, Ong, Nii, A.S. E. & Packer, L. al., (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research-Asia (Malaysia, Penang smell (September 1-3, 1994), (1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68), and Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN 508 0808. And all the other chemicals mentioned in the references cited therein, the metabolism and certain uses of these fine chemicals are described in detail below.
[0029]
A. Amino acid metabolism and usage
Since amino acids are the basic structural unit of any protein, they are important for the normal cellular function of all organisms. The term "amino acid" is conventionally known. There are 20 types of amino acids serving as protein sources, which have a role as a structural unit of a protein and are linked by peptide bonds. On the other hand, amino acids other than the original protein (100 types are known) are commonly found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, Vol. A2, p. 57-97 VCH: Weinheim (1985)). . The amino acid has a D- or L-optical configuration, and naturally occurring proteins usually have only L-amino acids. Each of the biosynthetic and degradation pathways of the twenty proto-amino acids has prominent features in both prokaryotic and eukaryotic cells (see, eg, Stryer, L. Biochemistry, 3rd edition, pp. 578-590, (1988)). . The essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) are so called because they are usually nutritionally essential components in complex biosynthesis, The biosynthetic pathway converts the remaining 11 "non-essential" amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine). Higher animals retain the ability to synthesize some of these amino acids, but essential amino acids must be supplied from the diet, which results in normal protein synthesis.
[0030]
Apart from their function in protein synthesis, these amino acids are naturally important chemicals, many of which are used variously in the food, feed, chemistry, cosmetics, agriculture, and pharmaceutical industries. Lysine is nutritionally important not only in humans but also in monogastric animals such as poultry and pigs. Glutamate is the most commonly used flavor additive (sodium glutamate, MSG) and is widely used throughout the food industry as well as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are all used in the cosmetics industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in both the pharmaceutical and cosmetic industries. Threonine, tryptophan and D / L methionine are common feed additives (Leuchtenberger, W., (1996) Amino aids-technical production and use, p. 466-502 (ed. By Rehm et al. (Ed.) Biotech. Vol., Chapter 14a, VCH: Weinheim) Additionally, these amino acids have been found to be used as synthetic amino acids and precursors to protein synthesis, including N-acetylcysteine, S-carboxymethyl- L-cysteine, (S) -5-hydroxytryptophan and (Ulmann's Encyclopedia of Industrial Chemistry, A2, p. 57-97, VCH: Wei There are other substances as set forth in heim 1985).
[0031]
Natural amino acids produced by organisms, such as bacteria, have remarkable characteristics (see bacterial amion biosynthesis and regulation thereof, Umberger, HE, (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate is synthesized by reductive amination of α-ketoglutaric acid, an intermediate in the citric acid cycle. Next, glutamine, proline, and arginine are produced from glutamine, respectively. Serine biosynthesis consists of three steps starting from 3-phosphoglycerate (intermediate of glycolysis), and the amino acid is obtained through oxidation, transamination, and hydrolysis steps. Both cysteine and glycerin are produced from serine, the former by the condensation of homocysteine and serine, and the latter by the transfer of the side chain β-carbon atom to tetrahydrofolate in a reaction catalyzed by transhydroxymethylase. Manufactured.
[0032]
Phenylalanine and tyrosine are synthesized from precursors of the glycolytic and pentose phosphate pathways, erythrose 4-phosphate and phosphoenolpyruvate in a nine-step biosynthetic pathway that differs only in the last two steps after prephenate synthesis. Tryptophan is also made from these two major molecules, but the synthesis is by an 11-step route. Tyrosine can be synthesized from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine and leucine are all biosynthetic products of pyruvate, the end product of glycolysis. Aspartate is produced from oxaloacetic acid, an intermediate in the citric acid cycle. Isoleucine is produced from threonine. Histidine is produced from the activated sugar 5-phosphoribosyl-1-pyrophosphate by a complex nine-step route.
[0033]
Amino acids in excess of that required for cellular protein synthesis degrade without storage and provide intermediates in the cell's major metabolic pathways (Stryer, L. Biochemistry, Third Edition, Chapter 21, "Amino Acid Degradation and the Urea Cycle "p. 495-516 (1988)). Although cells can convert unwanted amino acids into useful metabolic intermediates, synthetic amino acid synthesis is costly in energy, precursor molecules and required enzymes. That is, it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, and the presence of a special amino acid decreases the production rate or completely stops the production (the feedback mechanism in the amino acid biosynthetic pathway is described by Ullman. 's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, p. 443-613, VCH: Weinheim, 1996). Therefore, the release of a particular amino acid is controlled by the abundance of that amino acid in the cell.
[0034]
B. Metabolism and use of vitamins, cofactors, and functional foods
Vitamins, cofactors, and functional foods contain other types of molecules, and organisms such as bacteria can easily synthesize them, but higher animals lose their ability to synthesize and must be consumed . These molecules are either biologically active substances themselves, or act as electron carriers and intermediates in various metabolic pathways. Apart from their nutritional value, these compounds have important industrial value as colorants, antioxidants, catalysts or other processing aids (for their structure, activity and industrial use, for example, See Ullmann's Encyclopedia of Industrial Chemistry, Volume A27, "Vitamins", p. 443-613 (1996)). The term "vitamin" is as previously recognized, and this substance is necessary for the organism to perform its normal functions, but the organism itself contains nutrients that cannot be synthesized. Vitamins include cofactors and functional food compounds. The term cofactor usually includes non-proteinogenic compounds to obtain the required enzymatic activity. Such compounds may be organic or inorganic, but the cofactor molecule of the present invention is preferably organic. The term "functional food compound" includes supplements that benefit animal and plant health, especially animal health. Examples of such molecules are vitamins, antioxidants, and certain lipids (eg, polyunsaturated fatty acids).
[0035]
The biosynthesis of these substances in organisms capable of producing these substances, such as bacteria, has remarkable characteristics (Ullman's Encyclopedia of Industrial Chemistry, “Vitamins” Vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecule Biotechnology, Nihon University, Nik. "Nutrition, Lipids, Health, and Disease" Proceedings of the UN ESCO / Confederation of Scientific and Technological Associations in Malaya, and the Society for Free Radical Research Research, Asia, Pennsylvania, 1st to 3rd, Oc. .
[0036]
Thiamine (vitamin B₁) Is produced by the chemical coupling of a pyrimidine and a thiazole moiety. Riboflavin (vitamin B₂) Is synthesized from guanosine-5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin is used for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Collectively, "Vitamin B₆All of the families of compounds referred to as "" (e.g., pyridoxine, pyridoxyamine, pyridoxa-5'-phosphate, and conventional pyridoxine hydrochloride) are derivatives of the general structural unit 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R)-(+)-N- (2,4-dihydroxy-3,3-dimethyl-1-oxobutyl) -β-alanine) is produced by either chemical synthesis or fermentation. . The final step in pantothenic acid biosynthesis is the ATP driven condensation of β-alanine and pantoic acid. Enzymes involved in the biosynthesis step in the conversion to pantoic acid or alanine and in the condensation to pantothenic acid are known. The metabolically active form of pantothenic acid is coenzyme A, which is obtained by a five-step biosynthesis by the enzyme. Pantothenic acid, pyridoxal-5'-phosphate, cysteine and ATP are precursors of coenzyme A. These enzymes not only catalyze the production of pantotanic acid, but also (R) -pantonic acid, (R) -pantolactone, (R) -panthenol (provitamin B5), pantethein (and derivatives thereof) and coenzymes A is also manufactured.
[0037]
Biotin biosynthesis from the precursor molecule, pimeloyl-CoA, in microorganisms has been studied in detail and several genes involved have been recognized. Many of the corresponding proteins have been shown to be involved in Fe cluster synthesis and are members of the nifS protein. Lipoic acid is derived from octanoic acid, acts as a coenzyme for energy metabolism, and forms part of the pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex. Folate is a substance composed of all derivatives derived from folic acid, which is derived from L-glutaric acid, p-aminobenzoic acid and 6-methylpterin. The biosynthesis of folic acid and its derivatives has been studied in detail in certain microorganisms for the metabolic intermediates guanosine-5'-phosphate (GTP), L-glutaric acid and p-aminobenzoic acid.
[0038]
Corinoids such as cobalamin and especially vitamin B₁₂) And porphyrins belong to a class of chemicals characterized by the tetrapyrrole ring system. Vitamin B₁₂The biosynthesis of is so complex that it has not yet been fully characterized, but enzymes and substances related to this are known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives also called "niacin". Niacin is a precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and its reduced form.
[0039]
Mass production of these compounds is highly dependent on cell-free chemical synthesis. However, some of these chemicals, such as riboflavin, vitamin B₆, Pantothenic acid and biotin are also produced by large-scale culture of microorganisms. Vitamin B₁₂Is produced only by fermentation due to its complex synthesis. In vitro methods require considerable material and time, and are often expensive.
[0040]
C. Purine, pyrimidine, nucleoside and nucleotide metabolism and use
Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for use in the treatment of disease and viral infection by tumors. The term "purine" or "pyrimidine" includes nitrogen-containing bases, which are building blocks for nucleic acids, coenzymes, and nucleotides. A "nucleotide" is a basic structural unit of a nucleic acid molecule and is composed of a nitrogen-containing base, a pentose (the sugar in the case of RNA is ribose, and in the case of DNA it is D-deoxyribose) and phosphate. You. A “nucleoside” is a molecule that acts as a precursor of a nucleotide and does not have the phosphate moiety of the nucleotide. By inhibiting the biosynthesis of these molecules or the metabolism of nucleic acid molecules, it is possible to inhibit the synthesis of RNA and DNA. For example, if such activity in cancer cells is inhibited, it is believed that the ability of cancer cells to divide and replicate can be inhibited. In addition, there are nucleotides that do not form nucleic acid molecules but act as energy sources (eg, AMP) or coenzymes (eg, FAD and NAD).
[0041]
Several documents describe methods of using these medicinal chemicals by affecting purine and / or pyrimidine metabolism (see, eg, Christopherson, RI and Lyons, SD, (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10: 505-48. Studies of enzymes involved in purine and pyrimidine metabolism have focused primarily on the development of new drugs that are used, for example, as immunosuppressants or antiproliferatives (Smith, JL, (1995) "Enzymes in nucleotides"). Synthesis. "Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purines, pyrimidine bases, nucleosides and nucleotides can be used as intermediates in the biosynthesis of several fine chemicals (eg, thiamine, S-adenosylmethionine, folate or riboflavin) or as energy carriers for cells (eg, ATP or GTP). Or as a drug commonly used as a condiment (eg, IMP or GMP), or for multiple medical uses (eg, Kuninaka, A., (1996) Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al. VCH: Weinheim, p. 561-612). In addition, purines, pyrimidines, nucleosides and enzymes involved in nucleotide metabolism are also targeted, and chemicals for protecting crops, such as fungicides, herbicides and insecticides, are being developed.
[0042]
The metabolism of these compounds in bacteria has prominent features (see, for example, Zalkin, H. and Dixon, JE, (1992) "de novo purine nucleotide biosynthesis" (Progress in Nucleic Acid Medicine, Acidic Acid Medicine, Medical and Medical Sciences). Vol. 42, Academic Press, pp. 259-287; ). Phosphorus metabolism is an intensive study and is important for the normal functioning of cells: adverse effects on purine metabolism in higher organisms can lead to serious diseases such as gout. Guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP) is produced from phosphoric acid through an intermediate compound inosine-5'-phosphate (IMP) through a plurality of steps, They rapidly produce triphosphates used as nucleotides, which are also used as energy sources, which are degraded to provide energy to various biosynthetic pathways in cells. .
[0043]
Pyrimidine biosynthesis is performed by the production of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. This UMP is then converted to cytidine-5'-triphosphate (CTP). The deoxy form of all these nucleotides is achieved by a one-step reduction reaction of the nucleotide diphosphate ribose to the nucleotide diphosphate deoxyribose. These molecules may be involved in DNA synthesis in the phosphorylation reaction.
[0044]
D. Metabolism and use of trehalose
Trehalose is composed of two glucose molecules linked by an α, α-1,1 bond. Trehalose is used in the food industry for sweeteners, additives in dried or frozen foods, and for beverages. However, trehalose also has uses in the pharmaceutical, cosmetic, and biotechnology industries (eg, Nishimoto et al., (1998) US Patent No. 5,759,610, Singer, MA, and Lindquist, S .; (1998) Trends Biotech. 16: 460-467, by Paiva, CLA and Panek, AD, (1996) Biotech. Ann. Rev. 2: 293-314, and by Shiosaka, M. , (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from various microorganisms and is naturally released into a surrounding medium, and is collected by a known method.
[0045]
[II. Membrane biosynthesis and transmembrane transport]
Cell membranes perform various functions of cells. First, the membrane alters cellular components depending on the surrounding environment, thereby obtaining cellular integrity. The membrane acts as a barrier to the entry of harmful or inappropriate compounds and the exit of the desired compounds. Due to its structure, cell membranes originally do not allow the passage of hydrophilic compounds, such as proteins, water molecules and ions, unless they have been promoted and diffused. That is, the cell membrane consists of a bilayer of lipid molecules, in which the head-forming polar groups face outward (towards the outside and inside of the cell, respectively), and the tail-forming nonpolar groups A hydrophobic nucleus is formed toward the center of the double membrane (for the membrane structure and function, see Gennis RB, (1989), Biomembrans, Molecular Structure and Function, Springer: Heidelberg). ). This barrier allows the cell to maintain a relatively high concentration of the desired compound and a relatively low concentration of the unsuitable compound than are contained in the surrounding medium. This is because the diffusion of each compound is efficiently blocked by the cell membrane. However, this membrane also acts as an efficient barrier to entrap the desired compounds and release the effluent molecules. To perform this difficult operation, the cell membrane incorporates a variety of transporter proteins that can efficiently transport multiple compounds across the membrane. Such transporter proteins are roughly classified into two types. A pore or channel protein and a transporter protein. The former are integral membrane proteins that form regulated pores in the membrane, such as protein complexes. This modulation or "gating" is generally specific for the molecules transported by the pore or channel, and their transmembrane organization is made selectively permeable to specific groups of substances. For example, a potassium channel is configured to allow only ions having the same charge and size as potassium to pass through. Channels and pore proteins have discontinuous hydrophobic and hydrophilic domains, the hydrophobic part of the protein binds to the inside of the membrane, and the hydrophilic part covers the inside of the channel. A shielded hydrophilic environment is obtained which only allows passage. Many pores / channels are known in the art, examples of which are pores / channels for potassium, calcium, sodium and chloride ions.
[0046]
Facilitated diffusion pores and channel-regulation systems accommodate only very small molecules, such as ions. This is because the presence of large pores or channels that allow the entire protein molecule to pass through the facilitating nucleic acid makes it impossible to prevent the passage of small hydrophilic molecules. Transport of molecules by this method is sometimes referred to as "facilitated diffusion" because transport requires a driving force due to a concentration gradient. Permease allows permeation of large molecules, such as glucose or other saccharides, into cells when the molecular concentration on one side of the membrane is greater than the molecular concentration on the other side (also Uniport). Called). Unlike pores or channels, these integral membrane proteins (often with 6-14 membrane-bound α-helices) do not form open channels across the membrane and bind to target molecules at the membrane surface, This causes a conformational shift (conformational shift) such that the target molecule is released to the opposite side of the membrane.
[0047]
However, cells need to introduce or release molecules ("active transport") into existing concentration gradients, in which case facilitated diffusion does not occur. Two common mechanisms used by cells for such membrane transport are symport (isotropic transport) or antiport (counter transport), and energy-coupled transport regulated by the ABC transporter. The symport and antiport systems link the movement of two different molecules across the membrane (permease with two separate binding sites for the two different molecules), but in the symport both molecules are transported in the same direction. In the antiport, one molecule is taken in and the other molecule is released. This is energetically possible because one of the two molecules moves according to a concentration gradient. This energetically favorable phenomenon is only possible if the desired compound moves with it against the surrounding concentration gradient. In the energy run process, one type of molecule is transported across the membrane against a concentration gradient and utilized by the ABC transporter. In this system, the transport protein present at the membrane has an ATP-binding cassette, and upon binding of the target molecule, ATP is converted to ADP + Pi, and the resulting release of energy results in the transporter assisting the transport of the target molecule's membrane. Movement to the other side is caused. For further details of such transport systems, see Bamberg et al., (1993) {"Charge transport of ion pumps on lipid bilayer membranes", Q.A. Rev .. Biophys. 26: 1-25; Findlay, J. M .; B. C. (1991), "Structure and Function in Membrane Transport Systems", Curr. Opin. Struct. Biol. 1: 804-810; Higgins, C.I. F. (1992), "ABC transports from microorganisms to man", Ann. Rev .. Cell boil. 8: 67-113; Gennis, R .; B. (1989) "Portes, Channels and Transporters", (Biomembrans, Molecular Structure and Function, Springer: Review of Heidelberg, p. 270-322), and Nakao. And bSaier, H .; (1992) "Transport proteins in bacteria: common themes in the air design", Science 258: 936-942, and the references cited in each of the above references.
[0048]
Membrane synthesis is a characteristic step involving a number of components, the most important of which are lipid molecules. Lipid synthesis is divided into two parts. That is, lipid synthesis and addition to sn-glycerol-3-phosphate and addition or modification of a head polar group. Examples of common lipids used in bacterial membranes are phospholipids, glycolipids, sphingolipids and phosphoglycerides. Fatty acid biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase or to acetyl-ACP by acetyl transacylase. After the condensation reaction, the molecules thus formed undergo a series of condensation, reduction and dehydration reactions to jointly produce acetoacetyl ACP, resulting in saturated fatty acid molecules having a chain of the desired length. The production of unsaturated fatty acids from such molecules is carried out aerobically or anaerobically catalysed by specific desaturases in the presence of molecular oxygen. C. Neidhardt et al., (1996), E. coli and Salmonella.ASM Press: Washington, DC, p.612-636, and references described therein, Lengeller et al., Ed., (1999) Biology. of Procaryotes. Theme: Stuttgart, New York, and references cited therein, and Magnuson, K. et al., (1996) Microbiological Reviews 57: 522-542 and references cited therein. That). Cyclopropane fatty acids (CFA) are synthesized by specific EFA synthetases by using SAM as a co-substrate. Branched fatty acids are synthesized by the generation of branched 2-oxo acids by deamination of branched amino acids (eg, Langeler et al., Eds., (1999) Biology of Procaryotes. Thieme: Stuttgart, New York and in this document). References listed). Another important step in lipid synthesis is the step of transporting fatty acids onto the head polar group by glycerol-phosphate-acyltransferase or the like. The binding of biosynthetic enzymes to various precursor molecules leads to the production of various fatty acid molecules, which have a significant effect on membrane composition.
[0049]
[III. Elements and Methods of the Invention]
The present invention is based on novel molecules (referred to as MCT nucleic acid and protein molecules), whereby C.I. The production of cell membranes in Glutamicum is controlled and the movement of molecules through this membrane is governed. In one embodiment, the MCT molecule is C.I. It is involved in the metabolism of compounds required for the formation of cell membranes in Glutamicum, or in the transport of molecules across this membrane. In a preferred embodiment, the activity of the MCT molecules of the present invention to regulate membrane component production and transport affects the production of the desired fine chemical by the organism. In a particularly preferred embodiment, the M.C.T. The yield, production and / or production efficiency of the glutamicum metabolic pathway is regulated, and / or the production and transport efficiency of the compound across the membrane is altered, which is The activity of the MCT molecule of the present invention is adjusted so as to directly or indirectly adjust the yield, production and production and / or production efficiency of the desired fine chemical by Glutamicum.
[0050]
The expressions "MCT protein" or "MCT polypeptide" are used in C.I. It refers to proteins involved in the metabolism of compounds required for the formation of the cell membrane of Glutamicum or the transport of molecules across this membrane. Examples of MCT proteins include those proteins listed in Table 1 or encoded by the MCT gene indicated by an odd SEQ ID NO. "MCT gene" or "MCT nucleic acid sequence" consists of the nucleic acid sequence encoding the MCT protein and the corresponding untranslated 5 'and 3' sequence regions. Examples of MCT genes include those described in Table 1. “Production” and “productivity” are as conventionally understood, and a fermentation product (eg, a desired fine chemical) obtained in a predetermined fermentation amount (eg, product (kg) / hour / liter) within a predetermined time period Including the concentration of "Manufacturing efficiency" means the time required for a predetermined amount of production to be performed (for example, the time required for cells to produce fine chemicals at a predetermined ratio). "Yield (yield)" or "product (product) / carbon yield" is as conventionally understood and means the efficiency with which a carbon source is converted to a product (ie, a fine chemical). This is usually described, for example, as product (kg) / carbon source (kg). Increasing the yield or production of the compound increases the amount of molecules recovered or useful of the recovered molecules from a given amount of culture at a given time. The expression "biosynthesis" or "biosynthetic pathway" is as conventionally understood, and refers to compounds, especially organic compounds, by a multi-step (e.g.) advanced control (e.g.) of an intermediate compound by a cell. Refers to the synthesis of a compound. The terms “degradation” and “degradation pathway” are as conventionally understood, and the degradation products (generally, in general) of a compound, particularly an organic compound, by a multi-step (such as) advanced control (such as) by cells. (Small or less complex molecules). "Metabolism" is as conventionally understood, and refers to the entire body reaction taking place in an organism. Metabolism of a given compound (eg, the metabolism of an amino acid such as glycine) refers to the overall intracellular biosynthesis, modification, and degradation pathways associated with this compound.
[0051]
In another embodiment, the MCT molecule of the present invention comprises a desired molecule, such as C.I. It is possible to regulate production in microorganisms such as glutamicum. By altering the MCT protein of the present invention, such altered proteins can be transformed into C. elegans. By introducing the C. glutamicum, There are a number of mechanisms that directly affect the yield, production and / or production efficiency of glutamicum fine chemicals. Increases the number and activity of MCT proteins involved in the release of fine chemical molecules from cells, secretes larger amounts of such compounds into extracellular media and makes recovery of compounds from media easier than ever It is also possible to be performed. Similarly, these MCT proteins are involved in the uptake of nutrients required for the biosynthesis of one or more fine chemicals (eg, phosphate compounds, sulfate compounds, nitrogen compounds, etc.) and are improved in number and activity. It is also possible to increase the intracellular concentrations of these precursors, cofactors or intermediate products. In addition, fatty acids and lipids, which are important fine chemicals, may be used to optimize the activity and increase the number of one or more MCT proteins of the invention involved in their biosynthesis, or to degrade the aforementioned compounds. By reducing the action of one or more related MCR proteins, C.I. It is also possible to improve the yield, production and / or production efficiency of glutamicum-derived fatty acids and lipid molecules.
[0052]
By manipulating one or more MCT genes of the present invention, C.I. It is also possible to produce MCT proteins having altered activity that directly affects the production of one or more desired fine chemicals derived from Glutamicum. For example, normal metabolic excretion in cells, which may increase in quantity due to overproduction of the desired fine chemicals, may adversely affect nucleotides and proteins in the cell (eg, decrease cell viability). Of the present invention that are involved in the release of excreta so that they can be efficiently released from cells prior to disrupting the fine chemical biosynthetic pathway (which can reduce the yield, production or production efficiency of the fine chemical). The number or activity of MCT proteins may be increased. Furthermore, if the intracellular mass of the desired fine chemical is relatively large, this may have a toxic effect on cells, so that a transporter capable of releasing these compounds from cells may be used. By increasing the activity or the number, it is possible to increase the viability of the cells in the culture and obtain a large number of cells in the culture for producing the desired fine chemical. The MCT proteins of the present invention can also be engineered to provide relative amounts of various lipid and fatty acid molecules. Since lipids have different physical properties for each type, changing the lipid composition in the membrane significantly changes the fluidity of the membrane. Changes in membrane fluidity affect the transport of molecules across the membrane and the integrity of the cell, both of which affect C. elegans in large-scale fermentation cultures. Significantly affects the production of glutamicum-derived fine chemicals.
[0053]
The isolated nucleic acid sequence of the present invention is contained within the genome of Corynebacterium glutamicum marketed by the American Type Culture Collection, known as ATCC 13032. The isolated C. The nucleotide sequence of Glutamicum MCT DNA is an odd-numbered SEQ ID NO. The predicted amino acid sequence in the glutamicum MCT protein is shown as each even SEQ ID NO. Computer analysis was performed to classify and / or identify the nucleotide sequence as a sequence encoding a protein involved in the metabolism of cell membrane components, or a protein involved in the transport of compounds across this membrane.
[0054]
The present invention includes proteins having an amino acid sequence substantially homologous to the amino acid sequence of the present invention (for example, a sequence according to an even SEQ ID NO in the sequence listing). As used herein, a protein having an amino acid sequence substantially homologous to a selected amino acid sequence is at least 50% homologous to the selected amino acid sequence, eg, the entire selected amino acid sequence. A protein having an amino acid sequence substantially homologous to the selected amino acid sequence is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70%, relative to the selected amino acid sequence. % -80 &, 80% -90%, or 90% -95%, and more preferably at least about 96%, 97%, 98% or more than 99% homologous. Ranges of values indicated by intermediate values of the above values (for example, 75% to 80% identical, 85 to 87% identical, 91 to 92% identical) are also included in the present invention.
[0055]
The MCT proteins or biologically active proteins or fragments thereof of the present invention may be C.I. It is involved in the metabolism of compounds required for the construction of the cell membrane of Glutamicum, or in the transport of molecules across this membrane, or has one or more of the activities listed in Table 1.
[0056]
The details of the present invention are described further below.
[0057]
A. Isolated nucleic acid molecule
The present invention relates to an isolated nucleic acid molecule encoding an MCT polypeptide or a biologically active portion thereof, a hybridization probe used for recognition (identification) or amplification of a nucleic acid encoding SCT DNA (SRT DNA) or Includes nucleic acid molecule fragments that can be used as primers. As used herein, “nucleic acid molecule” is intended to include DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA and RNA generated using analogs of nucleotides. The term refers to the untranslated sequence present at both the 3 'and 5' ends of the coding region of the gene, ie, a sequence of at least about 100 nucleotides upstream from the 5 'end of the coding region of the gene, and 3' of the coding region of the gene. 'A sequence of at least about 20 nucleotides downstream from the terminus. The nucleotide molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. An “isolated” nucleic acid molecule is derived from the genomic DNA of an organism and naturally has sequences that flank the nucleic acid of the genomic DNA (ie, sequences located at the 5 ′ and 3 ′ ends). Preferably not. For example, in various embodiments, the isolated MCT nucleic acid molecule is naturally located on the side of the genomic DNA of the cell from which the nucleic acid was obtained (eg, a C. glutamicum cell) and has a size of about 5 kb, Consists of a nucleotide sequence of less than 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb. Further, an "isolated" nucleic acid molecule, eg, a DNA molecule, is free of other cellular materials, the media used when produced by recombinant techniques, and also contains a chemical precursor when chemically synthesized. Contains no body or other chemicals.
[0058]
A nucleic acid molecule of the invention, for example, a nucleic acid molecule having an odd SEQ ID NO nucleotide sequence in the sequence listing, or a portion thereof, is isolated by standard molecular biology techniques, the information of which is provided herein. . For example, C.I. Glutamicum SRT DNA can be C. mutated using standard hybridization techniques with all or part of any of the odd SEQ ID NO sequences in the Sequence Listing. Can be isolated from Glutamicum library
(For example, Sambrook, J., Fritsh, EF, Maniatis, T., co-authored, Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor, Colorado Harbor, Colorado Harbor Laboratory, California) , 1989). Further, a nucleic acid molecule comprising all or part of any of the nucleic acid sequences of the invention (eg, odd SEQ ID NO) is isolated by polymerase chain reaction using oligonucleotide primers designed based on this sequence. (For example, a nucleic acid molecule comprising all or a portion of any of the nucleic acid sequences of the invention (eg, odd SEQ ID NOs) is isolated by polymerase chain reaction using oligonucleotide primers designed based on similar sequences. Is possible). For example, mRNA is isolated from normal endothelial cells (eg, by the guanidinium-thiocyanate extraction procedure described by Chirgwin et al., (1979) Biochemistry 18: 5294-5299), and DNA is produced using reverse transcriptase. (Eg, Gibco / BRL, Bethesda, MD, Moloney MLV reverse transcriptase, or Seikagaku America, Inc., AMV, St. Petersburg, FL AMV, reverse transcriptase). Synthetic oligonucleotides used for amplification by the polymerase chain reaction are designed based on any of the nucleotide sequences described in the Sequence Listing. The nucleic acids of the invention are amplified using cDNA or genomic DNA as a template and oligonucleotide primers as appropriate in standard PCR amplification techniques. The nucleic acid so amplified is cloned into a suitable vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to MCT nucleic acid sequences are produced by standard synthetic techniques, eg, using an automated DNA synthesizer.
[0059]
In a preferred embodiment, the isolated nucleic acid molecule of the present invention comprises any of the nucleotide sequences set forth in the Sequence Listing. The nucleic acid sequence of the present invention described in the sequence listing corresponds to Corynebacterium glutamicum MCT DNA of the present invention. This DNA contains the sequence encoding the MCT protein (ie, the “coding region” shown in the odd SEQ ID NO sequence in the sequence listing), as well as the 5 coding sequence shown in the odd SEQ ID NO sequence in the sequence listing. Includes 'untranslated and 3' untranslated sequences. Further, the nucleic acid molecule may include only one of the coding regions of the nucleic acid sequence in the sequence listing.
[0060]
In the present invention, all nucleic acid and amino acid sequences described in the sequence listing include identifications including “RXA”, “RXN”, “RXS” or “RXC”, and a 5-digit numerical value thereafter. RXA, RXN, RXS, RXC numbers are assigned (eg, RXA02099, RXN03097, RXS00148 or RXC01748). Each nucleic acid sequence contains up to three portions: a 5 'upstream region, a coding region, and a downstream region. These three regions are each identified by a common RXA, RXN, RXS, RXC designation to prevent confusion. The phrase "any of the odd sequences in the Sequence Listing" indicates any of the nucleic acid sequences in the Sequence Listing that are recognizable by different RXA, RXN, RXS, RXC designations. The coding region of each sequence is translated as an even SEQ ID NO in the sequence listing into the corresponding amino acid sequence listed immediately below the corresponding nucleic acid sequence. For example, the coding region of RXA03097 is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is described in SEQ ID NO: 2. The nucleic acid sequences of the present invention are indicated by the same RXA, RXN, RXS, and RXC designations as the amino acid molecules they encode, and the correlation can be easily read. For example, the amino acid sequence labeled RXA02099 is a translation of the coding region of the nucleotide sequence of the nucleic acid molecule RXA02099, and the amino acid sequence labeled RXN03097 is a translation of the coding region in the nucleotide sequence of the nucleic acid molecule RXN03097, Further, the amino acid sequence designated as RXS00148 is a translation of the coding region in the nucleotide sequence of the nucleic acid molecule RXS00148, and the amino acid sequence designated as RXC01748 is obtained by translating the coding region in the nucleotide sequence of the nucleic acid molecule RXC01748. is there. The nucleotide and amino acid sequences by RXA, RXN, RXS or RXC of the present invention, and their correspondence to assigned SEQ ID NOs are shown in Table 1.
[0061]
Several types of genes in the present invention are “F-designated genes”. F-display genes have a “F” before the display of RXA, RXN or RXS and include the genes listed in Table 1. For example, as described in Table 1, SEQ ID NO: 7 is an F-display gene called “FRXA00498”, and SEQ ID NOs: 25, 33, and 37 are also F-display genes (FRXA01345 in Table 1, respectively). , F RXA02543, F RXA02282).
[0062]
In one embodiment, the nucleic acid molecules of the invention may not include those listed in Table 2. The sequence of the dapD gene is described in Wehrmann, A .; Et al., (1998) Bacteriol. 180 (12): 3159-3165. However, the sequences obtained by the inventor are considerably longer than those published by the above references. In addition, it is considered that the sequence published in the above-mentioned document shows a fragment of only the actual coding region due to the incorrect start codon.
[0063]
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule having complementarity to a nucleotide sequence of the invention (eg, an odd SEQ ID NO sequence in the Sequence Listing or a portion thereof). . A nucleic acid molecule complementary to any of the nucleotide sequences of the present invention has sufficient complementarity to any of the nucleotide sequences shown in the sequence listing (eg, the odd SEQ ID NO sequence in the sequence listing), It hybridizes to this to form a stable double strand.
[0064]
In still other embodiments, the isolated nucleic acid molecules of the present invention comprise at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%. Or 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, Or 90%, or 91%, 92%, 93%, 94%, more preferably at least about 95%, 96%, 97%, 98%, 99% or more of the nucleotide molecules of the invention (odd numbers in the sequence listing). SEQ ID NO :) or a portion thereof. Ranges of values indicated by intermediate values of the above values (for example, 75% to 80% identical, 85 to 87% identical, 91 to 92% identical) are also included in the present invention. For example, any of the above values described as the upper limit value and / or the lower limit value may be combined to form a value range. In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence that hybridizes, eg, hybridizes under stringent conditions, to any of the nucleic acid molecules of the invention or any portion thereof.
[0065]
Furthermore, the nucleic acid molecules of the present invention may encode only a portion of the coding region in any of the odd SEQ ID NO sequences of the Sequence Listing, for example, a fragment for a probe or primer, or a biologically active portion of an MCT protein. It may consist only of fragments that do. C. A probe or probe for recognizing and / or cloning MCTs homologous to cells and organisms of other species and MCT analogs from other Corynebacterium or related species, depending on the nucleotide sequence determined by cloning of the MCT gene from G. glutamicum. A primer is generated. Probes / primers usually include substantially purified oligonucleotides. The oligonucleotide generally comprises at least about 12, preferably about 25, more preferably 40, 50 or 75 contiguous nucleotide sequences of the invention under stringent conditions (eg, an odd SEQ ID NO: In any of the sequence listings, the antisense sequence in any of the sequence listings or naturally occurring variants thereof. Primers based on the nucleotide sequences of the present invention can be used in PCR reactions to clone MCT homologs. Probes based on the MCT nucleotide sequence detect sequences obtained by transcription or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probe further has a label group (labeling functional group). Examples of radio groups are radioisotopes, ultraviolet compounds, enzymes, enzyme cofactors. Such probes can be used, for example, to measure the level of MCT-encoding nucleic acid in a cell sample, for example, by detecting the level of MCT mRNA or confirming the mutation or deletion of the genomic MCT gene, to measure cells that mis-express the MCT protein. Used as part of a medical test kit for testing.
[0066]
In one embodiment, the nucleic acid molecule of the invention comprises an amino acid sequence of the invention (e.g., an even SEQ ID NO sequence in the sequence listing) comprising a protein or a portion thereof comprising C.I. It encodes a protein or protein portion with an amino acid sequence that is sufficiently homologous to maintain the metabolism of compounds required for the formation of the cell membrane of Glutamicum, or the ability to participate in the transport of molecules across this membrane. As used herein, the expression “sufficiently homologous” refers to the same or equivalent amino acid sequence of the present invention (for example, an amino acid residue having a side chain similar to an amino acid residue in any of the even SEQ ID NO sequences in the sequence listing). A protein or protein part having a minimum number of amino acid residues, wherein the protein or protein part It means that it is possible to participate in the metabolism of compounds required for the construction of the cell membrane of Glutamicum or the transport of molecules across this membrane. As mentioned above, protein members of the membrane component metabolic pathway or membrane transport system can play a role in the production or secretion of one or more chemicals. Examples of such activities are described herein. "Function of MCT protein" is directly or indirectly involved in the yield, production and / or production efficiency of one or more chemicals. Examples of MCT protein activity are described in Table 1.
[0067]
In another embodiment, the protein is at least about 50-60%, preferably about 60-70%, more preferably about the entire sequence of amino acids of the invention (eg, the sequence according to the even SEQ ID NO in the sequence listing). Is at least about 70-80%, 80-90%, 90-95%, and particularly preferably at least about 96%, 97%, 98%, 99% or more homologous.
[0068]
Preferably, the protein portion encoded by the MCT nucleic acid molecule of the present invention is a biologically active portion of any of the MCT proteins. As used herein, the expression “biologically active portion of the MCT protein” refers to C.T. It is involved in the metabolism of compounds required for the formation of the cell membrane of Glutamicum, or the transport of molecules across this membrane, or has a moiety having the activity described in Table 1, such as the domain / motif of the MCT protein. The MCT protein or a biologically active portion thereof is C. Enzyme activity is assessed to determine whether it is involved in the metabolism of compounds required for the formation of the cell membrane of Glutamicum or the transport of molecules across this membrane. Such evaluation methods are known to those skilled in the art, and specific examples are described in detail in Example 8.
[0069]
Other nucleic acid fragments encoding a biologically active portion of the MCT protein can be produced by isolating a portion of any of the amino acid sequences of the present invention (eg, even SEQ ID NOs in the Sequence Listing). And expressing the encoded portion of the MCT protein or peptide (eg, by recombinant expression in vitro) and assessing the activity of the encoded portion of the MCT protein or peptide.
[0070]
It has a sequence that differs from the nucleic acid sequence of the present invention (for example, the sequence of odd SEQ ID NO in the sequence listing) (and a part thereof) due to a change in the genetic code (degeneracy). Includes nucleic acid molecules encoding the same MCT protein as encoded. In another embodiment, it is preferred that the isolated nucleic acid molecule of the present invention is a nucleotide sequence encoding a protein having the amino acid sequence set forth in the Sequence Listing (eg, even SEQ ID NO). In yet another embodiment, the isolated nucleic acid molecule of the present invention comprises a C. subtilis substantially homologous to an amino acid sequence of the present invention. Encodes the entire glutamicum protein (eg, encoded by the open reading frame indicated by the odd SEQ ID NO in the sequence listing).
[0071]
It will be apparent to those skilled in the art that in one embodiment, the sequences of the present invention do not include the Benbank sequences described in Table 2 or 4 obtained prior to the present invention. In one embodiment, the present invention relates to the use of portions of the nucleotide or amino acid sequences of the present invention that are more identical to prior art sequences (eg, Genbank sequences (or proteins encoded by the sequences) as set forth in Tables 2 or 4). Includes large percentages of nucleotide and amino acid sequences. For example, the present invention provides a nucleotide sequence labeled RXA01420 (SEQ ID NO: 7) of 38% or more, a nucleotide sequence labeled RXA00104 (SEQ ID NO: 5) of 43% or more, and a nucleotide sequence labeled RXA02173 ( SEQ ID NO: 25) contains a nucleotide sequence that is 45% or more identical. One skilled in the art can determine the lower limit of the percentage of identity for a given sequence of the present invention by examining the identity score calculated by GAP from the top three data listed in Table 4 that hit for this given sequence, and calculated by GAP. It can be obtained by calculation by subtracting the highest value (%) of the same ratio from 100%. Nucleic acid and amino acid sequences with an identical percentage higher than the lower limit thus calculated (eg at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%) %, Or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%. %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, or 90%, or 91%, 92%, 93%, 94%, and more preferably at least about 95%, 96%, 97%, 98%, 99% or more) are included in the present invention. Will be clearly understood by those skilled in the art.
[0072]
C. listed in the sequence listing as odd SEQ ID NO. It is also understood by those skilled in the art that, in addition to the glutamicum MCT nucleic acid sequence, there may be DNA sequence polymorphisms (polymorphism) that cause changes in the amino acid sequence of the MCT protein in the population (C. glutamicum population). Such genetic polymorphism in the MCT gene may exist due to natural mutation. As used herein, the terms “gene” and “recombinant gene” refer to the MCT protein, preferably C.I. A nucleic acid molecule comprising an open reading frame encoding a Glutamicum MCT protein. Such natural variations usually vary from 1 to 5% in the nucleotide sequence of the MCT gene. Such or any other nucleotide variations and the resulting amino acid polymorphisms in MCTs are the result of natural mutations and do not alter the functional activity of the MCT protein, It is included in.
[0073]
C. of the present invention. Natural variants of G. glutamicum MCT DNA and and non-C. The nucleic acid molecule corresponding to the glutamicum homolog is C. glutamicum. C. glutamicum, or C. glutamicum described above with reference to a part thereof. Based on homology to glutamicum MCT nucleic acids, they are isolated as hybridization probes by standard hybridization techniques under stringent hybridization conditions. Thus, in another embodiment, the isolated nucleic acid molecule of the invention consists of 15 or more nucleotides and hybridizes under stringent conditions to a nucleic acid molecule comprising an odd SEQ ID NO nucleotide sequence in the Sequence Listing. I do. In other embodiments, the nucleic acid molecule has at least 30, 50, 100, or 250 or more nucleotides. "Hybridizes under stringent conditions" refers to hybridization and washing conditions in which nucleotides that are 60% or more homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences that are homologous to each other at least about 65%, more preferably at least about 70%, and more preferably at least about 75%, remain hybridized to each other. Such stringent conditions are known to those of skill in the art and are described in Current Protocols in Molecular Biology, John Wiley & Sons, NJ. Y. (1989), 6.3.1-6.3.6. It is described in. Preferred examples of stringent hybridization conditions include, but are not limited to, hybridization at about 45 ° C. with 6 × sodium chloride / sodium citrate (SSC), followed by 50 × with 0.2 × SSC, 0.1% SDS. Hybridization by washing at ６５65 ° C. at least once. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleic acid sequence of the invention corresponds to a naturally occurring nucleic acid molecule. As used herein, "naturally occurring" nucleic acid refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (eg, encoding a naturally occurring protein). In one embodiment, the nucleic acids of the invention are naturally occurring C. It encodes the glutamicum MCT protein.
[0074]
In addition to naturally occurring variants of the MCT sequence that may be present in a population, the nucleotide sequence of the present invention may be altered by mutation, resulting in a change in the amino acid sequence of the encoded MCT protein, It will be understood by those skilled in the art that the functional capabilities of this will not change. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can occur in the nucleotide sequences of the present invention. "Non-essential" amino acid residues are those residues that differ from any wild-type sequence of the MCT protein (eg, even SEQ ID NOs in the Sequence Listing) without altering the activity of the MCT protein. It is. On the other hand, “essential” amino acid residues are those amino acids required for MCT protein activity. However, other amino acid residues (eg, amino acids that are not conserved in the domain having MCT activity or are in a semi-conserved state) may not be essential for activity, and therefore may be altered without change in MCT activity. Cheap.
[0075]
Accordingly, the present invention further includes nucleic acid molecules that include MCT proteins that include changes in amino acid residues that are not essential for MCT activity. The amino acid sequence of such an MCT protein differs from the even SEQ ID NO sequence in the sequence listing, but contains one or more of the MCT activities described herein. In one embodiment, an isolated nucleic acid molecule comprises an amino acid sequence that is at least 50% homologous to an amino acid sequence of the present invention, and Nucleotide sequences encoding proteins that are capable of participating in the metabolism of compounds required for the construction of the cell membrane of Glutamicum, or for transporting molecules across this membrane, or that enhance one or more of the activities listed in Table 1. including. The protein encoded by the nucleic acid molecule may contain at least about 50-60%, preferably at least about 60-70%, more preferably about 70-80%, 80% of the amino acid sequence of any of the odd SEQ ID NOs in the sequence listing. It is preferred to have an amino acid sequence that is -90%, 90-95%, particularly preferably at least about 96%, 97%, 98% or 99% homologous.
[0076]
In order to measure the homology ratio of two kinds of amino acid sequences (for example, any one of the amino acid sequences of the present invention and its variant) or two kinds of nucleic acids, the sequences are determined so that optimal comparison can be performed ( A gap can be provided to obtain an optimal position (alignment) between the protein or nucleic acid of the present invention and the other protein or nucleic acid). Compare the corresponding amino acid position or nucleic acid position with the amino acid residue or nucleic acid. If a position in one sequence (eg, one of the amino acid sequences of the invention) has the same amino acid residue or nucleotide as the corresponding position in the other sequence (eg, a variant of the amino acid sequence), the molecule at this position Are homologous (ie, "homology" of an amino acid or nucleic acid is equivalent to "identity" of an amino acid or nucleic acid in the present invention). The percentage of homology between two sequences is a function of the number of identical positions between the sequences (ie, homology (%) = number of identical positions / total number of positions x 100).
[0077]
An isolated nucleic acid molecule encoding an MCT protein that is homologous to a protein sequence of the invention (eg, an even SEQ ID NO sequence in the Sequence Listing) can be obtained by substituting one or more nucleotides for a nucleotide sequence of the invention. An addition or deletion occurs when one or more amino acids of the encoded protein are replaced, added, or deleted. Standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis, result in mutations of any of the nucleotide sequences of the present invention. Conservative amino acid substitutions occur at one or more predicted non-essential amino acid residues. "Conservative amino acid substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), and amino acids with uncharged electrode side chains (eg, glycine, asparagine, glutamine). , Serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (eg, threonine, valine, Isoleucine), and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Preferably, the predicted nonessential amino acids in the MCT protein are replaced by other amino acid residues belonging to the same side chain family. In another preferred embodiment, mutations can be randomly introduced into all or a part of the MCT coding sequence by saturation mutagenesis or the like. The MCT activity of the obtained mutant is screened to confirm that the mutant retains the MCT activity. Mutagenesis of any of the odd SEQ ID NOs in the sequence listing results in recombinant expression of the protein encoded thereby, and the activity of this protein may be determined, for example, as described herein (see specific examples). (See Example 8 for evaluation).
[0078]
In addition to the nucleic acid molecules encoding the MCT proteins described above, the present invention includes isolated nucleic acid molecules that are antisense thereto. By “antisense” nucleic acid is meant a nucleotide sequence that is complementary to a “sense” nucleic acid that encodes a given protein, eg, a nucleotide sequence that is complementary to the coding strand or mRNA sequence of a cDNA molecule. Thus, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. An antisense nucleic acid can have complementarity to all MCT coding strands, or part of it. In one embodiment, the antisense nucleotide molecule is antisense "in the coding region" of the coding strand of the nucleotide sequence encoding the MCT protein. “Coding region” refers to a region of a nucleotide sequence that includes codons translated into amino acid residues (eg, the coding region of SEQ ID NO .: 5 (RXA00104) includes nucleotides 1 to 756). In another embodiment, the antisense nucleic acid molecule is antisense to the "non-coding region" of the coding strand (coding strand) of the nucleotide sequence encoding MCT. "Non-coding region" refers to the 5 'and 3' sequences flanking the coding region, which are not translated into amino acids (also referred to as 5 'and 3' untranslated regions).
[0079]
The antisense nucleic acid of the present invention can be designed as a coding strand encoding MCT in the present invention (for example, the sequence shown in the odd SEQ ID NO in the sequence listing) by the base pairing rule of Watson and Crick. The antisense nucleic acid molecule may have complementarity to the entire coding region of MCT mRNA, but is preferably an oligonucleotide that is antisense to only a part of the coding region or non-coding region of MCT mRNA. For example, an antisense oligonucleotide may have complementarity to a region surrounding the translation initiation site of MCT mRNA. Antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleotide of the present invention is constituted by a chemical synthesis and an enzymatic ligation reaction by a method known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides, but to improve the biological stability of the molecule, or to antisense and sense nucleic acids, For example, nucleotides with various modifications designed to improve the physical stability of the duplex between the phosphorothioate derivative and the acridine substituted nucleotide may be used. Examples of modified nucleotides used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxy (Hydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquinosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1- Methyl inosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino Methyl-2- Uracil, β-D-mannosyl quinosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, Quinosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl- 2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, an antisense nucleic acid can be biologically produced by using a predetermined nucleic acid that is subcloned in an antisense direction into an expression vector (that is, RNA transcribed from the inserted nucleic acid is It is placed in an antisense orientation with respect to the nucleic acid, as described in more detail below.)
[0080]
The antisense nucleic acid molecules of the invention are typically introduced into cells or generated in situ, so that they hybridize or bind to the mRNA and / or genomic DNA of the cells encoding the MCT protein, and are transcribed and synthesized. Protein expression is inhibited by inhibiting translation. This hybridization can be carried out by the complementarity of the original nucleotides forming a stable double strand. However, in the case of an antisense nucleic acid molecule binding to a DNA double strand, the hybridization is carried out at the main groove binding to the double helix. May be performed by the specific interaction of The antisense molecule binds to a peptide or antibody that binds to a receptor or antigen on the cell surface in advance so as to specifically bind to a receptor on the cell surface or an antigen expressed on the cell surface. Qualified. An antigen nucleic acid molecule can be introduced into a cell using, for example, a vector. To obtain a sufficient intracellular concentration of the antisense molecule, a vector construct in which the antisense nucleic acid molecule is placed under the strong control of a prokaryotic, viral or eukaryotic promoter is preferably used.
[0081]
In another embodiment, an α-anomeric nucleic acid molecule is used as the antisense nucleic acid molecule of the present invention. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with the complementary RNA. In this case, unlike normal β units, the nuclear strands extend parallel to each other (Gaultier et al., (1987) Nucleic Acids. Res. 15: 6625-6641). Antisense nucleic acid molecules can be 2'-o-methyl ribonucleotides (Inoue et al., (1987) @ Nucleic Acids Res. 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al., (1987) FEBS Lett). 215: 327-330).
[0082]
In another embodiment, the antisense nucleic acid of the present invention may be a ribozyme. A ribozyme is an RNA molecule having catalytic activity and has a ribonuclease activity capable of cleaving a single-stranded nucleic acid having a complementary region, for example, mRNA. Ribozymes (e.g., hammerhead ribozymes (described by Haselhoff and Gerlach, (1988) Nature 334: 585-591)) can be used to catalytically cleave MCT mRNA transcripts and inhibit MCT mRNA translation. A ribozyme having specificity for an MCT-encoding nucleic acid can be designed based on the nucleotide sequence of MCT DNA described herein (eg, SEQ ID NO: 5 (RXA00104). For example, Tetrahymena L-19 IVS RNA has an active moiety The nucleotide sequence is configured to have complementarity to a cleavable nucleotide sequence in the MCT-encoded mRNA, as described, for example, in Cech et al., U.S. Patent No. 4,987,071, and Cech et al. 116, 742. In addition, MCT mRNA can be used to select catalytically active RNAs having specific ribonuclease activity in a pool of RNA molecules, as described by Bartel, D. and Szostak, JW, co-author, 993) Science 261: 1411-1418 becomes the reference.
[0083]
In addition, the expression of the MCT gene can be inhibited by targeting a nucleotide sequence having complementarity to a regulatory region (eg, an MCT promoter and / or enhancer) of the MCT nucleotide sequence. For this, see Helene, C .; (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C.E. Et al., (1992) Ann. N. Y. Acad. Sci. 660: 27-36, and Maher, L. et al. J. Et al. (1992) Bioassays 14 (12): 807-15.
[0084]
B. Recombinant expression vectors and host cells
The invention further includes a vector, preferably an expression vector, containing a nucleic acid encoding the MCT protein (or a portion thereof). Here, “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which the nucleic acid molecule is bound. One example of a vector is a "plasmid," which is a circular double stranded DNA loop into which other DNA segments can be ligated. Another example of a vector is a viral vector, to which other DNA segments can be ligated to the viral genome. Introduction of a given vector into a host cell allows for autonomous replication in the host cell (eg, bacterial vectors derived from bacteria by replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are introduced into the genome of the host cell by introduction into the host cell, and replicate along with the host genome. In addition, certain vectors are capable of controlling the expression of genes to which they are cooperatively linked. These vectors are referred to herein as "expression vectors". Generally, expression techniques used in DNA recombination techniques often take the form of a plasmid. As used herein, "plasmid" and "vector" are used interchangeably, with plasmid being the most frequently used form of vector. However, the invention includes other forms of expression vectors having similar functions, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).
[0085]
The recombinant expression vector of the present invention contains a nucleic acid in a form suitable for expression in a host cell. Recombinant expression vectors contain one or more regulatory sequences selected based on the host cell used for expression, and are cooperatively linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "cooperatively linked" refers to regulatory sequences that permit expression of the nucleotide sequence (e.g., when the vector is introduced into an in vitro transcription / translation system or into a host cell). (In a host cell). The "regulatory sequence" includes a promoter, enhancer, and other expression control elements (eg, a polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Preferred regulatory sequences are, for example, cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI.^q, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, any, SPO2, [lambda] -P_R− Or λ-P_LAnd these are preferably used in bacteria. Examples of other regulatory sequences include promoters from yeast and fungi such as ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, CaMV / 35S, SSU, OCS, lib4, usp, STLS1, B33 and other plant-derived promoters, nos or ubiquitin- and phaseolin-promoters. It is also possible to use artificial promoters. It is understood by those skilled in the art that the design of an expression vector varies depending on factors such as selection of a host cell to be transformed and the degree of expression of a desired protein. The expression vector of the present invention can be introduced into a host cell, whereby a protein or peptide containing a fusion protein or peptide encoded by a nucleic acid (eg, an SRT protein, a mutant form of MCT protein, a fusion protein, etc.) is produced. .
[0086]
The recombinant expression vectors of the present invention are designed for expression of the MCT protein in prokaryotic or eukaryotic cells. For example, the MCT gene is C.I. Bacterial cells such as glutamicum, insect cells (using a baculovirus expression vector), yeast and other fungal cells (Romanos, MA, et al., (1992) “Foreign gene expression in yeast: areview”, Yeast 8). : 423-488; van den Honda, C.A.M.J.J., et al., (1991) "Heterologous gene expression in filamentous fungi" (Mole Gene Manipulations in W. F., J. F., and J. F. in Gene Gene. L. Lasure, ed., P.396-428: Academic Press: San Diego, and van den Honda, CA. MJJ & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi (Applied Molecular Genetics, etc., in Applied Molecular Genetics, Applied Molecular Genetics, etc.) Cambridge University Press: See Cambridge, Algae and Multicellular Plants (Schmidt, R., Willmitzer, L., 1988) High efficiency radiation adatomifation adatomifation adatomifation edifice explants "Plant Cell ReP .: 583-586) or suitable host cells for suitable host cells Goeddel, Gene Expression Technology: Method in Enzymology 185, Academic Medicine, Canada, Canada, Canada. Furthermore, the recombinant expression vector can be transcribed and translated in vitro by using, for example, a T7 promoter regulatory sequence and T7 polymerase.
[0087]
Expression of proteins in prokaryotes is most commonly carried out by vectors containing constitutive or inducible promoters that govern the expression of fusion or non-fusion proteins. Fusion vectors add a large number of amino acids to the encoded protein, usually at the amino terminus and also at the C-terminus, or are fused within an appropriate region of the protein. Such ligation vectors serve three roles. That is, it assists in the purification of recombinant proteins by 1) increasing the expression of the recombinant protein, 2) improving the solubility of the recombinant protein, and 3) acting as a ligand in affinity purification. In a fusion expression vector, a proteolytic cleavage site is often introduced into the junction between the fusion portion and the recombinant protein, and the fusion portion can be separated from the recombinant portion by purifying the fusion protein. Has been done. Examples of such enzymes and their cognate recognition sites include factor Xa, thrombin and enterokinase.
[0088]
Examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith, DB, Johnson, KS, co-authored, (1988) Gene 67: 31-40), pMAL (New England Biolabs, MA) and pRIT5 (Pharmacia, Piscataway, NJ), each of which fuses glutathione S-transferase (GST), maltose E binding protein, and protein A to the recombinant protein. In one embodiment, the coding sequence of the MCT protein is cloned into a pGEX expression vector to produce a vector encoding the fusion protein, comprising, in order from N-terminus to C-terminus, a GST-thrombin cleavage site-X protein. . The fusion protein is purified by affinity chromatography using glutathione-agarose resin. Recombinant MCT protein removed from the fusion with GST is recovered by cleavage of the fusion protein with thrombin.
[0089]
Suitable inducible non-fusion E. Examples of E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pMPL2, pMc2, pMc2, pM2 pIN-III 113-B1, λgt11, pBdC1, and pET11d (Studier et al., Gene Expression Technology: eds. Vectors Elsevier: New York IBSN 0 444 904018). Expression of the target gene by the pTrc vector is performed by host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Expression of the target gene by the pET11d vector is performed by transcription with a T7 gn10-lac fusion promoter mediated by co-expressing viral RNA polymerase (T7 gnl). This viral polymerase is supplied from host strains BL21 (DE3) or HMS174 (DE3) by a resident phage containing the T7gn1 gene under the transcriptional control of the lacUV5 promoter. For transformation of other various bacteria, suitable vectors can be selected. For example, plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be effective for transformation of Streptomyces, and plasmids pUB110, pC194 or pBD214 are suitable for transformation of Bacillus species. Examples of several plasmids used to transfer genetic information to Corynebacterium are pHM1519, pBL1, pSA77 or pAJ667 (eds. Pouwels et al., (1985) Cloning Vectors. Elsevier: New York IBSN 044404018).
[0090]
One way to maximize expression of a recombinant protein is to express the protein in a host bacterium that is defective in its ability to perform proteolytic cleavage of the recombinant protein (Gottesman, S., Gene Expression). Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another method is to change the nucleic acid sequence of the nucleic acid to be inserted into the expression vector and to use a given bacterium, C. elegans, which is used to express codons corresponding to each amino acid. Preferential use in glutamicum (Wada et al., (1992) Nucleic Acids Res. 20: 2111-2118). Such alterations in nucleic acid sequence are made by standard DNA synthesis techniques.
[0091]
In another embodiment, a yeast expression vector is used as the MCT protein expression vector. Yeast S. Examples of vectors for expression in S. cerevisiae include pYepSec1 (Baldari et al., (1987) Embo J. 6: 229-234), 2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan, Herskow82, ) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods used to construct vectors preferably used for other fungi such as filamentous bacteria are described in van den Honda, C. et al. A. M. J. J. & Punt, P.M. J. .. (1991) "Gene transfer systems and vector development for filamentous fungi, (Applied Molecular Genetics of Fungi) J.F Peberdy et al., Eds., P 1-28, Cambridge University Press: Cambridge, and Pouwels et al., Eds., (1985) Cloning Vectors Elsevier: New York (IBSN 0 444 904018).
[0092]
Furthermore, the MCT protein of the present invention is expressed in insect cells using a baculovirus expression vector. Examples of baculovirus vectors capable of expressing a protein in cultured insect cells (eg, Sf9 cells) include the pAc system (Smith et al., (1983) Mol. Cell. Biol. 3: 2156-2165) and the pVL system ( Lucklow and Summers, (1989) Virology 170: 31-39).
[0093]
In other embodiments, the MCT proteins of the invention can be expressed in unicellular plant cells (eg, algae) or plant cells from higher plants (eg, seed plants such as crops). For examples of plant expression vectors, see Becker, D.W. Kemper, E .; , Schell, J. et al. And Masterson, R.A. Authors, (1992) “New plant binary vectors with selectable markers located proximal to the left border”, Plant Mol. Biol. 20: 1195-1197, Bevan, M .; W. Authors, (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721, and furthermore, pLGV23, pGHlac +, pBIN19, pAK2004, and pDH51 (edited by Pouwels et al., (1985) Cloning Vectors. Elsevier: New York: Examples of IBSN 01844, which are listed as New York). .
[0094]
In another embodiment, the nucleic acids of the invention are expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors are pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al., (EMBO J. 6: 187-195) for use in mammalian cells. The control functions of the expression vector in some cases are often provided by viral regulatory elements, for example, commonly used promoters from polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems in both prokaryotic and eukaryotic cells, see Sambrook, J., Fritsh, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, 16th Edition. , Chapter 17, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
[0095]
In another embodiment, a recombinant mammalian expression vector is capable of directing the expression of a nucleic acid that is selective for a given cell type (eg, the expression of such a nucleic acid requires a tissue-specific regulatory element). Used). Tissue-specific regulatory elements are known in the art. Examples of tissue-specific promoters include albumin promoters (liver-specific: by Pinkert et al., (1987) Genes Dev. 1: 268-277) and lymphoid-specific promoters (by Calame and Eaton, (1988) Adv. Immunol). 43: 235-275), especially T cell receptor specific promoters (Winoto, Baltimore, (1989) EMBO J. 8: 729-733), immunoglobulins (Banerji et al., (1983) Cell 33: 729). -740, Queen, Baltimore, (1983) Cell 33: 741-748), a neuron-specific promoter (eg, the neurofilament promoter, Byrne and Ruddle, (1989) P AS 86: 5473-5577), pancreas-specific promoter (Edlund et al., (1985) Science 230: 912-916), mammary gland-specific promoter (e.g., whey promoter, U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166), but is not limited thereto. Examples of promoters that are regulated during development include the murine (mouse) hox promoter (Kessel, Gruss, (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes, Tilghman, (1989) Genes). Dev. 3: 537-546).
[0096]
The present invention further provides a recombinant expression vector comprising the DNA vector of the present invention cloned in an antisense direction into an expression vector. That is, the DNA molecule is cooperatively linked to a regulatory sequence such that expression (by transcription of the DNA molecule) of an RNA molecule antisense to MCT mRNA is possible. For example, a regulatory sequence is selected that binds cooperatively to the cloned nucleic acid in the antisense orientation, which governs the continuous expression of the antisense RNA molecule in various cell types. Examples of regulatory sequences are viral promoters and / or enhancers. Alternatively, regulatory sequences that govern the constitutive tissue-specific or cell type-specific expression of the antisense RNA may be selected. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus that produces the antisense nucleic acid under the control of a highly efficient regulatory region. The activity in this case is determined by the type of cell into which the vector has been introduced. For the regulation of antisense gene expression, see Weintraub, H. et al. Antisense RNA as a molecular tool for genetic analysis, Reviews @ Trends in Genetics, Vol. 1 (1) 1986.
[0097]
Furthermore, the present invention includes a host cell into which the recombinant expression vector of the present invention has been introduced. The expressions “host cell” and “recombinant host cell” are interchangeable herein. These expressions are used not only for specific cells, but also for those that may develop into next generation cells and cells of later generations. Such later generations may not actually be identical to the parent cell, since modifications may occur in later generations either due to mutations or environmental effects, but again in this case, as described above. Shall be included in the expression.
[0098]
A host cell can be any prokaryotic or eukaryotic cell. For example, the MCT protein is C.I. It is expressed in bacterial cells such as glutamicum, insect cells, yeast or mammalian cells (eg Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Table 3 shows microorganisms related to Corynebacterium glutamicum used as the conventional host cells, which are used for the nucleic acid and protein molecules of the present invention, which can be used as the conventional host cells.
[0099]
Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, transformation (transformation), transfection (transfection), pairing (conjugation), and transduction (transduction) refer to foreign nucleic acids (eg, linear DNA or RNA (eg, linearized vectors). Or various known techniques for introducing a vector-like nucleic acid (eg, a plasmid, a phage, a phsmid, a phagemid, a transporon or other DNA) into a host cell, such as calcium phosphate or calcium chloride. Shows co-precipitation, DEAE-dextran mediated transfection, lipofection, natural competence, chemical mediated transfer, or electroporation. Suitable methods for the transformation or transfection of host cells can be found in Sambrook et al., (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, Nd., USA). And other research manuals.
[0100]
For stable transfection of mammalian cells, it is known that foreign DNA may be integrated into the genome by a small portion of the cell, depending on the expression vector and transfection technique used. To recognize and select all of these components, a gene encoding a selectable marker (eg, resistance to an antibiotic) is introduced into the host cell along with the desired gene. Examples of suitable markers include those that confer resistance to the drug, such as G418, hygromycin, methotrexate. The nucleic acid encoding the selectable marker can be introduced into the same vector as the vector encoding the MCT protein in the host cell, but can also be introduced into another vector. Cells stably transfected with the introduced nucleic acid are recognized by drug selection or the like (for example, cells into which a selectable marker gene has been introduced are maintained, and other cells die).
[0101]
In order to obtain a homologous recombinant microorganism, a vector containing at least a part of the MCT gene into which a deletion, addition, or substitution has been introduced is produced, and thereby the MCT gene is changed due to, for example, functional disruption. The MCT gene is preferably the Corynebacterium glutamicum MCT gene, but may be homologous to the relevant bacteria or mammals, yeasts or insects. In a preferred embodiment, the vector is preferably designed such that upon homologous recombination, the endogenous MCT gene is functionally disrupted (ie, designed to not encode a functional protein. Are also known as "knockout" vectors). Alternatively, after homologous recombination, the endogenous MCT gene is mutated or altered but remains functionally encoding (eg, an upstream regulatory region is altered, thereby altering the endogenous MCT protein). It is also possible to design the vector to alter expression). In a homologous recombination vector, additional nucleic acids are placed 5 'and 3' to the side of the MCT gene, resulting in a modified portion. This vector allows for homologous recombination between the exogenous MCT gene provided by the vector and the endogenous MCT in the microorganism. This additionally laterally located MCT nucleic acid is of sufficient length to allow for sufficiently homologous recombination with foreign genes. Usually, several kb of laterally arranged DNA (both at the 5 'and 3' ends) is included in the vector (Thomas, KR and Capecchi, MR, co-authored, (1987) Cell 51. : 503 for a description of homologous recombination vector). The vector is introduced into a microorganism (eg, electroporation), and cells having a combination of the introduced MCT gene and the endogenous MCT gene are selected by known techniques.
[0102]
In another embodiment, a recombinant microorganism is produced having a selected system that allows for regulated expression of the introduced gene. By introducing the MCT gene into a vector under the control of the lac operon, it becomes possible to express the MCT gene only when IPTG is present. Such adjustment systems are known from the prior art.
[0103]
In other embodiments, the endogenous MCT gene in the host cell is disrupted (by homologous recombination or other genetic means by conventional techniques) so that the resulting protein product is not expressed. In other embodiments, the endogenous or introduced MCT gene in the host cell has been altered by one or more point mutations, deletions or inversions, but encodes a functional MCT protein. In still other embodiments, one or more regulatory regions (eg, promoter, repressor, or inducer) of the MCT gene in the microorganism are altered (eg, deleted) such that expression of the MCT gene is regulated. Loss, truncation, inversion, point mutation). Those skilled in the art will readily recognize that host cells having one or more of the above-described MCT gene and protein modifications are readily produced by the methods of the present invention and are also included in the present invention.
[0104]
MCT proteins can be produced (ie, expressed) using the host cells of the invention, eg, prokaryotic or eukaryotic cells in culture. Accordingly, the present invention further provides a method for producing an MCT protein using the host cell of the present invention. In a preferred embodiment, the method comprises the steps of introducing a cell of the invention (recombinant expression vector encoding the MCT protein or genome) into the wild-type or modified MCT protein until the MCT protein is obtained. Are cultured in a suitable medium. In another embodiment, the method of the invention comprises the step of isolating the MCT protein from the medium or the host cell.
[0105]
C. MCT protein isolated
The invention further includes the isolated MCT protein, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cytoplasmic material when produced by recombinant DNA technology, and when chemically synthesized. Is substantially free of chemical precursors or other chemicals. By "substantially free of cellular material" is meant the preparation of MCT proteins, produced by natural or recombinant techniques, separated from cellular components in cells. In one embodiment, "substantially free of cellular material" refers to less than about 30% (by dry weight) non-MCT protein (also referred to as "contaminating protein"), preferably about 20% Less than, more preferably less than about 10%, particularly preferably less than 5%. If the MCT protein or a biologically active part thereof can be produced recombinantly, it is preferred that it is substantially free of medium. That is, it is preferred that the medium comprises less than 20% of the volume of protein production, more preferably less than about 10%, and particularly preferably less than about 5%. In one embodiment, the phrase "substantially free of chemical precursors or other chemicals" refers to "less than about 30% (dry weight), preferably less than about 20%, more preferably about 10%. Refers to the preparation of an MCT protein containing less than about 5%, particularly preferably less than about 5%, of a chemical precursor or non-MCT chemical, wherein the isolated protein or biologically active portion thereof is Preferably, it is free of contaminating (source) proteins from the same organism as that produced, generally such proteins are produced by recombinant expression of the C. glutamicum MCT protein or the like in a microorganism such as C. glutamicum.
[0106]
The isolated MCT protein of the present invention, or a portion thereof, may be a C.I. It is involved in the metabolism of compounds required for the formation of the cell membrane of Glutamicum, or the transport of molecules across this membrane, or has the activities listed in Table 1. The protein or a part thereof contains an amino acid sequence sufficiently homologous to a sequence of the present invention (for example, a sequence according to an even SEQ ID NO in the sequence listing). Preferably, Glutamicum retains its ability to participate in the metabolism of compounds necessary for the formation of the cell membrane or the transport of molecules across the membrane. It is preferred that a part of the protein is a part having the above-mentioned biological activity. In another preferred embodiment, the MCT protein of the present invention has an even amino acid sequence according to SEQ ID NO in the sequence listing. In another preferred embodiment, the MCT protein is encoded by a nucleotide sequence that hybridizes to a nucleotide sequence of the invention (eg, a sequence according to an odd SEQ ID NO in the sequence listing), eg, hybridizes under stringent conditions. . In still other preferred embodiments, the MCT protein is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% relative to a nucleic acid sequence of the invention or a portion thereof. %, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least About 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, 91%, 92%, 93%, 94%, particularly preferably encoded by a nucleotide sequence that is at least about 95%, 96%, 97%, 98%, 99% or more homologous Having the amino acid sequence shown. The present invention also includes a range of values represented by intermediate values of the above values (for example, 70 to 90% identical or 80 to 95% identical). For example, any of the above-described values described as the upper limit value and / or the lower limit value may be combined to form a value range. Preferably, the MCT proteins of the present invention have at least one MCT activity described herein. For example, an MCT protein of the present invention is substantially homologous to an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleic acid sequence of the present invention, eg, hybridizes under stringent conditions; It is preferred that it is capable of participating in the metabolism of compounds required for the construction of the cell membrane of Glutamicum or the transport of molecules across this membrane, or has one or more of the activities listed in Table 1.
[0107]
In a preferred embodiment, the MCT protein is substantially homologous to an amino acid sequence of the invention (eg, a sequence according to an even SEQ ID NO in the sequence listing), as described in detail in column I above, Although the amino acid sequence differs due to natural changes and mutations, it retains the functional activity of the protein. In another embodiment, the MCT protein comprises at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% of the total amino acid sequence of the invention. , 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89% or 90%, 91%, 92%, 93%, 94%, particularly preferably at least about 95%, 96%, 97%, 98%, 99% or more of the above-mentioned MCT A protein that contains any of the activities. The present invention also includes a range of values represented by intermediate values of the above values (for example, 70 to 90% identical or 80 to 95% identical). For example, any of the above-described values described as the upper limit value and / or the lower limit value may be combined to form a value range. The present invention relates to C. elegans substantially homologous to the entire amino acid sequence of the present invention. Contains the entirety of glutamicum protein.
[0108]
The biologically active portion of the MCT protein may be derived from the amino acid sequence derived from the amino acid sequence of the MCT protein (eg, the amino acid sequence according to the even SEQ ID NO in the sequence listing) or the amino acid sequence of a protein homologous to the MCT protein. It contains the derived amino acid sequence (comprising less than the whole amino acid in the MCT protein or including the whole protein homologous to the MCT protein) and exhibits at least one activity of the MCT protein.
[0109]
Generally, biologically active moieties (peptides, such as peptides of 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) are MCT Contains at least one activity domain or motif of the protein. In addition, other biologically active proteins in which other portions of the protein have been deleted can be produced recombinantly and evaluated for one or more of the activities described herein. It is possible. The biologically active portion of an MCT protein is one or more selected domains / motifs or portions thereof that have biological activity.
[0110]
Preferably, the MCT protein is produced by recombinant DNA technology. For example, a nucleic acid molecule encoding a protein is cloned into an expression vector (see above), the expression vector is introduced into a host cell (see above), and the MCT protein is expressed in the host cell. MCT protein is isolated from cells by a suitable purification method using standard protein purification techniques. In methods other than recombinant expression, MCT proteins, polypeptides, or peptides are chemically synthesized using standard peptide synthesis techniques. In addition, native MCT protein can be isolated from cells (eg, endothelial cells) using, for example, anti-MCT antibodies produced by standard techniques using the MCT protein of the present invention or fragments thereof.
[0111]
The present invention further provides MCT chimeric or fusion proteins. Here, the MCT “chimeric protein” or “fusion protein” includes an MCT polypeptide that cooperatively binds to a non-MCT polypeptide. MCT “polypeptide” means a polypeptide having an amino acid sequence corresponding to MCT, and “non-MCT polypeptide” is a protein having no substantial homology to MCT protein, such as MCT protein, Or a polypeptide having an amino acid sequence corresponding to a protein derived from a different organism. With respect to a fusion protein, "cooperatively linked" means that the MCT and non-MCT polypeptides are fused together in frame. Non-MCT polypeptides can be fused to the C-terminal or N-terminal of the MCT polypeptide. For example, an example of a fusion protein is a GST-MCT fusion protein in which the MCT sequence is linked to the C-terminus of the GST sequence. Such a fusion protein is effectively used for purification of a recombinant MCT protein. In another embodiment, an MCT containing a heterologous signal sequence at the N-terminus is used as the fusion protein. In certain host cells (eg, mammalian host cells), expression and / or secretion of the MCT protein is improved by using a heterologous signal sequence.
[0112]
The MCT chimeric or fusion proteins of the present invention are produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together by conventional techniques. For example, treatment with blunt or cohesive ends for ligation, digestion with restriction enzymes to obtain preferred ends, packing to obtain suitable sticky ends, alkaline phosphatase treatment to avoid improper ligation, and enzymes It is preferred to be carried out by ligation. In another embodiment, the fusion gene is synthesized by conventional techniques, such as by using an automatic DNA synthesizer. In other cases, PCR amplification of the gene fragment is performed using an anchor primer that creates a complementary overhang between two parallel gene fragments. The two gene fragments in this case are subsequently annealed and reamplified to produce a chimeric gene sequence (eg, Molecular Biology, Ausubel et al., John Wiley & Sons: 1992). In addition, various expression vectors are commercially available that previously encode a fusion moiety (eg, a GST polypeptide). The nucleic acid encoding MCT is cloned into an expression vector as described above such that the fusion moiety binds in-frame to the MCT protein.
[0113]
Homologs of the MCT protein are generated by mutagenesis, eg, scattered point mutation or truncation of the MCT protein. As used herein, the term "homolog" refers to a variant of the MCT protein that acts as an agonist or antagonist of MCT protein activity. An agonist of the MCT protein can maintain a biological activity or subset that is equivalent to the MCT protein. MCT protein antagonists prevent the occurrence of translocation by competitively binding to downstream or upstream members of the cell membrane component metabolic cascade, including the MCT protein, or by binding to the MCT protein, which mediates the transport of compounds across the membrane can do.
[0114]
In another embodiment, homologs of the MCT protein are identified by screening a combinatorial library of mutant, eg, truncated, mutants of the MCT protein to obtain MCT protein agonist or antagonist activity. In one embodiment, a variant library of MCT variants is obtained by combinatorial mutagenesis at the nucleic acid level and is encoded by a variant gene library. A variant library of MCT variants may be, for example, a mixture of synthetic oligonucleotides in a gene sequence, a series of variants that may constitute the MCT sequence as individual polypeptides, or a large fusion protein containing a series of MCT sequences. (Eg, phage display) produced by enzymatic ligation. Various methods are used to form a library that may constitute an MCT homolog from the degraded oligonucleotide sequence. Chemical synthesis of the degraded gene sequence is performed in an automatic DNA synthesizer, and the synthesized gene is ligated to an appropriate expression vector. By using the degraded genes as a set, all sequences encoding the desired set that may constitute the MCT sequence are provided as a mixture. Methods for synthesizing degraded oligonucleotides are known in the art (eg, Narang, SA, (1983) Tetrahedron 39: 3, Itakura et al., (1984) Annu. Rev. Biochem. 53: 323). (1984) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477).
[0115]
Furthermore, it is possible to produce a heterogeneous population of MCT fragments by screening and selecting homologues of the MCT protein using the fragment library encoding the MCT protein. In another embodiment, the library of coding sequence fragments is obtained in the following manner. That is, a double-stranded PCR fragment of the MCT coding sequence is treated with a nuclease under conditions in which nicking occurs only once for each molecule to denature the double-stranded DNA, and a sense is obtained from a product having different nicking. / Restore the DNA containing the antisense pair to form double-stranded DNA, remove single-stranded proteins from the re-formed double strand by treatment with S1 nuclease, and express the resulting fragment library Ligation to the vector. By this method, an expression library is obtained which encodes N-terminal, C-terminal and internal fragments of MCT proteins of various sizes.
[0116]
A plurality of techniques for screening gene products of a combination library obtained by point mutation or truncation and screening a cDNA library of a gene product having predetermined properties are known. Such techniques are used for rapid screening of gene libraries obtained by combinatorial mutagenesis of MCT homologs. This most widely used method is used for high-throughput analysis to screen large gene libraries. In this method, generally, a gene library is cloned into a replicable expression vector, an appropriate cell is transformed to obtain a library of vectors, and a vector encoding the gene is isolated by detecting a desired function. The recombinant gene is expressed under conditions where the product from is detected. A new technique for increasing the incidence of recursive variants in libraries, namely recursive ensemble mutagenesis (REM), can be used in combination with screening methods to identify MCT homologs (Arkin, Youvan (1992) PNAS 89: 7811-7815, Delgrave et al., (1993) Protein Engineering 6 (3): 327-331).
[0117]
In another embodiment, a cell-based analysis is performed and the variant MCT library can be analyzed using known methods.
[0118]
D. Methods and uses of the invention
The nucleic acid molecules, proteins, protein homologs, homologous proteins, primers, vectors, and host cells described herein are used by one or more methods below. That is, C.I. Identification (recognition) of glutamicum and related organisms, C.I. Mapping of organisms related to G. glutamicum; Identification and location of glutamicum, study of evolution, determination of MCT protein region required for function, regulation of MCT protein activity, regulation of metabolism of one or more cell membrane components, transmembrane of one or more compounds It is used in regulating transport and in regulating the production of desired compounds, for example fine chemicals, by cells.
[0119]
The MCT nucleic acid molecules of the present invention have various uses. First, the molecule is used to identify Corynebacterium glutamicum or an organism closely related thereto. In addition, MCT nucleic acid molecules can be found in C. elegans in mixed populations of microorganisms. Used to recognize the presence of glutamicum or an organism closely related thereto. In addition, the present invention provides a method for transforming a genomic DNA extracted from a culture of a single or mixed population of microorganisms into C. elegans under stringent conditions. The presence of this organism is confirmed by examining with a probe spanning a region specific to the glutamicum gene.
[0120]
Corynebacterium glutamicum itself is not pathogenic, but has relevance to pathogenic species such as Corynebacterium diphtheria. Corynebacterium diphtheria is the causative agent of diphtheria and grows rapidly, causing acute and febrile infections associated with both local and systemic pathologies. In this disease, local lesions reach the upper respiratory tract and necrotic damage reaches the endothelial cells, bacilli secrete toxins, and the lesions spread to sensitive tissues distal to the body. Inhibition of protein synthesis in these tissues, such as the heart, muscle, peripheral nerves, adrenal glands, kidneys, liver and spleen, results in deformable changes, which are indicative of a systemic pathology of the disease. Diphtheria has a high incidence in many parts of the world, such as Africa, Asia, Eastern Europe and the former Soviet Union. Subsequent epidemics of diphtheria in the last two regions have resulted in 5,000 deaths since 1990.
[0121]
In one embodiment, the present invention provides a method for recognizing (identifying) the presence of Corynebacterium diphtheria activity in a subject (subject / patient). In this method, the presence of one or more activities of a nucleic acid or amino acid sequence of the invention (eg, an odd or even SEQ ID NO nucleotide sequence in the sequence listing) is detected in a subject, thereby providing a Corynebacterium diphtheria. Detect the presence or activity of. C. Glutamicum and C.I. Diphtheria is a related bacterium, Many of the nucleic acid and protein molecules of G. glutamicum are C. glutamicum. This method is homologous to nucleic acid and protein molecules of diphtheria, and therefore, the method is useful for C. elegans in a subject. It can also be used to detect diphtheria.
[0122]
The nucleic acid and protein molecules of the present invention also serve as markers for specific regions of the genome. This is not limited to genomic mapping, but also to C.I. It is also used for research on the function of glutamicum protein. For example, a specific C.I. To identify the region of the genome to which the glutamicum DNA-binding protein binds, C. glutamicum DNA-binding protein is It is possible to digest the glutamicum genome, but the fragments are incubated with the DNA-binding protein. The region that binds to the protein can also be examined in detail using the nucleic acid of the present invention, preferably with a label that is easily detectable. The location of the fragment in the glutamicum genomic map is made possible, and the nucleic acid sequence to which the protein is bound can be quickly determined by multiple attempts with various enzymes. In addition, the nucleic acid molecules of the invention have sufficient homology to the sequence of the relevant species and thus act as markers for the structure of the genomic map in related bacteria, such as Brevibacterium lactofermentum.
[0123]
The MCT protein molecules of the present invention are also useful in evolutionary and protein structural studies. The metabolic and transport processes to which the molecules of the present invention are related compare the sequence of the nucleic acid molecule of the present invention to the sequence of a molecule encoding a similar enzyme obtained from another organism, and the evolutionary relevance of this organism. Used in a variety of prokaryotic and eukaryotic cells. Similarly, such comparisons allow one to evaluate which parts of the sequence are converted and which are not, and are used to identify protein regions that are important for enzyme function. Such a specific method is important in protein engineering, and serves as an indicator of what resistance can be obtained by mutagenesis of a protein that mutates without losing its function.
[0124]
By processing the MCT nucleic acid molecule of the present invention, it becomes possible to produce an MCT protein having a function different from that of a wild-type MCT protein. With such a protein, it is also possible to improve its efficiency or activity, to make it present in a cell in a higher number than usual, or to reduce its efficiency or activity.
[0125]
The present invention provides a method for screening a molecule by measuring the activity of the MCT protein by interacting with the protein itself, a substrate or a binding target of the MCT protein, or regulating the transcription or translation of the MCT nucleic acid molecule of the present invention. In this method, a microorganism expressing one or more MCT proteins of the present invention is contacted with one or more test compounds, and the effect of each test compound on MCT protein activity or expression level is evaluated.
[0126]
The modification of the MCT protein of the present invention is characterized by the fact that the modified CCT protein incorporates the modified protein. There are a number of mechanisms directly involved in the yield, production and / or production efficiency of the fine chemical obtained from the glutamicum strain. C. Recovery of fine chemical compounds from large scale glutamicum cultures has been greatly improved. This is C.I. This is because when glutamicum produces the desired compound (as opposed to extraction when C. glutamicum cells are produced in large quantities), such compound is easily purified from the culture medium. By increasing the number or activity of transporter molecules that release fine chemicals from cells, it is possible to increase the amount of fine chemicals in the extracellular medium, thereby significantly facilitating recovery and purification. Conversely, to efficiently overproduce one or more fine chemicals, a suitable biosynthetic pathway requires the use of increasing amounts of cofactors, precursor molecules and intermediate compounds. Thus, by increasing the number and / or activity of transporter proteins involved in the introduction of carbon sources (eg, sugars), nitrogen sources (eg, amino acids, ammonium salts), phosphates (salts) and sulfur sugar nutrients, There are no restrictions on the nutrient supply in the synthesis process, which will lead to improved fine chemical manufacturing. Furthermore, because the fatty acids and lipids themselves are the desired fine chemicals, by optimizing or increasing the activity of one or more MCT proteins of the invention involved in the biosynthesis of these compounds, or By reducing the activity of MCT protein involved in the degradation of the compound of It is possible to improve the yield, production and / or production efficiency of glutamicum-derived fatty acids and lipid molecules.
[0127]
By processing one or more MCT genes of the present invention, it is possible to obtain an MCT protein having an altered activity. It has an indirect effect on the production of one or more desired fine chemicals from glutamicum. For example, the normal biosynthetic process of metabolism affects the production of various effluents (eg, hydrogen peroxide and other reactive oxygen species) and may positively affect similar metabolic processes ( For example, peroxynitrile is known as a nitrate tyrosine side chain, and the presence of tyrosine in the active site inactivates several enzymes (Groves, JT. (1999) {Cuur. Opin. Chem. Biol). 3 (2): 226-235) In large-scale culture production, when the C. glutamicum strain used is regulated so as to overproduce one or more fine chemicals, the wild type C. glutamicum is usually More excretion is secreted than is seen.The activity of one or more MCT proteins of the invention involved in the release of excretion molecules The regulation can improve cell viability and maintain efficient metabolic activity, and the presence of the desired fine chemical at a high intracellular concentration is actually harmful to the cell. In some cases, therefore, by improving the ability of cells to secrete these compounds, it is possible to improve cell viability.
[0128]
Furthermore, the MCT proteins of the present invention can be processed to change the relative amounts of the various lipid and fatty acid molecules produced. This can have a significant effect on the lipid composition of cell membranes. Since the various lipids have different physical properties, changing the lipid composition in the membrane significantly changes the fluidity of the membrane. Changes in the fluidity of the membrane affect the transport of molecules across the membrane, which in turn alters the release of effluents or manufactured fine chemicals and the introduction of required nutrients as described above. In addition, changes in membrane fluidity can have a significant effect on cell integrity, and cells with relatively weak membranes are sensitive to mechanical properties in large-scale fermentation environments, which can lead to cell damage or death. May be brought. The MCT protein involved in the production of fatty acids or lipids constituting the membrane is manipulated such that the composition of the obtained membrane is susceptible to the environmental conditions in the culture used for the production of fine chemicals, whereby C . Glutamicum will survive and proliferate more. More C. in cultures The presence of glutamicum leads to a fine chemical yield, production or production efficiency from the culture.
[0129]
C. Mutagenesis of the MCT protein that increases the yield of fine chemicals obtained from Glutamicum is not limited to the above, and it will be apparent to those skilled in the art that these procedures can be modified. By using these methods and incorporating the above mechanism w, the nucleic acid and protein molecule of the present invention can be transformed into C. elegans. A glutamicum or related bacterial strain expressing a mutant MCT nucleic acid and protein molecule is obtained with improved yield, production and / or production efficiency of the desired compound. The desired compound is C.I. It may be a natural product of G. glutamicum, such as an end product of a biosynthetic pathway, an intermediate of a naturally occurring metabolic pathway, or C. Although it does not occur naturally in the metabolism of glutamicum, the C. glutamicum of the present invention does not. Includes molecules produced by the Glutamicum strain.
[0130]
Examples of the present invention will be described below, but the present invention is not limited to these. The references, patent applications, patents, patent publications, tables, and sequence listings set forth herein are incorporated herein by reference.
[0131]
[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Table 28]

[Table 29]

[Table 30]

[Table 31]

[Table 32]

[Table 33]

[Table 34]

[Table 35]

[Table 36]

[Table 37]

[Table 38]

[Table 39]

[Table 40]

[Table 41]

[Table 42]

[Table 43]

[Table 44]

[Table 45]

[Table 46]

[Table 47]

[Table 48]

[Table 49]

[0132]
[Example]
Example 1: Production of whole genomic DNA of Corynebacterium glutamicum ATCC13032
Cultivation of Corynebacterium glutamicum (ATCC 13032) was carried out at 30 ° C. overnight in a vigorously stirred BHI medium (Difco). The cells were harvested by centrifugation and the supernatant was discarded, and the cells were treated with 5 ml of Buffer I (5% of the initial culture volume, all indicated volumes were calculated for a 100 ml culture volume) ). The composition of Buffer I was 140.34 g / l sucrose, 2.46 g / l MgSO₄x7H₂O, 10 ml / l KH₂PO₄Solution (100 g / l, adjusted to pH 6.7 with KOH), 50 ml / l M12 concentrate (10 g / l (NH₄)₂SO₄1 g / l NaCl, 2 g / l MgSO₄x7H₂O, 0.2 g / l CaCl₂0.5 g / l yeast extract (Difco), 10 ml / l trace-elements-mix (200 mg / l FeSO₄xH₂O, 10 mg / l ZnSO₄x7H₂O, 3 mg / l MnCl₂x4H₂O, 30 mg / l H₃BO₃, 20 mg / l CoCl₂x6H₂O, 1mg / l NiCl₂x6H₂O, 3 mg / l Na₂MoO₄x2H₂O, 500 mg / l complexing agent (EDTA or citric acid), 100 ml / l vitamin-mix (0.2 mg / l biotin, 0.2 mg / l folic acid, 20 mg / l p-aminobenzoic acid, 20 mg / l riboflavin, 40 mg / l calcium pantothenate, 140 mg / l nicotinic acid, 40 mg / l pyridoxol hydrochloride, 200 mg / l myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg / ml. After culturing at 37 ° C. for about 4 hours, the protoplasts obtained by degrading the cell wall were collected by centrifugation. The pellet was washed once with 5 ml Buffer I and once with 5 ml TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml of TE buffer and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5M) were added. After adding proteinase K to a final concentration of 200 μg / ml, the suspension was incubated for about 18 hours at 37 ° C. DNA was purified by standard procedures by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol. The DNA was then precipitated by adding 1/50 volume of 3M sodium acetate and 2 volumes of ethanol, then incubated for 30 minutes at -20 ° C and centrifuged at 12000 rpm in a high speed centrifuge using an SS34 rotor (Sorvall). Separation was performed for 30 minutes. The DNA was dissolved in 1 ml of TE buffer containing 20 μg / ml RNase A and dialyzed at 4 ° C. for at least 3 hours against 1000 ml of TE buffer. During this time, the buffer was changed three times. To the 0.4 ml portion of the dialyzed DNA solution, 0.4 ml of 2M LiCl and 0.8 ml of ethanol were added. After incubating for 30 minutes at -20 ° C, the DNA was collected by centrifugation (13000 rpm, Biofuge @ Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE buffer. The DNA produced by this procedure could be used for all purposes, Southern blotting or construction of genomic libraries.
[0133]
Example 2 Construction of Genomic Library of Corynebacterium glutamicum ATCC13032 in Escherichia Coli
Using DNA produced as described in Example 1, cosmid and plasmid libraries were constructed according to known and well-established methods (eg, Sambrook, J et al., (1989) 'Molecular Cloning: A Laboratory Manual ', Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994)' Current Protocols in Molecular Biology ', John Wileyand.
[0134]
Any plasmid or cosmid could be used. In particular, plasmid pBR322 (Sutcliffe, JG (1979) Proc. Natl. Acad. Sci. USA, 75: 3737-3741); pACYC177 (Change and Cohen (1978) J. Bacteriol 134: 1141-1156), pBS series of plasmids. (pBSSK +, pBSSK- and others; Stratagene LaJolla, USA), or SuperCos1 (Stratagene LaJolla, USA), Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H (1987) Gene 53: 283-286) was used. In particular, C.I. Gene libraries for use in G. glutamicum can be constructed using plasmid pSL109 (Lee, HS and AJ Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
[0135]
Example 3: DNA sequence and functional analysis by computer
The genomic library described in Example 2 was prepared using standard methods, in particular, a chain growth termination reaction using an ABI377 sequence machine (eg, Fleischman, RD, et al. (1995) 'Whole-genome Random Sequencing and Assembly of Haemophilus). Influenzae Rd. ', Science, 269: 496-512). Sequence primers of the following nucleotide sequence were used: 5'-GGAAACAGTATGACCATG-3 'or 5'-GTAAAACGACGGCCAGT-3'.
[0136]
Example 4 In Vivo Mutagenesis
In vivo mutagenesis of Corynebacterium glutamicum has been described in E. coli. E. coli or other microorganisms (eg, yeast such as Bacillus spp. or Saccharomyces cerevisiae), which have a reduced ability to maintain the integrity of their genetic information, by passage through a plasmid (or other vector). be able to. Typical mutator strains include DNA repair systems (see, for example, mutHLS, mutD, mutT, and others; Rupp, WD (1996) DNA repair mechanisms, Escherichia coli and Salmonella, p2277-2294, ASM: Washingon gene). Have a mutation. Such strains are well-known to those skilled in the art. The use of such strains is described, for example, in Greener, A .; And Callahan, M .; (1994) Strategies 7: 32-34.
[0137]
Example 5: DNA conversion between Escherichia coli and Corynebacterium glutamicum
Some Corynebacterium and Brevibacterium species contain an endogenous plasmid (eg, pHM1519 or pBL1) that replicates autonomously (see, eg, Martin, JF et al. (1987) Biotechnology, 5: 137-146). . The shuttle vector of Escherichia coli and Corynebacterium glutamicum is E. coli. coli standard vectors (Sambrook, J. et al. (1989), 'Molecular Cloning: A Laboratory Manual', Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. , John Wiley and Sons) and can be readily constructed by adding markers for initiation or replication and appropriate Corynebacterium glutamicum. Such an origin of replication preferably comes from an endogenous plasmid isolated from Corynebacterium and Brevibacterium species. Transformation markers for these species include kanamycin resistance gene (derived from the Tn5 or Tn903 transposon) or chloramphenicol resistance gene (Winnacker, EL (1987) 'From Genes to Clones-Introduction to Gene). (Technology, VCH, Weinheim) are particularly used. E. FIG. coli and C.E. The literature on the extensive construction of shuttle vectors that replicate in both G. glutamicum is numerous and can be used for several purposes, including gene overexpression (eg, Yoshihama, M. et al. (1985)). J. Bacteriol. 162: 591-597, Martin, JF et al. (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ. Et al. (1991) Gene, 102: 93-98. ).
[0138]
Using standard methods, the gene of interest can be cloned into one of the shuttle vectors as described above, and such a hybrid vector can be introduced into a Corynebacterium glutamicum strain. . C. glutamicum was transformed by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53: 399-303) and, if special vectors are used, can be achieved by conjugation (eg, as described in Schaeffer, A et al. (1990) J. Bacteriol. 172: 1663-1666). C. production of plasmid DNA from C. glutamicum (using well-known standard methods) and its E. coli. C. by transformation into E. coli. glutamicum from E. coli. The transfer of the shuttle vector to E. coli is also possible. This transformation step can be performed using standard methods, but can include MCT-deficient E. coli. It is advantageous to use E. coli strains, such as NM522 (Gough and Murray (1983) J. Mol. Biol. 166: 1-19).
[0139]
The gene may be pCG1 (U.S. patent No. 4617267) or a fragment thereof, optionally TN903 (Grindley, ND and Joyce, CM (1980) Proc. Natl. Acad. Scad. Sci. USA 77 (12). C.) using a plasmid containing the kanamycin resistance gene from 7176-7180). glutamicum strain can be overexpressed. In addition, the gene was cloned using the plasmid pSL109 (Lee, H.S. and AJ Sinskey (1994) J. Microbiol Biotechnol. 4: 256-263). It can be overexpressed in Glutamicum strains.
[0140]
In addition to replicating plasmids, overexpression of the gene can be achieved by integration into the genome. C. Gene integration in G. glutamicum or other Corynebacterium or Brevibacterium species can be performed by well-known methods, such as homologous recombination in genomic regions, restriction endonuclease-mediated integration (REMI) (eg, DE patent 198238334) or use of transposons. And so on. Regulatory regions (eg, promoters, repressors, and / or enhancers) can be modified, inserted, deleted by site-directed (eg, homologous recombination), or random event-based methods (eg, transposon mutagenesis or REMI). ) Can change the activity of the gene of interest. A nucleic acid sequence that functions as a transcription terminator can also be inserted 3 'into the coding region of one or more genes of the invention. Such terminators are well known and are described, for example, in Winnacker, E .; L. (1987) From Genes to Clones-Induction to Gene Technology. VCH: described in Weinheim.
[0141]
Example 6: Evaluation of mutant protein expression
Observation of the activity of the mutein in the transformed host cell is based on the fact that the mutein is expressed in a similar form and in a similar amount to the wild-type protein. A method useful for confirming the level of transcription of a mutant gene (an indicator of the amount of mRNA used for transcription for a gene product) is described in Northern blotting (Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York), a primer designed to bind to the gene of interest, uses detection tags (usually radioactive or chemiluminescent) to extract total RNA from the culture of the organism, run it on a gel, and migrate to a stable matrix And when incubated with the probe, the binding and the amount of binding of the probe are labeled to indicate the presence and amount of mRNA for this gene. This information is evidence of the degree of transcription of the mutated gene. Whole cell RNA can be obtained by several well-known methods, such as those described in Bormann, E .; R. et al. (1992) Mol. Microbiol. 6: 317-326, can be produced from Corynebacterium glutamicum.
[0142]
Standard techniques such as the Western Brod method can be used to assess the presence and comparative amount of proteins translated from this mRNA (eg, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New). York). In this method, whole cell proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe such as an antibody that specifically binds to the designed protein. The probe is generally tagged with a readily detectable chemiluminescent or colorimetric label. The presence and amount of the label observed indicates the presence and amount of the desired mutein in the cell.
[0143]
Example 7: Growth of genetically modified Corynebacteria-media and culture conditions
Genetically modified Corynebacterium glutamicum is cultured in synthetic or natural growth media. Many of the different growth media of Corynebacteria are both known and are readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32: 205-210; von der Osten et al. 1998) Biotechnology Letters, 11: 11-16; Patent DE 4,120,867; Liebl (1992).
'The Genus Corynebacterium, in: The Procaryotes, Volume II, Barrows, A .; et al. , Eds. Spring-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars such as mono-, di- or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply the medium with sugar by complex compounds such as molasses or other by-products of sugar refining. It is also advantageous to supply a mixture of different carbon sources. Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid, lactic acid. The nitrogen source is usually an organic or inorganic nitrogen compound or a material containing these compounds. As a nitrogen source, for example, ammonia gas, ammonium salt (eg, NH₄Cl or (NH₄)₂SO₄), NH₄Complex nitrogen sources such as OH, nitrate, urea, amino acids, corn steep liquor, soy flour, soy protein, yeast extract, meat extract and the like are included.
[0144]
The inorganic salt compound contained in the medium includes chloride, phosphoric acid, and calcium, magnesium, sodium, cobalt, molybdenum, potassium, magnesium, zinc, copper, and iron salts of sulfuric acid. Chelate compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols such as catechol and protocatechuate or organic acids such as citric acid. Mediums containing growth factors or growth promoters such as vitamins, for example, biotin, riboflavin, thiamine, folate, nicotinic acid, pantothenate, pyridoxine and the like are typical. Growth factors and salts are often derived from complex media components such as yeast extract, molasses, corn steep liquor. The exact composition of the media compound depends heavily on direct experimentation and is determined individually in each particular case. Information on media optimization can be found in the textbook Applied Microbiol. Physiology, A Practical Approach (eds. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 019 9635773). It is also possible to select the growth medium from commercial products, such as standard 1 (Merck) or BHI (grain heart infusion, DIFCO).
[0145]
All media components are heat (1.5 × 10⁵(Pa, 121 ° C. for 20 minutes) or filtration sterilization method. The components can be sterilized together or, if desired, in portions. All media components can be present at the beginning of growth or optionally added continuously or batchwise.
[0146]
Culture conditions are defined separately for each experiment. The temperature should be in the range 15 ° C to 45 ° C. The temperature can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be kept constant by adding a buffer to the medium. An example of a buffer for this purpose is a potassium phosphate buffer. MOPS, HEPES, ACES and other synthetic buffers can be used alternatively or simultaneously. NaOH or NH during growth₄It is also possible to maintain a constant culture pH by adding OH. If a complex media component such as yeast extract is used, no additional buffer may be needed due to the fact that many complex components have a high buffer capacity. When a fermenter is used for culturing microorganisms, the pH can be controlled by ammonia gas.
[0147]
Incubation times usually range from hours to days. This time is chosen to maximize the product that accumulates in the broth. The disclosed growth experiments can be performed in various vessels, such as microtiter plates, glass tubes, glass flasks, or various sizes of glass or metal fermenters. For screening of large numbers of clones, the microorganisms should be cultured in microtiter plates, glass tubes, shake flasks, with or without baffles. Preferably a 100 ml shake flask is used and is filled with 10% by volume of the desired growth medium. The flask should be shaken on a rotary shaker (25 mm shake width) using a speed range of 100 to 300 rpm. Evaporation loss can be reduced by keeping it in a humid atmosphere. Instead, a mathematical correction of the evaporation loss should be made.
[0148]
When testing genetically modified clones, unmodified control clones or control clones containing the basic plasmid without any insertions should also be tested. The medium is placed on an agar plate, for example a CM plate (10 g / l glucose, 2.5 g / l NaCl, 2 g / l urea, 10 g / l polypeptone, 5 g / l yeast extract, 5 g / l meat extraction OD of 0.5-1.5 using cells grown on agar, 22 g / l agar, pH 6.8 with 2M NaOH) at 30 ° C.₆₀₀Inoculate until The medium was inoculated with C.I. This is accomplished either by introducing a G. glutamicum cell line suspension or by adding a pre-culture of this bacterium.
[0149]
Example 8: In vitro analysis of the function of mutant proteins
The determination of enzyme activity and kinetic parameters is well established in the prior art. Experiments to determine the activity of the resulting modified enzyme must be performed on the specific activity of the wild-type enzyme within the skill of the art. A general description of enzymes, as well as specific details regarding structure, kinetics, principles, methods, applications and examples of determining the activity of a number of enzymes, can be found, for example, in the following references: Dixon, M .; , And Webb, E.A. C. , (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism. @ Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francesco; Price, N.M. C. , Stevens, L .; (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P .; D. , Ed (1983) The Enzymes, 3rd ed. Academic Press: New York; Bisswanger, H .; , (1994) Enzymetic, 2nd ed. VCH: Weinheim (ISBN 3527300325), Bergmeyer, H .; U. , Bergmeyer, J. et al. , Grassl, M .; , Eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed. , Vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, 'Enzymes'. VCH: Weinheim, p. 352-363. .
[0150]
The activity of a protein that binds to DNA can be measured by several well-established methods, such as a DNA band-shift assay (also called a gel retardation assay). The effect of such proteins on the expression of other molecules can be determined using reporter gene assays (eg, as described in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and references). Measured. Reporter gene test systems are well known and have established applications in both prokaryotic and eukaryotic cells using beta-galactosidase, green fluorescent protein and several other enzymes.
[0151]
Determination of the activity of membrane transport proteins is described, for example, in Gennis, R .; B. (1989) 'Ports, Channels and Transporters', in Biomembranes, Molecular Structure and Function, Springer; Heidelberg, p. 85-137; 199-234; and 270-322.
[0152]
Example 9: Analysis of sensitization of mutant proteins in the production of the desired product
C.I. in the production of the desired compound (eg, an amino acid) The effect of the genetic modification of Glutamicum can be assessed by analysis of modified microbial growth under appropriate conditions (eg, as described above) and production of the desired product (ie, amino acid) with increased media and / or cellular components. it can. Such analytical methods are well known to those skilled in the art, and analytical chromatography such as spectroscopy, thin layer chromatography, various staining methods, enzymatic and microbiological methods, high performance liquid chromatography (eg, Ullman, Encyclopedia of Industrial). Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) 'Applications of Chemistry's Pharmaceuticals in HPLC. Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology. , Vol.3, Chapter III: 'Product recovery and purification', page 469-714, VCH: Weinheim; Belter, PA, et al., Memorial, (reviewed). JF and Cabral, JMS (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. . Ullmann's Encyclopedia of Industrial Chemistry, vol B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, including the Noyes see Publications).
[0153]
In addition to measuring the final fermentation product, other components of the metabolic pathway used to produce the desired compound, such as intermediates and by-products, can also be analyzed to determine the efficiency of compound production. Analytical methods include measuring levels of nutrients (eg, sugars, hydrocarbons, nitrogen sources, phosphates and other ions) in the medium, measuring biomass composition and growth, and analyzing the production of metabolites in the normal biosynthetic pathway , Including the measurement of gas produced during fermentation. Standard methods for these measurements are described in Applied Micro Physiology, A Practical Approach, M. Rhodes and P.S. F. Stanbury, eds. , IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0196635773) and references cited therein.
[0154]
Example 10: C.I. Purification of the desired product from glutamicum culture
C. Recovery of the desired product from glutamicum cells or from the supernatant of the culture described above can be performed by various well-known methods. If the desired product is not hidden from the cells, the cells can be harvested from the culture by low speed centrifugation and the cells can be lysed by standard methods such as mechanical force or ultrasound. Cell debris is removed by centrifugation and the supernatant fraction containing the soluble protein is retained for further purification of the desired compound. The product is C.I. If hidden from glutamicum cells, the cells are removed from the culture by low speed centrifugation and the supernatant fraction is retained for further purification.
[0155]
The supernatant fraction from both purification methods is the appropriate resin, where the desired molecules are retained on the chromatographic resin and many impurities in the sample are not retained, or the impurities are retained by the resin and the sample is retained. Work up by chromatography on a resin that does not retain. Such chromatographic steps can be repeated, if desired, using the same or different chromatographic resins. Those skilled in the art will be familiar with selecting an appropriate chromatography resin and most efficiently applying it to the particular molecule being purified. The purified product can be concentrated by filtration or ultrafiltration and stored at a temperature at which the stability of the product is greatest.
[0156]
A wide range of purification methods are well known and the purification methods described above do not imply a reduction in range. Such purification methods are described, for example, in Bailey, J .; E. FIG. & Ollis, D.S. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
[0157]
The identity and purity of the isolated compound is assessed by standard methods in the prior art. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic or microbiological assays. Such an analysis method is described in Petek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Bioteknologiya 11: 27-32; and Schmidt et al. (1988) Bioprocess Engineer. 19: 67-70. See Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587; Michel, G .; (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A .; et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Technologies in Biochemistry and Molecular Biology, vol. 17.
[0158]
Example 11: Analysis of the gene sequence of the present invention
Comparison of sequences and determination of the percentage of homology between the two sequences are well known in the art and can be performed using mathematical algorithms, such as those described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA. 87: 2264-68, by the algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77. Such an algorithm is described in Altschul, et al. (1990) J. Am. Mol. Biol. 215: 403-10 incorporated into the NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequence homology to the MCT nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequence homology to MCT protein molecules of the present invention. For purposes of comparison, gapped BLAST was performed by Altschul et al. , (1997) Nucleic Acids Res. 25 (17): 3389-3402. In using the BLAST and gapped BLAST programs, one skilled in the art is familiar with how to optimize the parameters of the programs (eg, XBLAST and NBLAST) for the particular sequence being analyzed.
[0159]
Another example of a mathematical algorithm used for comparing sequences is the algorithm of Meyers and Miller ((1988) Comput. Appln. Biosci. 4: 11-17). Such an algorithm has been incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, can be used. Further, algorithms used for sequence analysis are known, and are described in ADVANCE and ADAM (described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10: 3-5) and FASTA (Pearson and Lipman (1988) P.N. A. S. 85: 2444-8).
[0160]
The percentage of homology between the two amino acid sequences was determined using the GAP program (available at http://www.gcg.com) in the GCG software package, either Blosum62 or PAM250 matrices, 12, 10 , 8, 6, or 4 gap weights, 2, 3, or 4 length weights. The percentage of homology between the two nucleic acid sequences is determined using the GAP program in the GCG software package, using standard parameters, such as 50 for gap weight and 3 for length weight. be able to.
[0161]
Comparative analysis of the gene sequences of the present invention with sequences present in the gene bank was performed using techniques known in the prior art (eg, Bexevanis and Oullette, eds. (1998) Bioinformatics: A Practical Guide to Analysis of Genesis of the Analysis). and Proteins. John Wiley and Sons: See New York). The gene sequence of the present invention was compared with the genes present in the gene bank in three steps. In the first step, BLASTN analysis (eg, local alignment analysis) was performed on each of the sequences of the present invention for nucleotide sequences present in the gene bank. Further analysis was performed on the first 500 hits. A FASTA search (eg, aligning restricted regions of the sequence with combined local and global alignment analysis) was then performed on these 500. Each inventive gene sequence is then generally aligned (using standard parameters) using the GAP program in the GCG software package for each of the three hits in the head of FASTA. To obtain the correct result, the length of the sequence extracted from the gene bank was adjusted to the length of the query sequence by a well-known method. The results of this analysis are summarized in Table 4. Although the results obtained were consistent with those that would be obtained when performing a GAP (global) analysis alone on each of the genes of the invention compared to each of the gene bank references, such a database was The calculation time was clearly shortened as compared to the GAP (global) analysis performed in an expanded manner. The sequences of the present invention in which an alignment exceeding the truncated value was not obtained are shown in Table 4 without alignment information. It is understood by those skilled in the art that the GAP alignment homology percentages listed in Table 4 under the heading '% Homology (GAP)' are ordered in the form of ',' where European numbers represent decimal points. It will be further understood. For example, the value of '40, 345 'in this column represents 40.345%.
[0162]
Example 12: Construction and operation of DNA microarray
In addition to the sequences of the present invention, DNA microarrays (design, methodology, use of DNA arrays are well known and described, for example, in Schena, M. et al. (1995) Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1467; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L. 78-76, et al. Can be used for construction and application.
[0163]
DNA microarrays are solid or flexible supports made of nitrocellulose, nylon, glass, silicone or other materials. Nucleic acid molecules can be attached to a surface in an ordered manner. After appropriate labeling, other nucleic acids or a mixture of nucleic acids can be hybridized to the immobilized nucleic acid molecule and labeled to detect and measure the individual signal strength of the hybridized molecule in a defined area. Can be used. This methodology makes it possible to simultaneously quantify the relative or absolute amounts of all or selected nucleic acids in the applied nucleic acid sample or mixture. For this reason, DNA microarrays make it possible to analyze the expression of a large number (6800 or more) of nucleic acids in parallel (see Schena, M. (1996) BioEssays 18 (5): 427-431).
[0164]
The sequences of the present invention may be prepared by a nucleic acid amplification reaction such as the polymerase chain reaction. It can be used to design oligonucleotide primers that can amplify a limited region of the glutamicum gene. The selection and design of 5 ′ or 3 ′ oligonucleotide primers or suitable linkers is described above on the support medium (eg, also described in Schena, M. et al. (1995) Science 270: 467-470). Allows the covalent binding of the resulting PCR product to
[0165]
Nucleic acid microarrays are described in Wodicka, L .; et al. (1997) Nature Biotechnology 15: 1359-1367 as described in in situ oligonucleotide synthesis. Photolithography exposes precisely defined areas of the matrix. The photolabile protecting group is thereby activated and nucleotide addition proceeds, while the region masked from light does not proceed with any modifications. Subsequent cycles of protection and photoactivation allow the synthesis of different oligonucleotides at defined locations. Small, confined regions of the gene of the invention can be synthesized by solid phase oligonucleotide synthesis on microarrays.
[0166]
Nucleic acid molecules of the invention present in a sample or mixture of nucleotides can hybridize to a microarray. These nucleic acid molecules can be labeled by standard methods. Briefly, nucleic acid molecules (eg, mRNA or DNA molecules) are labeled, for example, during reverse transcription or DNA synthesis, by incorporation of nucleotides labeled with radioisotopes or by fluorescence. Hybridization of labeled nucleic acids to microarrays is described, for example, in Schena, M .; et al. (1995) supra; Wodicka, L .; et al. (1997), supra; and DeSaizieu A. et al. et al (1998), supra. Detection and quantification of the hybridized molecule is performed by means of a specific incorporated label. Radioactive labels are described, for example, in Schena, M .; et al. (1995) can be detected as described in supra, and fluorescent labels are described, for example, in Shalon et al. (1996) Genome Research 6: 639-645.
[0167]
The application of the sequences of the present invention to DNA microarray technology is described in It allows the comparative analysis of different strains of G. glutamicum or other Corynebacteria. For example, identification of genes important for studying and identifying internal strain variants based on individual transcription characteristics and / or identification of desired strain characteristics, such as pathogenicity, productivity, and stress tolerance, can be accomplished by using nucleic acids. Driven by array methodology. Comparison of the expression characteristics of the genes of the present invention during the course of the fermentation reaction is possible using nucleic acid array technology.
[0168]
Example 13: Kinetic analysis of cellular protein populations (proteomics)
The genes, compositions, and methods of the present invention can be applied to study the interaction and kinetics of protein populations (defined as proteomics). The protein population of interest is C.I. glutamicum (eg, as compared to the protein population of other organisms), proteins or specific growths that are active under certain environmental or metabolic conditions (eg, at high or low temperatures, or during fermentation at high or low pH). And the total protein population of proteins active during the developmental phase.
[0169]
The protein population can be analyzed by various well-known techniques, for example, gel electrophoresis. Cellular proteins are obtained, for example, by cell lysis or extraction and can be separated from one another using various electrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) largely separates proteins based on their molecular weight. Isoelectric point polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also the post-translational modification of the protein). Other, more preferred methods of protein analysis are 2-D-gel electrophoresis (e.g., Hermann et al. (1998) Electrophoresis 19: 3217-3221; Fontourakis et al. (1998) Electrophoresis 19: 1193-1202; Langenet). al. (1997) Electrophoresis 18: 1184-1192; described in Antelmann et al. (1997) Electrophoresis 18: 1451-1463), which is a continuous combination of IEF-PAGE and SDS-PAGE. Other separation techniques, such as capillary gel electrophoresis, can also be used to separate proteins, and such techniques are well known.
[0170]
Proteins separated by these methodologies can be visualized by standard techniques, such as staining or labeling. Suitable stains are known and include Coomassie brilliant blue, silver stains, fluorescent dyes such as Sypro @ Ruby (Molecular @ Probes). C. radioactively labeled amino acids or other protein precursors in the medium of G. glutamicum (eg,³⁵S-methionine,³⁵S-cysteine,¹⁴C-labeled amino acids,^FifteenN-amino acids,^FifteenNO₃Or^FifteenNH₄ ⁺Or C_ThirteenThe inclusion of (labeled amino acids) makes it possible to label proteins from these cells prior to separation. Similarly, fluorescent labels can be used. These labeled proteins are extracted, isolated and separated according to the techniques described above.
[0171]
Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of protein obtained can be determined quantitatively, for example using optical methods, and can be compared to the amount of other proteins in a similar or other gel. Comparison of proteins on a gel can be performed, for example, through optical comparison, spectroscopy, image scanning and analysis of the gel, or through the use of photographic films and screens. Such techniques are well-known.
[0172]
Direct sequencing or other standard techniques can be used to identify the resulting protein. For example, using N- and / or C-terminal amino acid sequences (e.g., Edman degradation) as mass spectrometry (especially MALDI or ESI techniques (e.g., see Langen et al. (1997) Electrophoresis 18: 1184-1192)). it can. The protein sequences provided herein can be used to convert C. glutamicum protein can be used for identification.
[0173]
The information obtained by these methods can be used to determine the presence, activity, and the presence of different proteins in different samples from different biological conditions (eg, different organisms, time of fermentation, media conditions, or different biotopes, etc.). Can be used to compare the modes of modification. Data obtained from such experiments, alone or in combination with other techniques, yields a variety of applications, such as comparing the behavior of various organisms in a given (eg, metabolic) situation, producing fine chemicals. It can be used for increasing the productivity of a strain or increasing the efficiency of producing fine chemicals.
[0174]
Equivalent
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are included in the claims.

Claims (38)

A nucleic acid molecule isolated from Corynebacterium glutamicum encoding an MCT protein, wherein the nucleic acid molecule does not comprise any of the F-display genes listed in Table 1, or a portion thereof. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an MCT protein involved in the production of fine chemicals. An isolated nucleic acid molecule selected from the group consisting of the sequences shown in the odd numbered SEQ ID NOs (SEQ ID NO) of the Sequence Listing that does not contain any of the F-displayed genes listed in Table 1. The isolated Corynebacterium glutamicum nucleic acid molecule or a part thereof. A polypeptide sequence selected from the group consisting of the even numbered sequence numbers (SEQ ID NOs) of the sequence listing, wherein the nucleic acid molecule does not include any of the F-displayed genes listed in Table 1. An isolated nucleic acid molecule encoding The nucleic acid molecule is selected from the group of amino acid sequences consisting of the sequence shown in the even numbered SEQ ID NO (SEQ ID NO) of the sequence listing, which does not contain any of the F-display genes listed in Table 1. An isolated nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide. A nucleotide sequence selected from the group consisting of the sequences shown in the odd numbered SEQ ID NOs in the Sequence Listing (SEQ ID NO), wherein the nucleic acid molecule does not include any of the F-displayed genes listed in Table 1. An isolated nucleic acid molecule or a portion thereof comprising a nucleotide sequence having at least 50% homology. A nucleotide sequence selected from the group consisting of the sequences shown in the odd numbered SEQ ID NOs in the sequence listing (SEQ ID NO), wherein the nucleic acid molecule does not include any of the F-displayed genes listed in Table 1. An isolated nucleic acid molecule comprising a nucleic acid fragment of at least 15 nucleotides. An isolated nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule according to any of the preceding claims. An isolated nucleic acid comprising a nucleic acid molecule according to any of claims 1 to 8, or a portion thereof, and a nucleotide sequence encoding a heterologous polypeptide. A vector comprising the nucleic acid molecule according to claim 1. The vector according to claim 10, which is an expression vector. A host cell transfected with the expression vector according to claim 11. 13. The host cell according to claim 12, wherein said cell is a microorganism. 14. The host cell according to claim 13, wherein the cell belongs to the genus Corynebacterium or the genus Brevibacterium. 13. The host cell according to claim 12, wherein expression of the nucleic acid molecule regulates the production of fine chemicals from the cell. Fine chemicals include organic acids, proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatics, vitamins, cofactors, polyketides, and enzymes 16. The host cell according to claim 15, wherein the host cell is selected from the group consisting of: A method for producing a polypeptide, comprising culturing the host cell according to claim 12 in a suitable medium, and producing the polypeptide with the cultivation. An isolated MCT polypeptide from Corynebacterium glutamicum or a portion thereof. 19. The protein of claim 18, wherein the polypeptide is involved in the production of a fine chemical. The amino acid sequence is selected from the group consisting of the sequences shown in the even numbered SEQ ID NOs of the Sequence Listing (SEQ ID NO), which are not encoded by any of the F-displayed genes listed in Table 1. An isolated polypeptide comprising the amino acid sequence of claim 1. The amino acid sequence is selected from the group consisting of the sequences shown in the even numbered SEQ ID NOs of the Sequence Listing (SEQ ID NO), which are not encoded by any of the F-displayed genes listed in Table 1. Isolated polypeptides and naturally-occurring allelic variants of the polypeptides comprising the amino acid sequence and portions thereof. 22. The isolated polypeptide according to any of claims 18 to 21, further comprising a heterologous amino acid sequence. For nucleic acids selected from the group consisting of the sequences shown in the odd numbered SEQ ID NOs in the Sequence Listing (SEQ ID NO), wherein the nucleic acid molecule does not include any of the F-displayed genes listed in Table 1. An isolated polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 50% homology. The amino acid sequence is selected from the group consisting of the sequences shown in the even numbered SEQ ID NOs of the Sequence Listing (SEQ ID NO), which are not encoded by any of the F-displayed genes listed in Table 1. An isolated polypeptide comprising an amino acid sequence having at least 50% homology to the amino acid sequence. A method for producing a fine chemical, comprising culturing cells containing the vector according to claim 12 to produce a fine chemical. 26. The method of claim 25, further comprising recovering the fine chemical from the culture. The method according to claim 25, further comprising a step of transfecting a cell with the vector according to claim 11 to obtain a cell containing the vector. 26. The method of claim 25, wherein the cell belongs to the genus Corynebacterium or Brevibacterium. The cells are Corynebacterium-glutamicum, Corynebacterium-herculis, Corynebacterium-lilium, Corynebacterium-acetoacidophilium, Corynebacterium-acetoglutamicum, Corynebacterium-acetophyllium, Corynebacterium -Ammoniagen, Corynebacterium-fugiokens, Corynebacterium-nitrilofilius, Brevibacterium-ammonogen, Brevibacterium-butanicam, Brevibacterium-divacatum, Brevibacterium-flavin, Brevibacterium-hairy, Brevi Bacterium-ketoglutamicum, Brevibacterium-ketosoledactum, Brevibacterium-lactofermentum, Brevibacterium-linens, Brevibacterium 26. The method of claim 25, wherein the method is selected from the group consisting of Um-parafinolity cam and the strains listed in Table 3. The method according to claim 25, wherein the expression of the nucleic acid molecule by the vector regulates the production of the fine chemical. The fine chemicals include organic acids, proteinogenic amino acids and proteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes. 26. The method of claim 25, wherein the method is selected from the group consisting of: 26. The method of claim 25, wherein the fine chemical is an amino acid. The amino acid is selected from the group consisting of lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan; 33. The method according to claim 32. A method for producing a fine chemical, comprising a step of culturing a cell whose genomic DNA has been mutated by containing the nucleic acid molecule according to any one of claims 1 to 9. The presence of one or more of SEQ ID NOs from 1 to 676 of the Sequence Listing (SEQ ID NO) wherein the sequence is not any of the F-indicating sequences listed in Table 1 and is not encoded by any of the F-indicating sequences A method of diagnosing the presence or activity of Corynebacterium diphtheria, comprising the step of detecting from a patient, thereby diagnosing the presence or activity of Corynebacterium diphtheria in the patient. A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in the odd numbered SEQ ID NOs of the Sequence Listing (SEQ ID NO), wherein the nucleic acid molecule is confused. An odd-numbered SEQ ID NO in the sequence listing comprises a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in the odd-numbered SEQ ID NOs in the Sequence Listing (SEQ ID NO) A host cell comprising one or more nucleic acid modifications from the sequences set forth in 1. A nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in the odd-numbered SEQ ID NOs of the sequence listing (SEQ ID NO), wherein the regulatory region of the nucleic acid molecule corresponds to the wild-type regulatory region of the molecule. A host cell that has been modified in a related manner.