JP6522640B2 - プテロスチルベンの生合成製造のためにo‐メチルトランスフェラーゼを使用する方法 - Google Patents
プテロスチルベンの生合成製造のためにo‐メチルトランスフェラーゼを使用する方法 Download PDFInfo
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- JP6522640B2 JP6522640B2 JP2016552436A JP2016552436A JP6522640B2 JP 6522640 B2 JP6522640 B2 JP 6522640B2 JP 2016552436 A JP2016552436 A JP 2016552436A JP 2016552436 A JP2016552436 A JP 2016552436A JP 6522640 B2 JP6522640 B2 JP 6522640B2
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- pterostilbene
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- A—HUMAN NECESSITIES
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- A61P3/06—Antihyperlipidemics
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12N9/10—Transferases (2.)
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Description
[細胞システム]
[細胞システムの培養]
[遺伝子導入]
[修飾されたアミノ酸]
[4CL]
[STS]
[ROMT]
[材料および方法]
菌株、プラスミド、および培養条件
[DNA操作]
[RNA抽出およびcDNA合成]
当該メーカのマニュアルに従って、ROMTのPCR生成物がpETite N‐His SUMO Kan ベクター(ルシジェン)にクローニングされた。適切なインサートを持つ合成プラスミドは、シークエンシング、すなわちSumo−ROMTによって確認され、すなわちSumo‐ROMTは異種遺伝子の発現のためにBL21(DE3)に導入された。
4CL::STS遺伝子が、S.cerevisiaeアドバンストGatewayデスティネーションベクターpAG304GPD‐ccdB(マサチューセッツ州ボストンのアドジーン社)に導入され、ROMT遺伝子が、LRクロナーゼII酵素ミックスキット(インビトロゲン)によって、別のGatewayデスティネーションベクターpAG305GPD‐ccdB(アドジーン社)へ置換された。得られたプラスミドは、pAG304GPD‐4CLSTSおよびpAG305GPD‐ROMTと命名された。ベクターは、構成型プロモータ(GPD)の制御下、組み込みの組み換え側および発現カセットを含む。これらのベクターは、発酵試験のためにWAT11に変換された。
[酵母形質転換]
[ROMTの基質結合残基の推定のためのホモロジーモデリングおよびドッキング]
[大腸菌および出芽酵母における4CL::STS融合タンパク質を用いたp‐クマル酸からレスベラトロルへのバイオコンバージョン]
[大腸菌および出芽酵母におけるROMTのタンパク質を用いるレスベラトロルからプテロスチルベンへのバイオコンバージョン]
[大腸菌および出芽酵母におけるROMTおよび4CL::STS融合タンパク質のタンパク質を用いるp‐クマル酸のプテロスチルベンへのバイオコンバージョン]
[生成物の抽出]
[HPLC分析]
[結果]
[4CLおよびSTSの融合タンパク質を用いるp‐クマル酸のレスベラトロルへのバイオコンバージョン]
[ROMTのタンパク質を用いるレスベラトロルのプテロスチルベンへのバイオコンバージョン]
[4CL::STSおよびROMTの共発現を用いるp‐クマル酸のプテロスチルベンへのバイオコンバージョン]
[ROMTの従来型の飽和突然変異誘発]
[参照文献]
Schmidlin L、Poutaraud A、Claudel P、Mestre P、Prado E、Santos−Rosa M、Wiedemann− Merdinoglu S、Karst F、Merdinoglu D、Hugueney P (2008) A stress−inducible resveratrol O−methyltransferase involved in the biosynthesis of pterostilbene in grapevine. Plant Physiol.l48(3):1630‐1639。
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Grosdidier A、Zoete V、Michielin O.(2011) SwissDock、a protein−small molecule docking web service based on EADock DSS.JNucleic Acids Res. 39:W270‐277。
Claims (11)
- 細胞システムにおいて4‐クマル酸:コエンザイムAリガーゼ(4CL)を発現させる段階と、
前記細胞システムにおいてスチルベンシンターゼ(STS)を発現させる段階と、
前記細胞システムにおいてレスベラトロルO‐メチルトランスフェラーゼ(ROMT)を発現させる段階と、
前記細胞システムにp‐クマル酸を供給する段階と、
前記細胞システムを培地で培養する段階と、
プテロスチルベンを生成する段階と、を備える、プテロスチルベンを生成する生合成方法であって、
前記4‐クマル酸:コエンザイムAリガーゼを発現させる段階および前記スチルベンシンターゼを発現させる段階は、4CL::STS融合遺伝子を遺伝子導入する段階を含む、
プテロスチルベンを生成する生合成方法。 - 前記4‐クマル酸:コエンザイムAリガーゼは、シロイヌナズナ(Arabidopsis thaliana)(生態型 Columbia‐0)からクローニングされた4CL遺伝子(配列番号14)から発現される、請求項1に記載のプテロスチルベンを生成する生合成方法。
- 前記スチルベンシンターゼは、ぶどう(Vitis vinifera)からクローニングされたSTS遺伝子(配列番号16)から発現される、請求項1または2に記載のプテロスチルベンを生成する生合成方法。
- 前記レスベラトロルO‐メチルトランスフェラーゼは、ぶどう(Vitis vinifera)からクローニングされた遺伝子(配列番号12)から発現される、請求項1から3のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 前記レスベラトロルO‐メチルトランスフェラーゼを発現させる段階は、ROMT遺伝子を遺伝子導入する段階を含む、請求項1から4のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 前記細胞システムは、少なくともバクテリア、酵母、植物細胞、動物細胞およびそれらの組み合わせから成る群から選択される、請求項1から5のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 前記細胞システムは酵母である、請求項1から6のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 前記細胞システムは、異所性生合成反応を可能にする、請求項1から7のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 前記細胞システムは、in vitro変換システムを含む、請求項1から8のいずれか一項に記載のプテロスチルベンを生成する生合成方法。
- 細胞システムにおいて4‐クマル酸:コエンザイムAリガーゼ(4CL)を発現させる段階と、
前記細胞システムにおいてスチルベンシンターゼ(STS)を発現させる段階と、
前記細胞システムにp‐クマル酸を供給する段階と、
前記細胞システムを培地で培養する段階と、
レスベラトロルを生成する段階と、を備える、レスベラトロルを生成する生合成方法であって、
前記4‐クマル酸:コエンザイムAリガーゼを発現させる段階および前記スチルベンシンターゼを発現させる段階は、4CL::STS融合遺伝子を遺伝子導入する段階を含む、
レスベラトロルを生成する生合成方法。 - 第1の細胞システムにおいて4‐クマル酸:コエンザイムAリガーゼ(4CL)を発現させる段階と、
前記第1の細胞システムにおいてスチルベンシンターゼ(STS)を発現させる段階と、
前記第1の細胞システムにp‐クマル酸を供給する段階と、
前記第1の細胞システムを培地で培養する段階と、
レスベラトロルを生成する段階と、
第2の細胞システムにおいてレスベラトロルO‐メチルトランスフェラーゼ(ROMT)を発現させる段階と、
前記第2の細胞システムにレスベラトロルを供給する段階と、
前記第2の細胞システムを培地で培養する段階と、
プテロスチルベンを生成する段階と、を備える、プテロスチルベンを生成する生合成方法であって、
前記4‐クマル酸:コエンザイムAリガーゼを発現させる段階および前記スチルベンシンターゼを発現させる段階は、4CL::STS融合遺伝子を遺伝子導入する段階を含む、
プテロスチルベンを生成する生合成方法。
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