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WO1996027678A1 - Procede d'hydroxylation d'alcanes a longue chaine, d'acides gras et d'autres composes alkyle - Google Patents

Procede d'hydroxylation d'alcanes a longue chaine, d'acides gras et d'autres composes alkyle Download PDF

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Publication number
WO1996027678A1
WO1996027678A1 PCT/DE1996/000410 DE9600410W WO9627678A1 WO 1996027678 A1 WO1996027678 A1 WO 1996027678A1 DE 9600410 W DE9600410 W DE 9600410W WO 9627678 A1 WO9627678 A1 WO 9627678A1
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WO
WIPO (PCT)
Prior art keywords
long
fatty acids
alkyl compounds
reductase
cytochrome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DE1996/000410
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German (de)
English (en)
Inventor
Thomas Zimmer
Kristina Kaminski
Wolf-Hagen Schunck
Eva KÄRGEL
Ulrich Scheller
Stephan Mauersberger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Original Assignee
Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Publication of WO1996027678A1 publication Critical patent/WO1996027678A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12N9/0042—NADPH-cytochrome P450 reductase (1.6.2.4)
    • 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)
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Definitions

  • the invention relates to a process for the hydroxylation of long-chain alkanes and alkyl compounds, in particular fatty acids. It also relates to a vector for implementing this method.
  • the field of application of the invention is biotechnology, in particular the extraction of fatty acid oxidation products such as hydroxy fatty acids and long-chain dicarboxylic acids.
  • Monooxygenases of the cytochrome P450 type are common in organisms on the entire phylogenetic scale. They catalyze NAD (P) H- and 0_-dependent reactions in various biosynthetic and catabolic pathways.
  • NAD NAD
  • the primary sequences of more than 220 different P450 forms are currently known. These are combined into a superfamily based on characteristic sequence features (review by Nelson et al., DNA Cell Biol. 12 [1993] 1).
  • yeasts such as the alkane-utilizing species Candida maltosa (Vogel et al., Eur. J. Cell Biol. 57 [1992] 285) and in higher eukaryotes, the majority of the P450 forms are localized in the endoplasmic reticulum. The electron transfer required in the reaction cycle takes place via a membrane-based NADPH cytochrome P450 reductase. P450 and reductase components together form the active monooxygenase system.
  • Various strategies have been described in scientific publications and in the patent literature for the functional heterologous expression of such two-component systems (Murakami et al., DNA 5 [1986] 1; Urban et al., Biochem. Soc. Trans.
  • the CYP52 family is the most extensive P450 family in microorganisms.
  • 8 different alkane-inducible P450 forms have already been identified for the yeast C. maltosa, which differ significantly in their substrate specificity from various alkyl compounds (Schunck et al., DD-WP 271 339; Schunck et al., DD-WP 292 022) .
  • the associated reductase component was also cloned from this organism.
  • the object of the invention is achieved by monooxygenase systems which consist of cytochrome P450 and NADPH-cytochrome P450 reductase, which are expressed simultaneously in Saccharomyces.
  • the long-chain alkanes, fatty acids or alkyl compounds are brought into cultivation solutions from such genetically modified Saccharomyces, the expressed enzymes causing a regioselective hydroxylation. After the reaction, the hydroxylation products are separated off.
  • a preferred yeast species for the invention is Saccharomyces cerevisiae
  • a preferred enzyme system are cytochrome P450 forms of the CYP 52 family and Candida maltosa NADPH cytochrome P450 reductase.
  • the process is carried out at low temperature (30-40 ° C), the hydroxylation is usually largely complete after one hour. First of all, regioselective monohydroxylation occurs, predominantly at the terminal or subterminal carbon atom. The point of entry of the OH group is determined by the P450 form used and by the reaction time.
  • the monohydroxylated product is further hydroxylated during further cultivation.
  • hydroxy compounds hydroxycarboxylic acids or dicarboxylic acids are obtained.
  • ⁇ , ⁇ * -dicarboxylic acid is obtained from an alkane.
  • the respective turnover is checked by means of thin layer chromatography, the reaction is preferably terminated with acids, for example with dilute sulfuric acid, as soon as the desired product is formed in sufficient quantity.
  • the essence of the invention is the vector according to claim 7. It is based on the YEp ⁇ l vector and contains reductase cDNA between the restriction sites Sall and BamHI. A second
  • Expression cassette consisting of the GALIO promoter, the coding sequence of cytochrome P450 and the ADH1 terminator, is ligated into the restriction site Nrul of this vector. Starting materials and the structure of the vector are shown in Figure 1.
  • the process according to the invention is distinguished by high hydroxylation rates.
  • lauric acid is hydroxylated about 20 times faster than in the single expression of natural P450 forms, in which only a molar ratio of P450: reductase of approx. 32: 1 is given due to the low expression of the endogenous reductase (see Table 2).
  • CGGGATCCAAGGGAGAGCGTCGAC-3 'amplified, digested by means of restrictionases Kpn I and BamH I and ligated into the restriction sites Kpn I and BamH I of the vector pUCBM21 (Boehringer Mannheim).
  • the resulting vector is used to amplify the ADH1 terminator fragment which is primed from the vector pAAH5 (Ammerer, Meth. Enzym. in the BamH I / EcöR I location.
  • the Sal I site must finally be destroyed by digestion with EcöR V, treatment with T4 DNA polymerase and religation.
  • the restriction sites Sal I and BamH I the cDNAs of P450Cml and P450Cm2 are integrated between the GALIO promoter and the ADH1 terminator.
  • the microsomes obtained after P450 and reductase co-expression can increase the lauric acid hydroxylase activity by up to 20 times and increase the n- Hexadecane hydroxylase activity can be measured.
  • the increase in the reaction rate can be attributed to an optimized molar P450: reductase ratio, which is 1: 3 for coexpression, but only 32: 1 (YEp51Cml) or 14: 1 (YEp51Cm2) for single expression.
  • Fig. 1 Construction of plasmids for the simultaneous expression of cytochrome P450 forms and the NADPH cytochrome P450 reductase.
  • Each of the complete P450 expression cassettes can be cleaved from the vector pUCBM21-P450 using the restriction sites Asc I and Not I and ligated into the vector YEp ⁇ lR.
  • the specified restriction sites are: A - Asc I, B - BamH I, N - Not I, S- Sal I.
  • Fig.2 Thin-layer chro atographic analysis of the product pattern in the biotransformation of lauric acid using intact yeast cells.
  • the plasmids used to transform Saccharomyces cerevisiae and the reaction times are: YEp51 - 20 min (lanes 1 and 2), YEp51Cm2-R - 20 min (lanes 3 and 4), YEp51Cm2-R - 1.5 h (lanes 5 and 6 ).
  • the product analysis can be carried out separately according to cell pellet (lanes 1, 3 and 5) and supernatant (lanes 2, 4 and 6).

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé d'hydroxylation microbienne qui sert à oxyder sélectivement des régions d'alcanes à longue chaîne, d'acides gras et d'autres composés alkyle. Le procédé doit permettre d'obtenir de manière aisée un bon rendement en produits d'oxydation, notamment des acides gras hydroxylés et des acides dicarboxyliques à longue chaîne. L'invention a pour objet de modifier par génie génétique des levures de sorte qu'elles expriment les enzymes requises lorsqu'elles sont cultivées. Le procédé se caractérise en ce que l'on traite les alcanes à longue chaîne, les acides gras et les autres composés alkyle avec des systèmes à monooxygénase constitués de cytochrome P450 et de NADPH-cytochrome P450-réductase, les produits d'hydroxylation étant ensuite isolés. Les systèmes à monooxygénase sont produits dans le mélange de réaction par expression simultanée de leurs composants dans des levures, de préférence la saccharomyces cerevisiae. L'invention porte essentiellement sur un vecteur de modification par génie génétique de saccharomyces, qui sur la base de la structure fondamentale Yep 51 contient l'ADNc de la réductase entre les sites de restriction Sa1I et BamHI et une deuxième cassette d'expression liée dans le site de restriction NruI et constituée du promoteur GAL10, de la séquence de codage de cytochrome P450 et du terminateur ADH1.
PCT/DE1996/000410 1995-03-03 1996-03-01 Procede d'hydroxylation d'alcanes a longue chaine, d'acides gras et d'autres composes alkyle Ceased WO1996027678A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19507546.3 1995-03-03
DE19507546A DE19507546C2 (de) 1995-03-03 1995-03-03 Verfahren zur regioselektiven Hydroxylierung von langkettigen Alkanen, Fettsäuren und anderen Alkylverbindungen

Publications (1)

Publication Number Publication Date
WO1996027678A1 true WO1996027678A1 (fr) 1996-09-12

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PCT/DE1996/000410 Ceased WO1996027678A1 (fr) 1995-03-03 1996-03-01 Procede d'hydroxylation d'alcanes a longue chaine, d'acides gras et d'autres composes alkyle

Country Status (2)

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DE (1) DE19507546C2 (fr)
WO (1) WO1996027678A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065061A3 (fr) * 1999-04-24 2001-03-15 Max Delbrueck Centrum Sequences d'acide nucleique issues de levures de candida, codant des polypeptides b5 cytochromes
EP1326984B1 (fr) * 2000-10-16 2012-03-28 Basf Se Mono-oxygenases de type cytochrome p450 constituees de bacteries thermophiles

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL133931A0 (en) * 1997-07-21 2001-04-30 Du Pont Transformed yeast strains and their use for the production of monoterminal and diterminal aliphatic carboxylates
EP1273663A3 (fr) * 1997-07-21 2004-02-04 E.I. Dupont De Nemours And Company Souches de lévure transformées et leur uitlisation dans la production de carboxylates aliphatiques à terminaison unique et à terminaison double
WO2000003008A2 (fr) * 1998-07-10 2000-01-20 Technische Universität Dresden Cellules yarrowia lipolytica haploïdes ou diploïdes recombinees pour l'expression heterologue fonctionnelle de systemes du cytochrome p450
MY126592A (en) * 1999-07-27 2006-10-31 Basf Ag Novel cytochrome p450 monooxygenases and their use for the oxidation of organic compounds
RU2285044C2 (ru) * 1999-07-27 2006-10-10 Басф Акциенгезельшафт Новые цитохром р450-монооксигеназы и их применение для окисления органических соединений

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001564A1 (fr) * 1992-07-03 1994-01-20 Orsan Souche de levure permettant la co-expression d'une activite mono-oxygenase de cytochrome p450 de plante et d'une nadph-cytochrome p450-reductase endogene ou heterologue et son utilisation a des fins de bioconversion

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD292022A5 (de) * 1990-02-16 1991-07-18 Adw Zi F. Molekularbiologie,De Verfahren zur gewinnung des strukturgens eines fettsaeure- und n-alkanehydroxylierenden cytochrom p-450
WO1991014781A1 (fr) * 1990-03-19 1991-10-03 Henkel Research Corporation METHODE POUR AUGMENTER L'ACTIVITE D'OMEGA-HYDROXYLASE CHEZ LE $i(CANDIDA TROPICALIS)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001564A1 (fr) * 1992-07-03 1994-01-20 Orsan Souche de levure permettant la co-expression d'une activite mono-oxygenase de cytochrome p450 de plante et d'une nadph-cytochrome p450-reductase endogene ou heterologue et son utilisation a des fins de bioconversion

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MICHAIL A. ALTERMAN ET AL.: "Fatty Acid Discrimination and Omega-Hydroxylation by Cytochrome P450 4A1 and a Cytochrome P4504A1/NADPH-P450 Reductase Fusion Protein", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 320, no. 2, July 1995 (1995-07-01), XP002060065 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065061A3 (fr) * 1999-04-24 2001-03-15 Max Delbrueck Centrum Sequences d'acide nucleique issues de levures de candida, codant des polypeptides b5 cytochromes
EP1326984B1 (fr) * 2000-10-16 2012-03-28 Basf Se Mono-oxygenases de type cytochrome p450 constituees de bacteries thermophiles

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Publication number Publication date
DE19507546A1 (de) 1996-09-12
DE19507546C2 (de) 2001-05-03

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