JP2005518210A - 二次代謝物同属種分布の調節 - Google Patents
二次代謝物同属種分布の調節 Download PDFInfo
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- JP2005518210A JP2005518210A JP2003571418A JP2003571418A JP2005518210A JP 2005518210 A JP2005518210 A JP 2005518210A JP 2003571418 A JP2003571418 A JP 2003571418A JP 2003571418 A JP2003571418 A JP 2003571418A JP 2005518210 A JP2005518210 A JP 2005518210A
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- Prior art keywords
- epothilone
- host cell
- oxygen
- polyketide
- production
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Abstract
Description
本出願は、2002年2月25日提出の米国特許出願番号60/359,821に対する優先権を主張する。本特許出願は、1998年11月20日提出の米国特許出願番号09/724,878に対する優先権を主張し、かつこの出願に関連し、また米国特許第6,303,342 B1に関連する。これら文献はそれぞれ参照によってその全体が本明細書に取り込まれる。
本発明は、組換えDNA技術によってポリケチドを産生させるための組換え方法と材料、及び酸素圧を調節することでポリケチド同属種分布を調節する方法を提供する。本発明は、農業、動物管理、化学、医薬品化学、医学、分子生物学、薬理学、及び獣医学の分野に関する。
ポリケチド(Polyketides)は、一連の縮合とその後の修飾によって2-炭素単位から合成される多様な化合物の大ファミリーを表す。ポリケチドは、真菌及び菌糸細菌、特に放線菌を含む多くのタイプの生物内に存在する。多種多様なポリケチド構造があり、ポリケチドの分類は多様な活性を有する多数の化合物を包含する。エリスロマイシン、FK-506、FK-520、メガロマイシン、ナルボマイシン、オレアンドマイシン、ピクロマイシン、ラパマイシン、スピノシン(spinocyn)、及びチロシンは、このような化合物の例である。伝統的な化学的方法論でポリケチド化合物を生成することの困難性及び野生型細胞内でのポリケチドの典型的に低い生産量を考えると、ポリケチド化合物を生成する改良された或いは代替手段を見いだすことに相当の関心がある。PCT公開番号WO93/13663;WO95/08548;WO96/40968;97/02358;及び98/27203;米国特許第4,874,748;5,063,155;5,098,837;5,149,639;5,672,491;5,712,146;及び5,962,290;及びFuら, 1994, Biochemistry 33:9321-9326;McDanielら, 1993, Science 262:1546-1550;及びRohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8):881-888(それぞれ参照によって本明細書に取り込まれる)を参照せよ。
以下の参考文献は、本発明に関する背景情報を提供し、参照によってその全体が本明細書に含まれるものとする。
Schneider, S.; Wubbolts, M.; Oesterhelt, G.; Sanglard, D.; Witholt, B. 様々な溶解酸素濃度下での全細胞産生性チトクロームP450BM-3モノオキシゲナーゼによる脂肪酸酸化の制御された領域選択性.Biotechnol. Bioeng. 1999, 64, 333-341.
Jensen, N.; Melchiorsen, C.; Jokumsen, K.; Villadsen, J. 低希釈率の微好気性連続培養におけるLactococcus lactis MG1363の代謝挙動.Appl. Environ. Microbiol. 2001, 67, 2677-2682.
Liefke, E.; Kaiser, D.; Onken, U. 高めた全圧と酸素分圧で培養した放線菌の生育及び産物生成.Appl. Environ. Microbiol. 1990, 32, 674-679.
Kaiser, D.; Onken, U.; Sattler, I.; Zeeck, A. マヌマイシン(manumycin)産生性Streptomyces parvulusによる二次代謝物の生成に及ぼす高めた溶解酸素濃度の影響.Appl. Environ. Microbiol. 1994, 41, 309-312.
Dick, O.; Onken, U.; Sattler, I.; Zeeck, A. Streptomyces griseoflavusのコラボマイシン(colabomycin)産生性株のクラスターの生産性及び選択性に及ぼす高めた溶解酸素濃度の影響.Appl. Environ. Microbiol. 1994, 41, 373-377.
Lambalot, R.T.; Cane, D.E.; Aparicio, J.J.; Katz, L. エリスロマイシンC-12ヒドロキシラーゼ、EryKの過剰生産と特徴づけ.Biochemistry 1995, 34, 1858-1866.
Carreras, C.; Frykman, S.; Ou, S.; Cadapan, L.; Savala, S.; Woo, E.; Leaf, T.; Carney, J.; Burlingame, M.; Patel, S.; Ashley, G.; Licari、P.J. 新規エリスロマイシンの生産用6-デオキシエリスロノライドB類似体のSaccharopolyspola erythraea触媒生合成.J. Biotechnol. 2002, 92, 217-228.
Bollag、D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C.M. タキソール様機構の作用を有する新分類の微小管-安定化剤、エポチロン.Cancer Res. 1995, 55, 2325-2333.
Gerth, K.; Bedorf, N.; Hofle, G.; Irschik, H.; Reichenbach, H. エポチロンA及びB:Sorangium cellulosum(粘液細菌)由来の抗真菌及び細胞毒性化合物−生産、物理化学的、及び生物学的性質.J. Antibiot. 1996, 49, 560-563.
Kowalski, R.J.; Giannakakou, P.; Hamel, E. 精製チュブリンを有し、かつパクリタキセル抵抗性の細胞内における微小管-安定化剤エポチロンA及びBの活性.J. Biol. Chem. 1997, 272, 2534-41.
Su, D.S.; Meng, D.; Bertinato, P.; Balog, A.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. エポチロンの構造−活性の関係及びパクリタキセルとの最初のインビボ比較.Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096.
Chou, T.C.; Zhang, X.G.; Balog, A.; Su, D.S.; Meng, D.; Savin, K.; Bertino, J.R.; Danishefsky, S.J. デスオキシエポチロンB:エポチロンBと比較して有望なインビボプロフィルを有する有効な微小管-標的抗腫瘍薬.Proc. Natl. Acad. Sci. USA 1998, 95, 9642-9647.
Chou, T.C.; O'Connor, O.A.; Tong, W.P.; Guan, Y.; Zhang, Z.; Stachel, S.J.; Lee, C.; Danishefsky, S.J. 微小管安定化剤の高度に有望な分類の合成、発見、及び開発:デスオキシエポチロンB及びFのヌードマウス内におけるヒト腫瘍異種移植片に対する治療効果.Proc. Natl. Acad. Sci. USA 2001, 98, 8113-8118.
Gerth, K.; Steinmetz, H.; Hofle, G.; Reichenbach, H. エポチロンの生合成に関する研究:炭素骨格の生合成起点.J. Antibiot. 2000, 53, 1373-1377.
Julien, B.; Shah, S.; Zierman, R.; Goldman, R.; Katz, L.;Khosla, C. Sorangium cellulosum由来のエポチロン生合成遺伝子クラスターの単離及び特徴づけ.Gene. 2000, 249, 153-160.
Gerth, K.; Steinmetz, H.; Hofle, G.; Reichenbach, H. エポチロンの生合成に関する研究:PKSとエポチロンC/Dモノオキシゲナーゼ.J. Antibiot. 2001, 54, 144-148.
Serizawa, N.; Matsuoka, T. 3-ヒドロキシ-3-メチルグルタリル補酵素Aレダクターゼの組織選択性阻害薬、プラバスタチンへのML-236Bのヒドロキシル化を触媒する原核生物内の2成分型チトクロームP-450モノオキシゲナーゼ系.Biochem. Biophys. Acta. 1991, 1084, 35-40.
Tudzynski, B.; Holter, K. Gibberella fujikuroi内のジベレリン生合成経路:遺伝子クラスターの証拠.Fungal Genet. Biol. 1998, 25, 157-170.
Keller, N.; Watanabe, C.; Kelkar, H.; Adams, T.; Townsend, C. Aspergillus nidulansによるステリグマトシスチン生合成のためのモノオキシゲナーゼ媒介ステップの要求.Appl. Environ. Microbiol. 2000, 66, 359-362.
Lomovskaya, N.; Otten, S.; Doi-Katayama, Y.; Fonstein, L.; Liu, X.; Takatsu, T.; Inventi-Solari, A.; Filippini, S.; Torti, F.; Columbo, A.; Hutchinson, C.R. Streptomyces peucetius内でのドキソルビシン過剰生産:dnrUケトレダクターゼ及びdnrV遺伝子及びdoxAチトクロームP-450ヒドロキシラーゼ遺伝子のクローニングと特徴づけ.J. Bacteriol. 1999, 181, 305-318.
Walczak, R.; Dickens, M.; Priestley, N.; Strohl, W. 組換えStreptomyces sp.株C5 DoxA、ドキソルビシン生合成で複数ステップを触媒するチトクロームP-450の精製、特性、及び特徴づけ.J. Bacteriol. 1999, 181, 298-304.
Julien, B.; Shah, S. Myxococcus xanthus内のエポチロン生合成遺伝子の異種発現.Antimicrob. Agents Chemo. 印刷中.
Pirt, S.J.; 酸素要求と供給.Principles of Microbe and Cell Cultivation.John Wiley and Sons, New York, 1975; pg. 84.
Arras, T.; Schirawski, J.; Unden, G. 好気性及び微好気性条件下における細菌の細胞質内での基質としてのO2の有効性.J. Bacteriol. 1998, 180, 2133-2136.
Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Khosla, C.; Julien, B. エポチロン遺伝子クラスターのクローニング及び異種発現.Science 2000, 287, 640-642.
Frykman, S.; Tsuruta, H.; Lau, J.; Regentin, R.; Ou, S.; Reeves, C.; Carney, J.; Santi, D.; Licari, P. 培地設計によるエポチロン類似体生産の調節.J. Ind. Microbiol. Biotechnol. 2002, 28, 17-20.
一実施形態では、本発明は、ポリケチドシンターゼ(PKS)遺伝子又は仕立て酵素の遺伝子操作なしで、異種宿主細胞内のポリケチド同属種分布を調節する方法を提供する。
一実施形態では、本発明は、宿主細胞の培養中酸素圧を制御することによって特定分布のポリケチド同属種を産生するように操作できる、代謝的に操作された異種宿主細胞を提供する。
一実施形態では、本発明は、調節した酸素圧条件下で培養した場合、同属種の生産量を変化させる組換えポリケチド産生性宿主細胞を提供する。一実施形態では、組換え宿主細胞はエポチロンA及びBを産生するSorangium cellulosum宿主細胞であり、欠乏酸素条件下で培養すると、生産物をエポチロンC及びDに変える。
一実施形態では、エポチロン同属種分布が酸素圧で調節された結果、過剰酸素圧下では、エポチロンC同属種に対してより高率のエポチロンD同属種となる。別の実施形態では、欠乏酸素条件下でエポチロンCのエポチロンD同属種に対する比率が増す。一実施形態では、エポチロン同属種が異種Myxococcus xanthus株K111-32.25によって産生される。一実施形態では、株K111-32.25内過剰酸素圧下ではエポチロンA及びBが産生され、かつ欠乏酸素圧下ではエポチロンC及びDが産生される。
一実施形態では、本発明は、活性なEpoKモノオキシゲナーゼを有する代謝的に操作した異種宿主細胞によって産生される新規なポリケチドを提供する。
一実施形態では、異種宿主細胞の培養中酸素圧を調節することによって産生される新規なポリケチドがエポチロン506である。
本発明は、仕立て酵素のいかなる遺伝子操作をも必要とせずに、主要産物としてポリケチド中間体を産生させる代替方法を提供する。本発明の方法は、ブダペスト条約下、American Type Culture Collection(ATCC)によって寄託され、かつ寄託指定番号が割り当てられている宿主細胞を利用する。Myxococcus xanthus株K111-32.25は、寄託指定PTA-1700で2000年5月1日に寄託され、Myxococcus xanthus株K111-40.1は、寄託番号PTA-2712で2001年1月18日に寄託された。Sorangium cellulosum株K111-150.17は、寄託指定 で
に寄託された。
欠乏酸素培養内の酸素密度の増加は、(i)細胞が、酸素制限によって強いられる遅い増殖時に、異なる速度又は異なる選択パターンで栄養素を利用し、それによって臨界的な栄養素の消耗を延期し得る、(ii)培養が、酸素制限時に過剰酸素化下で排出されるのとは異なるタイプ又はレベルの強力な増殖-阻害代謝物を産生する、又は(iii)酸素の制限が、細菌の増殖及び呼吸プロセスに関与する遺伝子の発現及び酵素の活性を高め或いは抑制することに起因し得る。
エポチロンポリケチドシンターゼ配列を操作した株によって新規なエポチロンが創造される(Julienら, 2000, Tangら, 2000)。アセテートやプロピオネートのような特有の前駆体の産生培地への添加と組み合わせた溶解酸素圧の調節は、活性なEpoK酵素を有する単一のM. xantus株から主要産物として4つの主要エポチロン同属種A、B、C、又はD(図2)のそれぞれの産生を可能にする(Frykmanら, 2002)。
上述した本発明の詳細な説明、以下の実施例は、本発明を説明する目的のために与えられ、本発明又はクレイムの範囲の制限であると解釈すべきでない。
調節した酸素圧条件下での宿主細胞の培養
細菌株。エポチロンポリケチドシンターゼをSorangium cellulosum SMP44からクローン化し、Myxococcus xanthus株DZ1中に導入した(Julienら, 印刷中)。M. xantusエポチロンB産生株(K111-32.25)は機能性EpoKエポキシダーゼ(EpoK+)を有するが、この酵素はエポチロンD産生性株(K111-40.1)内では遺伝的に不活性化(EpoK-)されている。エポチロンPKSのエノイルレダクターゼ(ER5)ドメインの活性をK111-32.25及びK111-40.1の両株内の点突然変異の導入によって除去し、それぞれ株K165-79.7及びK165-76.2を生成した。株K165-79.7及びK165-76.2を操作してそれぞれ10,11-ジデヒドロ-エポチロンB及びDを生成した(図2)。
新規エポチロンの産生
エポチロンPKSを遺伝子的に変えて、腫瘍細胞系に対して高められた水溶性及び/又は効力を示す可能性のある新規なエポチロンを産生する本発明の株を創造した。遺伝子操作した操作Myxococcus xanthus産生株(株K165-79.7, EpoK+)に本発明の酸素化制御法を適用して新規エポチロン、10,11-ジデヒドロ-エポチロンBを生じさせた(図2)。その一対の片方(K165-76.2K-)を過剰酸素化条件下で培養した場合、10,11-ジデヒドロ-エポチロンC及びDが単独の産物であることが分かった。株K165-79.7内で活性なEpoK酵素は、過剰酸素の存在下でこの10,11-ジデヒドロ-エポチロンD中間体の10,11-ジデヒドロ-エポチロンBへのエポキシド化(図6a)を触媒すると予想された。しかし、この化合物は検出されず、代わりに、新規異性体(Epo506)がこれら培養の主要産物だった(図6b)。高分解能質量分析法と、一及び二次元NMRを利用してこのテトラヒドロフラン産物の構造を確立した(実施例1参照)。
Epo506の分子式は、m/z 506.25887の[M+H]+ピークのHREIMS測定によってC27H39NO6であると決定し(C27H39NO6で計算すると506.25709)、10,11-ジデヒドロ-エポチロンDについて1つの酸素原子の取り込みを示している。13Cスペクトルは、Epo506が10,11-ジデヒドロ-エポチロンDより少ない1つの二重結合を有することを示し、Epo506が付加環を含むことを示唆している。HSQC、HMBC、COSY、及びTOCSYデータは、炭素結合性の疑いの余地のない割当てを与えた。炭素3、7、10及び13における化学シフトは、これら4つの炭素すべてが酸素原子に結合していることを示した。これら酸素化炭素の2つは分子式を考慮して環状エーテルの一部であると想定された。炭素3と13のヒドロキシルプロトンは1-D及び2-Dスペクトルの両方で見られ、それらの隣接原子に対するHMBC及びCOSY相関関係はそれらの割当てを確証した。従って、環状エーテルは、炭素7及び10を含んでいなければならず、テトラヒドロフラン構造を示している。質量スペクトルの有意なNa+及びK+付加物も、LC/MSによって506.3で観察されるイオンが実は該分子のイオンであり、仮想の親ジオールのイオン化によって生成されたフラグメントでないことを確証した。
Sorangium cellulosum株Soce90 K111-150.17内で調節されたエポチロン同属種の産生
異種宿主細胞の培養中の溶解酸素の調節は、ポリケチド同属種分布を変化させることが分かった。この実施例では、酸素欠乏条件下で培養した場合、エポチロン産生性株を酸素圧にさらした結果、エポチロン同属種分布の変化となった。Sorangium cellulosum株Soce90 K111-150.17(ATCC寄託指定 )は、Soce90親株からUV突然変異によって誘導されたリファマイシン-抵抗性突然変異体である(B. Julien, 未発表結果)。この実施例で用いる培養方法論及び分析法は、上記実施例1で述べたとおりに行った。
前記実施例は本発明の説明のためであり、かつ本技術の当業者は、前記実施例が特許請求の範囲で発明者らが請求するとおりの本発明の非限定的な実施形態であると解釈するものである。
Claims (20)
- 制御された酸素圧条件下で培養された組換え粘液細菌宿主細胞であって、前記宿主細胞は発現制御配列の制御下のポリケチドシンターゼ(PKS)遺伝子を含み、前記ポリケチド産生をもたらす条件下で前記宿主細胞を培養した場合は前記PKS遺伝子の発現はポリケチドの産生となり、前記宿主細胞は、活性な酸素感受性チトクロームP-450モノオキシゲナーゼ仕立て酵素をコードする遺伝子をも含むことを特徴とする粘液細菌宿主細胞。
- 前記ポリケチドがエポチロンである、請求項1の粘液細菌宿主細胞。
- 前記宿主細胞がSorangium cellulosum株K111-150.17であり、かつ前記酸素感受性チトクロームP-450モノオキシゲナーゼがEpoKエポキシダーゼである、請求項2の粘液細菌宿主細胞。
- 宿主細胞内でエポチロンポリケチドシンターゼ(PKS)のマロニル-CoAからメチルマロニル-CoAにアシルトランスフェラーゼ(AT)ドメインエクステンダー単位特異性を変える方法であって、エポチロンの産生をもたらす条件下、増殖培地内で前記宿主細胞を培養する工程と、酸素圧を過剰溶解酸素に調節する工程を含む方法。
- 粘液細菌宿主細胞内のポリケチド同属種分布産生比を調節する方法であって、ポリケチドの産生をもたらす条件下、増殖培地内で前記宿主細胞を培養する工程と、前記宿主細胞の培養中酸素圧を調節して、前記ポリケチド同属種の産生の種類を変化させる工程を含む方法。
- さらに前記増殖培地をセリンで補充する工程を含む、請求項5の方法。
- さらに前記増殖培地をアセテートで補充する工程を含む、請求項5の方法。
- さらに前記増殖培地をプロピオネートで補充する工程を含む、請求項5の方法。
- 前記ポリケチドがエポチロンである、請求項5の方法。
- 前記酸素圧を低酸素圧に調節し、かつ産生される前記ポリケチド同属種がエポチロンDである、請求項9の方法。
- 前記宿主細胞が、活性なEpoK+モノオキシゲナーゼを有するMyxococcus xanthusである、請求項5の方法。
- 前記宿主細胞がSorangium cellulosum株Soce90 K111-150.17である、請求項5の方法。
- 前記宿主細胞がMyxococcus xanthus株K111.32.25である、請求項5の方法。
- 産生される前記エポチロンD同属種が低酸素圧下で得られる、請求項10の方法。
- 前記エポチロンD同属種が、過剰酸素圧条件下より4倍まで高濃度で存在する、請求項10の方法。
- 欠乏酸素培養下、エポチロン同属種産生の比率が、エポチロンA及びBからエポチロンC及びD同属種分布に変化する、請求項5の方法。
- 過剰酸素条件下、エポチロンD対Cの同属種産生比が少なくとも3対1である、請求項5の方法。
- 前記粘液細菌宿主細胞がEpoK-である、請求項17の方法。
- 前記粘液細菌宿主細胞がMyxococcus xanthus株K111-40.1である、請求項17の方法。
- 前記酸素圧が50%溶解酸素未満である、請求項19の方法。
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DK2276485T3 (da) | 2008-04-24 | 2014-10-13 | Bristol Myers Squibb Co | Anvendelse af epothilon d i behandling af tau-associerede sygdomme,herunder alzheimers sygdom |
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WO2003072730A2 (en) | 2003-09-04 |
CN1639319B (zh) | 2010-04-28 |
US7220560B2 (en) | 2007-05-22 |
EP1485462A2 (en) | 2004-12-15 |
KR20040088531A (ko) | 2004-10-16 |
EP1485462A4 (en) | 2005-07-06 |
JP4280810B2 (ja) | 2009-06-17 |
ATE380861T1 (de) | 2007-12-15 |
NZ535515A (en) | 2007-12-21 |
WO2003072730A3 (en) | 2004-06-03 |
ES2295574T3 (es) | 2008-04-16 |
AU2003223190A1 (en) | 2003-09-09 |
DE60318020D1 (de) | 2008-01-24 |
EP1485462B1 (en) | 2007-12-12 |
CN1639319A (zh) | 2005-07-13 |
AU2003223190A8 (en) | 2003-09-09 |
US20040014183A1 (en) | 2004-01-22 |
DE60318020T2 (de) | 2008-11-13 |
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