DK3097113T3 - Hidtil ukendt cytochrom p450-polypeptid med forøget enzymatisk aktivitet - Google Patents

Hidtil ukendt cytochrom p450-polypeptid med forøget enzymatisk aktivitet Download PDF

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DK3097113T3
DK3097113T3 DK15702160.1T DK15702160T DK3097113T3 DK 3097113 T3 DK3097113 T3 DK 3097113T3 DK 15702160 T DK15702160 T DK 15702160T DK 3097113 T3 DK3097113 T3 DK 3097113T3
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val
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DK15702160.1T
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Marine Bertin
Bruno Dumas
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Sanofi Sa
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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.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0079—Steroid 11 beta monooxygenase (P-450 protein)(1.14.15.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • C12P33/00—Preparation of steroids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15004—Steroid 11-beta-monooxygenase (1.14.15.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y118/00—Oxidoreductases acting on iron-sulfur proteins as donors (1.18)
    • C12Y118/01—Oxidoreductases acting on iron-sulfur proteins as donors (1.18) with NAD+ or NADP+ as acceptor (1.18.1)
    • C12Y118/01002—Ferredoxin-NADP+ reductase (1.18.1.2)

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
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  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Steroid Compounds (AREA)

Claims (14)

1. Isoleret cytochrom P450-monooxygenase, der omfatter eller består af en aminosyresekvens, der er mindst 80 % identisk med SEQ ID NO: 1, hvor sekvensen omfatter en threonin i en position svarende til position 225 i SEQ ID NO: 1 og en asparaginsyre i en position svarende til position 289 i SEQ ID NO: 1.
2. Enzym ifølge krav 1, der består af sekvensen SEQ ID NO: 1.
3. Isoleret nukleinsyre, der omfatter eller består af en nukleotidsekvens, der koder for et enzym ifølge krav 1 eller 2.
4. Vektor, der omfatter en nukleinsyre ifølge krav 3, som er operativt forbundet med ekspressionskontrolsekvenser.
5. Vektor ifølge krav 4, der yderligere omfatter en nukleinsyresekvens, der koder for adrenodoxin (Adx), og/eller en nukleinsyresekvens, der koder for adrenodoxinreductase (AdR).
6. Værtscelle, der indeholder en nukleinsyre ifølge krav 3 eller en vektor ifølge krav 4 eller 5.
7. Værtscelle ifølge krav 6, som er en genmodificeret mikroorganisme.
8. Værtscelle ifølge krav 6 eller 7, som er Saccharomyces cerevisiae.
9. Genmodificeret mikroorganisme, der er i stand til at omdanne et substrat valgt fra gruppen, der består af polycykliske og umættede monoalkoholer med en alifatisk sidekæde, såsom kolesterol, en kolesterolanalog og et kolesterolderivat, til et steroidhormonforstadie, hvor mikroorganismen omfatter en nukleinsyre ifølge krav 3 og eventuelt en nukleinsyresekvens, der koder for adrenodoxin (Adx), og/eller en nukleinsyresekvens, der koder for adrenodoxinreductase (AdR).
10. In vitro-fremgangsmåde til fremstilling af et enzym ifølge krav 1 eller 2, hvilken fremgangsmåde omfatter trinene: a) dyrkning af en værtscelle ifølge et hvilket som helst af kravene 6 til 8 under betingelser, der er egnet til opnåelse af ekspression af enzymet ifølge krav 1 eller 2, og b) indvinding af det udtrykte enzym.
11. Anvendelse af et enzym ifølge krav 1 eller 2 til fremstilling af et steroidhormonforstadie.
12. Fremgangsmåde til fremstilling af et steroidhormonforstadie, som omfatter trinene: a) tilvejebringelse af en mikroorganisme ifølge et hvilket som helst af kravene 7 til 9, b) dyrkning af mikroorganismen under betingelser, der åbner mulighed for ekspression af et enzym ifølge krav 1 eller 2, c) etablering af kontakt mellem mikroorganismekulturen opnået i trin b) og et substrat valgt fra gruppen, der består af polycykliske og umættede monoalkoholer med en alifatisk sidekæde, såsom kolesterol, en kolesterolanalog og et kolesterolderivat, under betingelser, der åbner mulighed for fremstilling ved hjælp af mikroorganismen af et steroidhormonforstadie fra substratet, og d) indvinding af det dannede steroidhormonforstadie.
13. Fremgangsmåde til fremstilling af et steroidhormonforstadie, som omfatter trinene: a) etablering af kontakt mellem et enzym ifølge krav 1 eller 2 og et isoleret adrenodoxin (Adx)-polypeptid, et isoleret adrenodoxinreductase (AdR)-polypeptid og et substrat valgt fra gruppen, der består af polycykliske og umættede monoalkoholer med en alifatisk sidekæde, såsom kolesterol, en kolesterolanalog og et kolesterolderivat, under betingelser, der åbner mulighed for transformation af substratet til et steroidhormonforstadie, og b) indvinding af det opnåede steroidhormonforstadie.
14. Fremgangsmåde ifølge krav 12 eller 13, hvor steroidhormonforstadiet er pregnenolon.
DK15702160.1T 2014-01-20 2015-01-19 Hidtil ukendt cytochrom p450-polypeptid med forøget enzymatisk aktivitet DK3097113T3 (da)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14305071 2014-01-20
PCT/EP2015/050866 WO2015107185A1 (en) 2014-01-20 2015-01-19 Novel cytochrome p450 polypeptide with increased enzymatic activity

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DK3097113T3 true DK3097113T3 (da) 2019-04-23

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US (2) US9765307B2 (da)
EP (1) EP3097113B1 (da)
JP (1) JP6608372B2 (da)
KR (1) KR20160108540A (da)
CN (1) CN106061996B (da)
AU (1) AU2015207519B2 (da)
BR (1) BR112016016586A2 (da)
CA (1) CA2936954A1 (da)
DK (1) DK3097113T3 (da)
ES (1) ES2724373T3 (da)
HU (1) HUE043216T2 (da)
IL (1) IL246801A0 (da)
MX (1) MX2016009466A (da)
RU (1) RU2677997C2 (da)
SG (1) SG11201605774QA (da)
TW (1) TW201613959A (da)
WO (1) WO2015107185A1 (da)

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CN111019913B (zh) * 2019-12-30 2023-07-07 苏州汇桢生物技术有限公司 一种p450scc突变体及核苷酸、表达载体、宿主细胞
CN112457412B (zh) * 2020-11-26 2022-10-14 浙江工业大学 一种人工电子传递系统及其在促进p450酶羟基化反应中的应用

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US4861719A (en) 1986-04-25 1989-08-29 Fred Hutchinson Cancer Research Center DNA constructs for retrovirus packaging cell lines
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
IL90207A (en) * 1988-05-06 1994-07-31 Roussel Uclaf Biochemical oxidation of steroids and genetically engineered cells to be used therefor
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AU6248994A (en) 1993-02-22 1994-09-14 Rockefeller University, The Production of high titer helper-free retroviruses by transient transfection
FR2712812B1 (fr) 1993-11-23 1996-02-09 Centre Nat Rech Scient Composition pour la production de produits thérapeutiques in vivo.
IL116816A (en) 1995-01-20 2003-05-29 Rhone Poulenc Rorer Sa Cell for the production of a defective recombinant adenovirus or an adeno-associated virus and the various uses thereof
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CN1343779A (zh) * 2000-09-19 2002-04-10 上海博德基因开发有限公司 一种新的多肽——含有过氧化物酶特征的蛋白12.21和编码这种多肽的多核苷酸
CN1283800C (zh) * 2004-11-29 2006-11-08 浙江大学 能催化吲哚生成靛蓝的P450BM－3Glu435Asp变体基因及其用途
CN101333521B (zh) * 2008-04-25 2010-12-15 浙江大学 细胞色素p450bm-3d168w变体酶以及应用其制备靛玉红的方法
US20140011206A1 (en) * 2011-02-09 2014-01-09 The Regents Of The University Of California Determination of oocyte quality

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BR112016016586A2 (pt) 2017-10-03
WO2015107185A1 (en) 2015-07-23
TW201613959A (en) 2016-04-16
HUE043216T2 (hu) 2019-08-28
EP3097113B1 (en) 2019-01-02
MX2016009466A (es) 2017-04-25
ES2724373T3 (es) 2019-09-10
AU2015207519B2 (en) 2018-11-08
CN106061996A (zh) 2016-10-26
KR20160108540A (ko) 2016-09-19
EP3097113A1 (en) 2016-11-30
JP2017504334A (ja) 2017-02-09
RU2016133705A (ru) 2018-03-05
US20170342389A1 (en) 2017-11-30
RU2016133705A3 (da) 2018-08-03
JP6608372B2 (ja) 2019-11-20
IL246801A0 (en) 2016-08-31
AU2015207519A1 (en) 2016-08-18
US9765307B2 (en) 2017-09-19
US20150225703A1 (en) 2015-08-13
RU2677997C2 (ru) 2019-01-22
CN106061996B (zh) 2021-03-09
US10155934B2 (en) 2018-12-18
CA2936954A1 (en) 2015-07-23
SG11201605774QA (en) 2016-08-30

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