JP2023500967A - ケトレダクターゼ突然変異体及びキラルアルコールの生産方法 - Google Patents
ケトレダクターゼ突然変異体及びキラルアルコールの生産方法 Download PDFInfo
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
Description
R1及びR2は、それぞれ独立して、C1~C8アルキル基、C5~C10シクロアルキル基、C5~C10アリール基又はC5~C10ヘテロアリール基であり、若しくは、R1及びR2は、カルボニル基上の炭素とともにC5~C10複素環式基、C5~C10炭素環式基又はC5~C10ヘテロアリール基を形成し、C5~C10複素環式基及びC5~C10ヘテロアリール基中のヘテロ原子は、それぞれ独立して、窒素、酸素及び硫黄から選ばれる少なくとも1種であり、C5~C10アリール基のうちのアリール基、C5~C10ヘテロアリール基のうちのヘテロアリール基、C5~C10炭素環式基のうちの炭素環式基又はC5~C10複素環式基のうちの複素環式基は、それぞれ独立して、未置換又はハロゲン、アルコキシ基又はアルキル基のうちの少なくとも1つの基によって置換され、
好ましくは、ケトン系化合物は下記構造式を有し、
より好ましくは、ケトン系化合物は下記構造式を有する。
「ケトレダクターゼ」及び「KRED」は、本出願において相互に交換して使用することができ、ケトン基を対応するアルコールに還元することができるポリペプチドを意味する。具体的には、本出願のケトレダクターゼポリペプチドは、ケトン化合物を対応するアルコール産物に立体特異的に還元することができる。このポリペプチドは、通常、還元剤として補因子である還元型ニコチンアミドアデニンジヌクレオチド(NADH)又は還元型ニコチンアミドアデニンジヌクレオチドリン酸を利用する。本出願において、ケトレダクターゼには、天然に存在する(野生型)ケトレダクターゼと、人工的な処理により生産された非天然に存在するケトレダクターゼ突然変異体とが含まれる。
MARVASKVAIVSGAANGIGKATAQLLAKEGAKVVIGDLKEEEGQKAVAEIKAAGGEAAFVKLNVTDEAAWKAAIGQTLKLYGRLDIAVNNAGIAYSGSVESTSLEDWRRVQSINLDGVFLGTQVAIEAMKKSGGGSIVNLSSIAGMVGDPMYAAYNASKGGVRLFTKSAALHCAKSGYKIRVNSVHPGYIWTPMVAGQVKGDAAARQKLVDLHPIGHLGEPNDIAYGILYLASDESKFVTGSELVIDGGYTAQ
好ましくは、ケトン系化合物の構造は、下記構造式を有し、
より好ましくは、ケトン系化合物は下記構造式を有する。
基質としての(R)-1-(2,4-ジクロロアセトフェノン)母液60mMは、下記の通りである:56.7mgを秤量して0.1Mリン酸塩緩衝液(PB)pH7.0 Buffer5mLに溶解し、50℃の水浴鍋に入れて溶解した。
まず酵素を加え、次に、基質である(R)-1-(2,4-ジクロロアセトフェノン)、NADH及びBufferの混合物を加え、マイクロプレートリーダに入れて、30℃で、340nm波長で酵素活性を検出した。
「テンプレート」及び突然変異体をそれぞれ45℃、53℃で1h保温した後に、その活性を測定し、保温していない場合と比較した。安定性は、残存活性と初期活性とのパーセンテージとして表される。全ての突然変異体の熱安定性は53℃で測定され、結果は表2に示される。
飽和突然変異を組み合わせることで、突然変異部位同士に相乗作用がある突然変異体が得られ、且つそのアミノ酸の組成を最適化して組み合わせることができる。Q44R+N156S+K200H+G201Dをテンプレートとして、突然変異点を組み合わせ、このとき、酵素液の処理条件として65℃で1h処理した。次にその活性を測定し、保温していない場合と比較した。安定性は、その残存活性と初期活性とのパーセンテージとして表される。
Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39Iをテンプレートとして突然変異を続けた。このときの酵素液処理条件として71℃で1h処理し、その後、活性を測定し、保温していない場合と比較し、安定性はその残存活性と初期活性とのパーセンテージとして表される。
酵素の安定性が向上する過程で、酵素全体の剛性が向上するので、安定性が向上した突然変異体はグルタルアルデヒドなどの化学物質、メタノールなどの有機溶媒に対する耐性が同時に向上することを示す。これは突然変異体全体の剛性向上の外的表現でもある。
突然変異体Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+G36Cを用いて、様々な基質反応検証を行い、結果を表6に示す。
Claims (18)
- SEQ ID NO:1に示される配列においてアミノ酸突然変異が起こった配列を有し、前記突然変異の部位はK200Hを含むことを特徴とするケトレダクターゼ突然変異体。
- 前記突然変異の部位は、さらに、少なくとも、A15、K28、G36、K39、G43、Q44、A46、V47、F59、K61、T65、K71、A94V、A144、M146、Y152、N156、I86、K208、及びK237のうちの1つを含み、又は前記ケトレダクターゼ突然変異体のアミノ酸配列は、突然変異が起こったアミノ酸配列における突然変異部位を有し、且つ突然変異が起こったアミノ酸配列と95%以上の相同性を有するアミノ酸配列であることを特徴とする請求項1に記載されたケトレダクターゼ突然変異体。
- 前記突然変異の部位は、さらに、少なくとも、A15C、K28A/E/M/Q/R/S、G36C、K39I/V、G43C/M、Q44R、A46C、V47C、F59C、K61E/H、T65A、K71R、A94V、A144T、M146I、Y152F、N156S、I86V、K208R、及びK237Eの突然変異のうちの1つを含む、ことを特徴とする請求項2に記載されたケトレダクターゼ突然変異体。
- 前記アミノ酸突然変異は、Q44R+N156S+K200H、Q44R+N156S+K200H+G201D、Q44R+N156S+K200H+G201D+M146I、Q44R+N156S+K200H+G201D+M146I+K61H、Q44R+N156S+K200H+G201D+M146I+K61H+I86V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43Mという部位組み合わせ突然変異のいずれかを含む、ことを特徴とする請求項3に記載されたケトレダクターゼ突然変異体。
- 起こった前記アミノ酸突然変異は、I86V+M146I+K200H、M146I+K200H、M146L+N156S+K200H、M146L+K200H、K200H+G201D、Q44R+M146I+K200H、K61E+K200H+K237E、I86V+M146I+K200H、M146I+K200H+G201D、M146L+K200H+G201D、N156S+K200H+G201D、K200H+G201D+K237E、K28E+M146I+K200H+G201D、K28E+M146L+K200H+G201D、K28E+N156S+K200H+G201D、Q44R+M146L+K200H+G201D、Q44R+N156S+K200H+G201D、I86V+M146I+K200H+K61H、I86V+M146I+K200H+K208R、I86V+M146I+K200H+G201D、I86V+M146L+K200H+G201D、Q44R+N156S+K200H+G201D、Q44R+N156S+K200H+G201D+M146I、Q44R+N156S+K200H+G201D+M146I+K61H、Q44R+N156S+K200H+G201D+M146I+K61H+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+T65A、Q44R+N156S+K200H+G201D+M146I+K61H+I86V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R、Q44R+N156S+K200H+G201D+M146I+K61H+I86V+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+I86V+K39I、Q44R+N156S+K200H+G201D+M146I+K61H+I86V+T65A、Q44R+N156S+K200H+G201D+M146I+K61H+I86V+A94V、Q44R+N156S+K200H+G201D+M146I+K61H+I86V+K208R、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+K39I、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+T65A、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+V47C+F59C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+G43C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28E、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28R、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28Q、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28M、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28A、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+K28S、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+I86V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+G36C、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+I39V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+K71R、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+A144T、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+Y152F、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+K28E、及びQ44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+G36Cという部位組み合わせ突然変異のいずれかを含む、ことを特徴とする請求項1に記載されたケトレダクターゼ突然変異体。
- 請求項1から請求項5のいずれか1項に記載されたケトレダクターゼ突然変異体をコードする、ことを特徴とするDNA分子。
- 請求項6に記載されたDNA分子が連結されている、ことを特徴とする組換えプラスミド。
- pET-22a(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18又はpUC-19である、ことを特徴とする請求項7に記載された組換えプラスミド。
- 請求項7又は8に記載された組換えプラスミドを含有する、ことを特徴とする宿主細胞。
- 原核細胞又は真核細胞を含み、好ましくは、前記原核細胞は大腸菌である、ことを特徴とする請求項9に記載された宿主細胞。
- プロキラルケトン系化合物の還元反応をケトレダクターゼで触媒してキラルアルコールを製造するステップを含むキラルアルコールの製造方法であって、
前記ケトレダクターゼは請求項1から請求項5のいずれか1項に記載されたケトレダクターゼ突然変異体である、ことを特徴とするキラルアルコールの製造方法。 - 前記キラルケトン系化合物は、下記構造式を有し、
R1及びR2は、それぞれ独立して、C1~C8アルキル基、C5~C10シクロアルキル基、C5~C10アリール基又はC5~C10ヘテロアリール基であり、若しくは、R1及びR2は、カルボニル基上の炭素とともにC5~C10複素環式基、C5~C10炭素環式基又はC5~C10ヘテロアリール基を形成し、前記C5~C10複素環式基及びC5~C10ヘテロアリール基中のヘテロ原子は、それぞれ独立して、窒素、酸素及び硫黄から選ばれる少なくとも1種であり、前記C5~C10アリール基のうちのアリール基、C5~C10ヘテロアリール基のうちのヘテロアリール基、C5~C10炭素環式基のうちの炭素環式基又はC5~C10複素環式基のうちの複素環式基は、それぞれ独立して、未置換又はハロゲン、アルコキシ基又はアルキル基のうちの少なくとも1つの基によって置換され、
好ましくは、前記ケトン系化合物は下記構造式を有し、
より好ましくは、前記ケトン系化合物は下記構造式を有する
- ケトレダクターゼを用いてケトン系化合物を還元反応させてキラルアルコールを生産する反応系には、補酵素、補酵素再生系及び緩衝液がさらに含まれている、ことを特徴とする請求項11に記載された方法。
- 前記反応系において、前記ケトン系化合物の濃度が1g/L~200g/Lである、ことを特徴とする請求項13に記載された方法。
- 前記反応系のpH値が5~9であり、前記反応系の反応温度が4℃~60℃である、ことを特徴とする請求項13に記載された方法。
- 前記補酵素はNADHである、ことを特徴とする請求項13に記載された方法。
- 前記補酵素再生系は、イソプロパノール、補酵素NAD+、及びケトレダクターゼを含む、ことを特徴とする請求項16に記載された方法。
- 前記緩衝液は、リン酸塩緩衝液、Tris-塩酸緩衝液、バルビタールナトリウム-塩酸緩衝液又はクエン酸-クエン酸ナトリウム緩衝液である、ことを特徴とする請求項13に記載された方法。
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WO2021087894A1 (zh) | 2021-05-14 |
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