JP6828038B2 - 低分子量物質およびタンパク質の抗生物質を含まない発酵製造のための微生物株および方法 - Google Patents
低分子量物質およびタンパク質の抗生物質を含まない発酵製造のための微生物株および方法 Download PDFInfo
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Description
天然プロモーター領域を含むpyrH遺伝子をコードする大きさ約1.0kbのDNAフラグメントを、プライマーpyrH−NcoI−fw(配列番号5)およびpyrH−NcoI−rev(配列番号6)を用いて増幅した。PCR反応に使用した鋳型は、大腸菌(E. coli)株W3110(ATCC27325)由来の染色体DNAであった。
温度感受性複製開始点を有するプラスミドpAF−ts−pyrHおよびpAF−ts−plsCの構築に使用した出発プラスミドは、プラスミドpKD46(Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645)であった。プラスミドpKD46の制限酵素および機能マップを図1に示す。PCR産物がそれ独自のプロモーターを有するpyrH遺伝子またはplsC遺伝子をコードする実施例1に記載およびNcoIを用いて消化した該PCR産物をpKD46のNcoI制限酵素部位にクローニングした。「DH5α(商標)−T1R E. coli cells」(Life Technologies GmbH)にライゲーション試料を形質転換し、前記細胞内で増殖し、単離したプラスミドのDNA配列をシークエンシングによって検証した。この様に作成した全部で4つの可能性あるコンストラクトの2つは、pAF−ts−pyrHおよびpAF−ts−plsCという記号表示を有する(図2および3参照)。
実施例2に記載した温度感受性複製開始点を有するプラスミドpAF−ts−pyrHおよびpAF−ts−plsCを、当業者に公知のCaCl2法を用いて、2つの大腸菌(E. coli)株W3110(ATCC27325)およびW3110lpp3(「リーキー」株として米国特許出願公開第2008/076158(A1)号に記載)に形質転換した。形質転換した細胞を100mg/lアンピシリンを含有するLB寒天プレート上で選択した。この様に作成した株は、W3110/pAF−ts−pyrH、W3110lpp3/pAF−ts−pyrH、W3110/pAF−ts−plsCおよびW3110lpp3/pAF−ts−plsCという記号表示を有する。
A)遺伝子pyrHの欠失
DatsenkoおよびWanner(Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645)により開発された「λ赤法」に従って、大腸菌(E. coli)の酵素ウリジル酸キナーゼ(pyrH)をコードする遺伝子pyrHを、大腸菌(E. coli)株W3110/pAF−ts−pyrHおよびW3110lpp3/pAF−ts−pyrHで欠失した。プライマーpyrH−fw(配列番号9)およびpyrH−rev(配列番号10)を用いて、カナマイシン耐性マーカー遺伝子(kanR)をコードするDNAフラグメントを増幅した。プライマーpyrH−fwは、pyrH遺伝子の5’末端と相同である30ヌクレオチドからなる配列およびプラスミドpKD13(大腸菌遺伝子ストックセンター(CGSC)番号7633)上の2つのFRT部位(FLP認識標的)の1つをコードするDNA配列と相補である20ヌクレオチドを含んでなる配列をコードする。プライマーpyrH−revは、pyrH遺伝子の3’末端と相同である30ヌクレオチドからなる配列およびプラスミドpKD13上の第二FRT部位をコードするDNA配列と相補である20ヌクレオチドを含んでなる配列をコードする。
pyrH遺伝子と同様に、大腸菌(E. coli)内で酵素1−アシルグリセロール−3−リン酸−O−アシルトランスフェラーゼ(PlsC)をコードする遺伝子plsCを、大腸菌(E. coli)株W3110/pAF−ts−plsCおよびW3110lpp3/pAF−ts−plsCにおいて欠失した(Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645)。プライマーplsC−fw(配列番号13)およびplsC−rev(配列番号14)を用いて、カナマイシン耐性マーカー遺伝子(kanR)をコードするDNAフラグメントを増幅した。プライマーplsC−fwは、plsC遺伝子の5’末端と相同である30ヌクレオチドからなる配列およびプラスミドpKD13(大腸菌遺伝子ストックセンター(CGSC)番号7633)上の2つのFRT部位(FLP認識標的)の1つをコードするDNA配列と相補である20ヌクレオチドを含んでなる配列をコードする。プライマーplsC−revは、plsC遺伝子の3’末端と相同である30ヌクレオチドからなる配列およびプラスミドpKD13上の第二FRT部位をコードするDNA配列と相補である20ヌクレオチドを含んでなる配列をコードする。
遺伝子cysEX(セリンアシルトランスフェラーゼのフィードバック耐性多様体をコードする:CysE)およびorf306(O−アセチルセリン/システインエキスポーターをコードする:EamA)のクローニングおよび発現用に使用した出発プラスミドは、欧州特許第0885962(B1)号に記載の基本プラスミドpMT1および産生プラスミドpACYC184−LH−cysEX−orf306であった。
クレブシエラ・ニューモニエ(Klebsiella pneumoniae)M5a1(ジーンバンク番号M15264)由来のシクロデキストリングリコシルトランスフェラーゼ(CTGアーゼ)遺伝子のクローニングおよび発現用に使用した出発プラスミドは、再び、米国特許出願公開第2008/076158(A1)号に記載のプラスミドpMT1およびプラスミドpCGTであった。
抗リゾチームFabフラグメントのための遺伝子のクローニングおよび発現用に使用した出発プラスミドは、再び、米国特許出願公開第2008/0076158(A1)号に記載のプラスミドpMT1およびpFab−抗リゾチームであった。
マーカー遺伝子として産生プラスミドpyrHまたはplsCを含有する産生プラスミド作成用に使用した出発プラスミドは、実施例5〜7に記載されているように、プラスミドpcysEX−GAPDH−ORF306_tetR、pCGT_tetRおよびpFab−anti−Lysozyme_tetRであった。全てのこれらのプラスミドは、個々のNcoI制限酵素部位を有する(図5〜7参照)。この汎用的NcoI制限酵素部位を、遺伝子pyrHまたはplsCのクローニングまたは組込みのために使用した。
pcysEX−GAPDH−ORF306_pyrH1_tetR (図8参照)およびpcysEX−GAPDH−ORF306_pyrH2_tetR
pCGT_pyrH1_tetR (図9参照)およびpCGT_pyrH2_tetR
pFab−anti−Lysozyme_pyrH1_tetR (図10参照)およびpFab−anti−Lysozyme_pyrH2_tetR
pcysEX−GAPDH−ORF306_plsC1_tetR(図11参照)およびpcysEX−GAPDH−ORF306_plsC2_tetR
pCGT_plsC1_tetR (図12参照)およびpCGT_plsC2_tetR
pFab−anti−Lysozyme_plsC1_tetR(図13参照)およびpFab−anti−Lysozyme_plsC2_tetR
抗生物質耐性遺伝子tetRを含まず、マーカー遺伝子としてpyrHまたはplsCを含む産生プラスミドの作成用に使用した出発プラスミドは、実施例8に記載されているように、プラスミドpcysEX−GAPDH−ORF306_pyrH1_tetR、pCGT_pyrH1_tetR、pFab−anti−Lysozyme_pyrH1_tetR、pcysEX−GAPDH−ORF306_plsC1_tetR、pCGT_plsC1_tetRおよびpFab−anti−Lysozyme_plsC1_tetRであった。実施例8に記載されているプラスミドpFab−anti−Lysozyme_pyrH1_tetRおよびpFab−anti−Lysozyme_plsC1_tetR由来の抗生物質耐性遺伝子tetRの取り出しを、制限酵素ClaIおよびその後の再ライゲーションを用いた消化により行った。
pyrHまたはplsCをコードする産生プラスミドを含み、かつ、染色体pyrHまたはplsC欠失を有するこのように作成しチェックした抗生物質耐性のない大腸菌(E. coli)株は、以下の記号表示を有する:
・W3110ΔpyrH/pcysEX−GAPDH−ORF306_pyrH
・W3110lpp3ΔpyrH/pCGT_pyrH
・W3110lpp3ΔpyrH/pFab−anti−Lysozyme_pyrH
・W3110ΔplsC/pcysEX−GAPDH−ORF306_plsC
・W3110lpp3ΔplsC/pCGT_plsC
・W3110lpp3ΔplsC/pFab−anti−Lysozyme_plsC
予備培養1:
エルレンマイヤーフラスコ(100ml)内で、LB培地20mlを、特定の大腸菌(E. coli)株W3110/pcysEX−GAPDH−ORF306_tetR、W3110ΔpyrH/pcysEX−GAPDH−ORF306_pyrHまたはW3110ΔplsC/pcysEX−GAPDH−ORF306_plsCと一緒にインキュベートし、7時間振盪機(150rpm、30℃)上でインキュベートした。従来技術、すなわち選択薬物として抗生物質を用いた従来技術と関連して、コンストラクトW3110/pcysEX−GAPDH−ORF306_tetRの培養のため、培地にテラサイクリン15mg/Lを添加した。
その後、予備培養液1を、SM1培地(12g/lのK2HPO4、3g/lのKH2PO4、5g/lの(NH4)2SO4、0.3g/lのMgSO4x7H2O、0.015g/lのCaCl2x2H2O、0.002g/lのFeSO4x7H2O、1g/lのクエン酸Na3x2H2O、0.1g/lのNaCl、0.15g/lのNa2MoO4x2H2O、2.5g/lのH3BO3、0.7g/lのCoCl2x6H2O、0.25g/lのCuSO4x5H2O、1.6g/lのMnCl2x4H2O、0.3g/lのZnSO4x7H2Oからなる1ml/lの微量元素溶液)100mlに完全に移動し、これに5g/lのグルコースおよび5mg/lのビタミンB1を添加した。培養液を150rpm、17時間、30℃でエルレンマイヤーフラスコ(1l)ないで振盪した。このインキュベーション後、600nmにおける光学密度(OD600)は、3〜5であった。従来技術、すなわち選択薬物として抗生物質を用いた従来技術と関連して、W3110/pcysEX−GAPDH−ORF306_tetRの培養のため、培地にテラサイクリン15mg/Lを添加した。
発酵槽、Sartorius Stedim社のBIOSTAT Bモデル内で発酵を行った。総容量2lの培養槽を使用した。発酵培地(900ml)は、15g/lのグルコース、10g/lのトリプトン(Difco)、5g/lの酵素抽出物(Difco)、5g/lの(NH4)2SO4、1.5g/lのKH2PO4、0.5g/lのNaCl、0.3g/lのMgSO4x7H2O、0.015g/lのCaCl2x2H2O、0.075g/lのFeSO4x7H2O、1g/lのクエン酸Na3x2H2Oおよび微量元素溶液(上記参照)1mlおよび0.005g/lのビタミンB1を含有する。25%NH4OH溶液をポンプで注入することにより、発光槽内のpHを最初6.5に調節した。発酵中、pHを、25%NH4OHを用いて自動補正により6.5のレベルに維持した。接種のため、100mlの予備培養液2を発酵槽にポンプで注入した。従って、開始体積は、約1lであった。培養液を最初400rpmで撹拌し、細菌ろ過器を通して滅菌した圧縮空気2vvmで曝気した。これらの開始条件下、接種前に、酸素プローブを100%飽和に補正した。発酵中のO2飽和に対する基準値を50%に設定した。O2飽和が基準値より下に落ちた後、O2飽和を基準値に戻すために制御カスケードを開始した。この関連で、先ず、気体供給を連続的に増加(最大5vvmまで)してから、撹拌速度を連続的に上昇(最大1500rpmまで)させた。
大腸菌(E. coli)のlpp変異体を用いて、例えば、CGTアーゼなどのバイオテクノロジー的に関連ある酵素を産生し、培地に分泌することができる(米国特許出願公開第2008/076158(A1)号)。
大腸菌(E. coli)のlpp変異体を用いて、機能性Fab抗体フラグメントも細胞外で製造することができる(米国特許出願公開第2008/076158(A1)号)。この場合、細胞は、ドメインVLおよびCLを含んでなる軽鎖、ならびにドメインVHおよびCH1を含んでなる重鎖の対応するフラグメントを同時に合成し、それから、周辺質および最終的に発酵培地に分泌しなければならない。それから、細胞質の外側では、2つの鎖は会合して機能性Fabフラグメントを形成する。
プラスミド試料を用いて、続く制限酵素消化でプラスミド安定性をチェックした。この目的のため、生産菌株の培養の完了(例えば、発酵72時間後)後にLB寒天プレート上に様々な希釈の培養液を蒔いた。プラスミド安定性のその後の確認、すなわち、プラスミド担持細胞(コロニー)の同定のため、個々のコロニーを含むLBプレートのみを評価に使用した。
Claims (7)
- 株の栄養要求性を引き起こす遺伝子変異をそのゲノム内に含み、該遺伝子がpyrH遺伝子またはplsC遺伝子であり、かつ
低分子量物質または少なくとも1つの組換えタンパク質の生産のための少なくとも1つの酵素をコードする産生プラスミドも含み、かつ
該産生プラスミド上に含まれる前記遺伝子の機能性コピーも含み、
前記遺伝子の染色体不活性化が前記栄養要求性を引き起こす、低分子量物質またはタンパク質の生産のための微生物株であって、
前記栄養要求性が供給不能な栄養要求性であり、かつ前記産生プラスミド上に含まれる前記遺伝子の機能性コピーをコードする遺伝子がpyrH遺伝子もしくはplsC遺伝子であり、ここで該pyrH遺伝子が酵素UMPキナーゼ(EC:2.7.4.22)をコードし、かつ該plsC遺伝子が酵素1−アシルグリセロール−3−リン酸 O−アシルトランスフェラーゼ(EC:2.3.1.51)をコードすることを特徴とする、微生物株。 - 前記株の供給不能な栄養要求性を引き起こす遺伝子の変異が、前記遺伝子の不活化をもたらすことを特徴とする、請求項1に記載の微生物株。
- 前記株の供給不能な栄養要求性を引き起こす遺伝子の変異が前記遺伝子によりコードされる遺伝子産物の活性の不活化をもたらすことを特徴とする、請求項1または2に記載の微生物株。
- 変異または欠失により前記株の供給不能な栄養要求性を引き起こす遺伝子に変換することができる遺伝子を有する微生物株に、温度感受性プラスミドを導入することを特徴とし、前記温度感受性プラスミドが変異または欠失する前記遺伝子の機能性コピーを有し、その上、前記株が遺伝子内に前記株の供給不能な栄養要求性をもたらす変異を有し、かつ前記遺伝子の機能性コピーが前記温度感受性プラスミド上に存在し、かつその上、前記温度感受性プラスミドが前記株内で非許容温度において産生プラスミドに交換され、ここで前記産生プラスミドは低分子量物質または組換えタンパク質の製造のための酵素をコードする遺伝子を含み、かつ前記栄養要求性を引き起こす前記遺伝子の機能性コピーも含む、請求項1〜3のいずれか一項に記載の株の製造方法。
- 前記温度感受性プラスミドが温度感受性複製開始点を有することを特徴とする、請求項4に記載の方法。
- 前記産生プラスミドを用いた形質転換直後に、細胞を30〜90分間47〜55℃で温度ショックに暴露し、その後に37〜45℃の非許容温度においてさらなるインキュベーションを行うことを特徴とする、請求項5に記載の方法。
- 発酵培地中の微生物株を用いた低分子量物質またはタンパク質の製造方法であって、該方法が、請求項1〜3の一項以上に記載の微生物株を使用し、かつ、抗生物質を含まない発酵培地を使用することを特徴とする、方法。
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