JP2020508651A - スクアレンホペンシクラーゼ、およびアンブロキサンを生成するためのその使用 - Google Patents
スクアレンホペンシクラーゼ、およびアンブロキサンを生成するためのその使用 Download PDFInfo
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- JP2020508651A JP2020508651A JP2019543227A JP2019543227A JP2020508651A JP 2020508651 A JP2020508651 A JP 2020508651A JP 2019543227 A JP2019543227 A JP 2019543227A JP 2019543227 A JP2019543227 A JP 2019543227A JP 2020508651 A JP2020508651 A JP 2020508651A
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
- C12Y504/99017—Squalene--hopene cyclase (5.4.99.17)
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Abstract
Description
V45XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、WまたはYであり;
E46XにおけるXは、A、C、D、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
Q54XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、WまたはYであり;
S86XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、T、V、WまたはYであり;
F139XにおけるXは、A、C、D、E、G、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
Y142XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、VまたはWであり;
Q178XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、WまたはYであり;
M184XにおけるXは、A、C、D、E、F、G、H、I、K、L、N、P、Q、R、S、T、V、WまたはYであり;
R194XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、S、T、V、WまたはYであり;
G239XにおけるXは、A、C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
I278XにおけるXは、A、C、D、E、F、G、H、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
T326XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、V、WまたはYであり;
L335XにおけるXは、A、C、D、E、F、G、H、I、K、M、N、P、Q、R、S、T、V、WまたはYであり;
E386XにおけるXは、A、C、D、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
I455XにおけるXは、A、C、D、E、F、G、H、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
F460XにおけるXは、A、C、D、E、G、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
Q603Xは、A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、WまたはYであり;
G623XにおけるXは、A、C、D、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
F624XにおけるXは、A、C、D、E、G、H、I、K、L、M、N、P、Q、R、S、T、V、WまたはYであり;
L656XにおけるXは、A、C、D、E、F、G、H、I、K、M、N、P、Q、R、S、T、V、WまたはYであり;
Y658XにおけるXは、A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、またはWである。
GmSHCの酵素活性を増加させる1つのアプローチとして、pelBリーダー配列を、GmSHCをコードする核酸を含有するpET28b(+)ベクター中に挿入した。pelBリーダー配列をコードするオリゴヌクレオチドを、pET28b(+)ベクター中への挿入のためにNcoIおよびNdeI適合性末端と共に調製した。pET28b(+)ベクターをNcoIおよびNdeIで消化し、pelBリーダー配列のオリゴヌクレオチドを伴う一晩の連結反応において使用した。連結反応物を精製し、エレクトロコンピテント大腸菌(E.coli)の形質転換において使用した。このように得られた形質転換体である大腸菌(E.coli)のランダム標本を、相補性オリゴヌクレオチドプライマー「pelB−SHC−Fw」および「pET−XhoI−Rev」を伴うコロニーPCRによってアセスメントし、連結反応が成功したかどうかを決定した。クローンは、正確なサイズの挿入断片を含有すると同定された。それに続くDNA配列分析によって、pET28b(+)ベクターへのpelBリーダー配列の挿入を確認した。
pelB−GmSHCクローンを含有するプラスミドを使用して、融合タンパク質の発現のために大腸菌(E.coli)BL21(DE3)を形質転換させた。形質転換に続いて、単一のコロニークローンを単離し、10mLのLB培地+カナマイシンを播種するために使用した。10mLの培養物を、200rpmで一晩振盪しながら37℃にてインキュベートした。一晩培養物を使用して、1LのLB培地+カナマイシンを含有するフラスコを播種し、これを、誘導の前に6時間、振盪しながら37℃にて200rpmでインキュベートした。タンパク質発現の誘導は、1mLのIPTG(1M)を加えることによって開始させた。誘導に続いて、インキュベーター温度を25℃に低下させ、培養物を振盪しながら200rpmで一晩静置した。一定分量(1.5mL)の25℃の一晩培養物をSDS−PAGEによる発現分析のために取り出し、残りの培養物は、さらなる処理のために遠心分離によって収集した。SDS−PAGE分析から、正確なサイズのpelB−GmSHC融合タンパク質が発現したことが観察された。
pelBリーダー配列を含め、大腸菌(E.coli)細胞周辺腔中へのGmSHC酵素の輸送を促進し、それによって、細胞を取り巻く環境において、GmSHC酵素を基質にとってより利用可能であるようにした。このように、スクリーニングアッセイを行い、GmSHCと比較するように、pelB−GmSHCを含有する全細胞懸濁液によってアンブロキサンへのホモファルネソールの変換を分析した。反応物は、全細胞、100μlのクエン酸ナトリウム(1M)、pH4.9、可溶化緩衝液(0.05MのTris−Cl、pH8.0、0.01MのMgCl2、1%v/v TRITON X−100)中の100μlのホモファルネソール(100mM)、および800μlの可溶化緩衝液を含んだ。反応物を37℃、200rpmでインキュベートし、試料を16時間および80時間後に取り出した。GC分析の前に、試料を2容のn−ヘプタンで抽出した。1mgの全細胞当たりの平均変換面積%を計算した。結果によれば、野生型GmSHC細胞懸濁液は、1時間当たり1mgの全細胞当たり平均0.033面積%を実現した一方、pelB−GmSHC細胞懸濁液は、アンブロキサンへのホモファルネソールの変換をもたらさなかったことが示された。したがって、pelBリーダー配列は、GmSHCの活性に悪影響を与えたようであった。
上記で示したように、GmSHCを発現している全細胞は、ホモファルネソールをアンブロキサンへと生物変換した。したがって、発酵における変換が達成することができるかを決定した。野生型GmSHCまたはR.パルストリス(R.palustris)SHC(RpSHC;国際公開第2010/139719号パンフレット)を発現している細胞を、下記の様式で成長および発現させた。一晩、GmSHCおよびRpSHCをコードする核酸を担持する細胞の10mLの(LB培地+カナマイシン、37℃、200RPM)開始培養物を使用して、1LのLB培地+カナマイシンを播種した。1Lの培養物を37℃、200rpmにて概ね4時間インキュベートした。それに続いて、1mLのIPTG(1M)を、培養物に加えて、SHCタンパク質発現を誘発し、培養物のインキュベーション温度を一晩のインキュベーションのために25℃に低減させた。
ホモロジーモデリング。ホモロジーモデリングを使用して、GmSHCの三次元構造を構築した。使用したテンプレートは結晶1GSZ(Lenhart,et al.(2002)Chem.Biol.9:639−45)および3SQC(Wendt,et al.(1999)J.Mol.Biol.286:175−87)であったが、これらはGmSHC配列の95%と44%および43%の配列同一性を共有する。
SHCは、二量体の3Dアレンジメントを採択する内在性モノトピック膜タンパク質である。各モノマーは、2つの高度に安定なα/α−バレルドメインを構築する多数のα−ヘリックスを密接に接続するQWモチーフによって特性決定される(Wendt et al.(1999)J.Mol.Biol.286:175−87)。活性中心空洞は2つのα/α−バレルドメイン内に埋没され、酵素の膜浸漬領域(membrane-immersed region)であることが示唆される内側の疎水性チャネルを通してそのアクセスは可能である(Lenhart,et al.(2002)Chem.Biol.9:639−45)。チャネルおよび活性中心空洞は、基質認識に関与している狭い狭窄部によって分離されている(Lenhart,et al.(2002)Chem.Biol.9:639−45)。GmSHCについて、これらの残基は、Phe176、Met184、Phe457およびCys458に対応する。保存DXDDモチーフを構成する残基(Wendt et al.(1999)J.Mol.Biol.286:175−87)は、活性中心空洞の上部において見出される。それらの残基の1つは、第1のプロトン供与体であるAsp396であり、これは二重結合C2=C3へとプロトンを供与することによってホモファルネソールの環化を開始させる。DXDDモチーフの後に、強力なカチオン−π相互作用によってカチオン性中間体を安定化させることに関与しているトリプトファン、チロシンおよびフェニルアラニン残基が続く(Dougherty (1996)Science 271:163−8)。空洞の底上には、最後のプロトン受容体である負に帯電している残基があり、これはヒドロキシル基からプロトンを受け、それによって、第3の環の閉環およびアンブロキサンの形成がもたらされる。
Claims (8)
- スクアレンホペンシクラーゼ(SHC)をコードする核酸分子を含む組換えベクターであって、前記スクアレンホペンシクラーゼ(SHC)のアミノ酸配列が、配列番号2、または配列番号2と少なくとも90%の配列同一性を有するアミノ酸配列である、組換えベクター。
- 前記SHCが、配列番号2に対して、45位、46位、54位、86位、139位、142位、178位、184位、194位、239位、278位、326位、335位、386位、455位、460位、603位、623位、624位、656位、658位またはこれらの組合せにおいてアミノ酸置換を含む、請求項1に記載の組換えベクター。
- 請求項1に記載の組換えベクターを含む組換え宿主細胞。
- 配列番号2と少なくとも90%の配列同一性を含み、かつ配列番号2に対して、45位、46位、54位、86位、139位、142位、178位、184位、194位、239位、278位、326位、335位、386位、455位、460位、603位、623位、624位、656位、658位またはこれらの組合せにおいてアミノ酸置換を含む、組換えスクアレンホペンシクラーゼ(SHC)。
- アンブロキサン(ambroxan)を生成する方法であって、
(a)スクアレンホペンシクラーゼ(SHC)を発現している組換え宿主細胞へとホモファルネソールを提供することであって、前記スクアレンホペンシクラーゼ(SHC)のアミノ酸配列が、配列番号2、または配列番号2と少なくとも90%の配列同一性を有するアミノ酸配列であることと、
(b)前記SHCによって生成されたアンブロキサンを収集することと、
を含む、方法。 - 前記ホモファルネソールが、可溶化剤の存在下で提供される、請求項5に記載の方法。
- 前記可溶化剤が、非イオン性界面活性剤を含む、請求項6に記載の方法。
- 前記ホモファルネソールが、(3E,7E)ホモファルネソールを含む、請求項5に記載の方法。
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GB201507207D0 (en) | 2015-04-24 | 2015-06-10 | Givaudan Sa | Enzymes and applications thereof |
US11091752B2 (en) * | 2017-02-24 | 2021-08-17 | International Flavors & Fragrances Inc. | Squalene hopene cyclase and use thereof for producing ambroxan |
CN109402100B (zh) * | 2018-11-12 | 2021-10-01 | 中国海洋大学 | 一种新型角鲨烯何帕烯环化酶及其应用 |
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GB201917694D0 (en) | 2019-12-04 | 2020-01-15 | Givaudan Sa | Enzyme mediated process |
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WO2018157021A1 (en) | 2018-08-30 |
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US20200385700A1 (en) | 2020-12-10 |
US11091752B2 (en) | 2021-08-17 |
BR112019015747A2 (pt) | 2020-03-17 |
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CN110312796A (zh) | 2019-10-08 |
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