WO2004013314A1 - Cellules secretant de l'acide r-alpha-lipoique et procede de production par fermentation de l'acide r-alpha-lipoique - Google Patents

Cellules secretant de l'acide r-alpha-lipoique et procede de production par fermentation de l'acide r-alpha-lipoique Download PDF

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Publication number
WO2004013314A1
WO2004013314A1 PCT/EP2003/008178 EP0308178W WO2004013314A1 WO 2004013314 A1 WO2004013314 A1 WO 2004013314A1 EP 0308178 W EP0308178 W EP 0308178W WO 2004013314 A1 WO2004013314 A1 WO 2004013314A1
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lipoic acid
gene
cell
culture medium
cells
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PCT/EP2003/008178
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German (de)
English (en)
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Tobias Dassler
Thomas Maier
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Consortium für elektrochemische Industrie GmbH
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Priority to AU2003260329A priority Critical patent/AU2003260329A1/en
Publication of WO2004013314A1 publication Critical patent/WO2004013314A1/fr

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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
    • C12N9/93—Ligases (6)
    • 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
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • 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
    • C12P11/00—Preparation of sulfur-containing organic compounds

Definitions

  • the invention relates to cells that secrete R- ⁇ -lipoic acid and a method for the fermentative production of R- ⁇ -lipoic acid using these cells.
  • R- ⁇ -lipoic acid is an essential cofactor of certain multienzyme complexes in a large number of pro and eukaryotes.
  • the R- ⁇ -lipoic acid is in each case covalently bound to the ⁇ -amino group of a specific lysine residue of the corresponding enzyme.
  • the R- ⁇ -lipoic acid is part of the E2 subunit of pyruvate dehydrogenase (PDH) [EC 2.3.1.12] and ⁇ -ketoglutarate dehydrogenase (KGDH) [EC 2.3.1.61] and plays there as Redox partners and acyl group carriers play a crucial role in the oxidative decarboxylation of ⁇ -keto acids.
  • PDH pyruvate dehydrogenase
  • KGDH ⁇ -ketoglutarate dehydrogenase
  • Lipoic acid also acts as an aminomethyl carrier in glycine cleavage enzyme systems.
  • ⁇ -Lipoic acid is an optically active molecule with a chiral center at the carbon atom C ⁇ .
  • the R configuration of ⁇ -lipoic acid is the naturally occurring enantiomer. Only this form shows physiological activity as a cofactor of the corresponding enzymes.
  • ⁇ -Lipoic acid can occur both in an oxidized (5- [1, 2] -dithiolan-3-yl-pentanoic acid) and in a reduced form (6, 8-dimercapto-octanoic acid).
  • ⁇ -lipoic acid both forms and the respective salts of ⁇ ⁇ lipoic acid, such as. B. the calcium, potassium, magnesium, sodium or ammonium salt to understand.
  • octanoic acid which is covalently bound to the acyl carrier protein (ACP) serves as a specific precursor in lipoic acid synthesis.
  • ACP acyl carrier protein
  • two sulfur atoms are transferred to the octanoic acid (octanoyl-ACP) activated in this way, producing R- ⁇ -lipoyl-ACP.
  • This reaction will catalyzed by the sulfur transferase lipoic acid synthase [EC 2.8.1.-], the lipA gene product.
  • the amino acid L-cysteine ultimately serves as the sulfur donor.
  • ⁇ -lipoic acid In addition to its relevance as an essential component of enzymes with a central role in metabolism, the importance of ⁇ -lipoic acid for pharmacotherapy and for food supplementation (nutraceutical) was recognized early on: Due to its two thiol groups, ⁇ -lipoic acid has a pronounced effectiveness as an antioxidant and can therefore protect the organism from harmful processes that are induced by oxidative stress. In addition, ⁇ -dihydro-lipoic acid, the reduced form of ⁇ -lipoic acid, is able, due to its property as a strong reducing agent, to regenerate other oxidized natural antioxidants in the body, such as ascorbic acid or ⁇ -tocopherol, directly or indirectly or, if they are deficient, to do so replace.
  • ⁇ -lipoic acid is of central importance in interaction with ascorbic acid, ⁇ -tocopherol and glutathione, the so-called "network of antioxidants".
  • ⁇ -Lipoic acid is also used for the prevention and control of type II diabetes mellitus and its consequential damage, such as. B. polyneuropathy, cataract or cardiovascular disease.
  • the different biological activity of the two enantiomers of ⁇ -lipoic acid is currently the subject of intensive investigations, although it is becoming increasingly clear that the application of the pure R enantiomer of ⁇ -lipoic acid has clear advantages over the S form. It was shown in the in vitro experiment that only the natural R- ⁇ -lipoic acid leads to the formation of functional ⁇ -keto acid dehydrogenases.
  • the S enantiomer even had an inhibitory effect on the stimulation of enzyme activity by R- ⁇ -lipoic acid.
  • the reduction of ⁇ -lipoic acid and thus the regeneration of the antioxidative ⁇ -dihydroliponic acid in the mitochondria is essential for the cell.
  • the mitochondrial NADH-dependent lipoamide reductase of mammals shows an almost 20-fold higher activity with the R enantiomer than with the S form.
  • R- ⁇ -lipoic acid has a significantly stronger effect on insulin-mediated glucose uptake and the glucose metabolism of skeletal muscle cells in insulin-resistant rats.
  • the R form also had an anti-inflammatory effect, while the S form had an analgesic effect. In order to avoid undesirable side effects, it is therefore extremely desirable to apply ⁇ -lipoic acid only in the enantiomerically pure form.
  • ⁇ -lipoic acid takes place exclusively by means of chemical processes, the racemate always being formed from the R and S forms as the end product (Yadav et al., 1990, J. Sei. Ind. Res. 49: 400-409 ).
  • Various processes have been developed to obtain enantiomerically pure R- ⁇ -lipoic acid.
  • the racemate of ⁇ -lipoic acid or one of the synthetic intermediates can either be chemically by means of chiral auxiliary substances (Walton et. Al. 1954, J. Amer. Chem. Soc. 76: 4748; DE 4137773) or enzymatically (Adger et al., 1995, J. Chem.
  • An object of the present invention was to use cells for
  • This object is released by cells which are characterized thereby are that they overexpress a lipoic acid synthase gene (Iip ⁇ gene).
  • Overexpression in the sense of the present invention is preferably to be understood to mean that the lipoic acid synthase gene is at least a factor of 2, preferably at least a factor, in comparison to the respective wild-type cell from which the lipoic acid synthase gene was obtained 5, is increasingly expressed.
  • the lipoic acid synthase gene is preferably a gene with the sequence SEQ ID NO: 1 or a functional variant of this gene.
  • a functional variant is to be understood as a DNA sequence which is derived from the sequence shown in SEQ ID NO: 1 by deletion, insertion or substitution of nucleotides, the enzymatic activity of those encoded by the gene Lipoic acid synthase is retained.
  • the copy number of the lipA gene in a cell can be increased and / or the expression of the lip ⁇ gene can be increased, preferably by suitable promoters.
  • Overexpression of a lip ⁇ gene increases the cell's lipoic acid synthase activity by at least the same factor.
  • a cell according to the invention preferably overexpresses a lipoic acid synthase gene which codes for a protein comprising the sequence ID NO: 2 or functional variants with a sequence homology to SEQ ID NO: 2 greater than 40%.
  • sequence homology to SEQ ID NO: 2 is preferably greater than 60%, particularly preferably the sequence homology to SEQ ID NO: 2 is greater than 80%.
  • a lipA gene can be cloned into a plasmid vector with a multiple copy number per cell (eg pUC19, pBR322, pACYC184 for Escherichia coli) and introduced into the cell.
  • a lip ⁇ gene can be integrated several times into the chromosome of a cell.
  • the known systems with temperate bacteriophages, in- tegrative plasmids or integration via homologous recombination can be used (eg Hamilton et al., 1989, J. Bacteriol. 171: 4617-4622).
  • the invention thus also relates to a plasmid characterized in that it contains a Üp ⁇ gene under the functional control of a promoter.
  • the natural promoter and operator region of the lipA gene can serve as the control region for the expression of a plasmid-encoded lip ⁇ gene, but the enhanced expression of a lipA gene can also be effected in particular by means of other promoters.
  • Corresponding promoter systems which either allow a permanent or a controlled, inducible expression of the lipoic acid synthase gene, as for example in
  • Escherichia coli the constitutive GAPDH promoter of the gap ⁇ gene or the inducible lac, tac, trc, lambda, ara or tet promoters are known to the person skilled in the art (Makrides S. C, 1996, Microbiol. Rev. 60 : 512-538). Such constructs can be used in a manner known per se on plasmids or chromosomally.
  • a plasmid which already contains a promoter for enhanced expression, such as, for example, the inducible tet promoter / repressor system from Escherichia coli.
  • translation start signals such as. B. the ribosome binding site or the start codon of the gene are present in an optimized sequence on the respective construct, or that according to the "codon usage", rare codons are exchanged for more common codons.
  • Cells according to the invention preferably contain a plasmid with a JipA gene and the aforementioned modifications of the regulation signals.
  • the cloning of a lipA gene in a plasmid vector is effected for example by specific 'amplification of a lipA gene by the polymerase chain reaction using specific primers which detect the complete 2ipA gene and subsequent ligation with vector DNA fragments.
  • Cells according to the invention which have an increased expression of a lipA gene in relation to a starting cell and, in connection therewith, an increased lipoic acid synthase activity, can be generated from a starting cell using standard techniques of molecular biology.
  • Lipoic acid synthase genes were identified in a large number of cells.
  • Cells according to the invention can thus preferably be produced from cells of pro- or eukaryotic organisms which are able to synthesize R- ⁇ -lipoic acid themselves (starting cell), which are accessible to recombinant methods and which can be cultured by fermentation. Plant or animal cells that can be grown in cell culture are thus also suitable for producing cells according to the invention.
  • the cells according to the invention are preferably microorganisms, such as, for example, yeast or bacterial strains.
  • microorganisms such as, for example, yeast or bacterial strains.
  • the strains of the Enterobacteriaceae family are particularly preferred, and strains of the type Escherichia coli are very particularly preferred.
  • the lipA-containing plasmids are transformed into a starting cell by a common transformation method (eg electroporation) introduced and selected for example by means of antibiotic resistance on plasmid-bearing clones.
  • a common transformation method eg electroporation
  • the invention thus also relates to methods for producing a cell according to the invention, characterized in that a plasmid according to the invention is introduced into an output cell.
  • Another object of the invention was to provide a fermentation process which enables the production of enantiomerically pure R- ⁇ -lipoic acid.
  • This object is achieved by a method which is characterized in that a cell according to the invention is cultivated in a culture medium, the cell excreting enantiomerically pure R- ⁇ -lipoic acid in the culture medium in free form and the enantiomerically pure R- ⁇ -lipoic acid from the culture medium is separated.
  • R- ⁇ -lipoic acid can be obtained from the culture medium by methods known to those skilled in the art, such as centrifugation of the medium to separate the cells and subsequent extraction or precipitation of the product.
  • the method according to the invention is the first method in which the entire synthesis of R- ⁇ -lipoic acid takes place exclusively in a fermentation process with living cells.
  • the advantage of the method according to the invention over the conventional chemical processes lies in the enantiomer-specific biosynthesis of R- ⁇ -lipoic acid, which is why there is no need for complex racemate separation during or at the end of the synthesis chain. Furthermore, the environmental impact in the production process according to the invention for R- ⁇ -lipoic acid is considerably lower in comparison to chemical synthesis.
  • the cells according to the invention for the production of R- ⁇ -lipoic acid are preferably cultivated in a minimal salt medium known from the literature (Herbert and Guest, 1970, Meth. Enzymol. 18A, 269-272).
  • all usable sugars, sugar alcohols or organic acids or their salts can be used as the carbon source.
  • Aspartic acid, malic acid, succinic acid, pyruvic acid, fumaric acid, glutamic acid, glucose, glycerol or oxaloacetic acid are preferably used.
  • Succinic acid and oxaloacetic acid are particularly preferred.
  • Combined feeding of several different carbon sources is also possible.
  • short-chain fatty acids with a chain length of C2-C8, preferably with a chain length of C6-C8 (hexanoic or octanoic acid) can be added to the medium as specific precursors for the ⁇ -lipoic acid synthesis.
  • the concentration of the carbon source added is preferably 1-30 g / l.
  • the cells according to the invention are preferably incubated under aerobic cultivation conditions over a period of 16-150 h and in the range of the optimal growth temperature for the respective cells. 15 - 55 ° C is preferred as the optimal temperature range. A temperature between 30 and 37 ° C. is particularly preferred.
  • the detection and quantification of the R- ⁇ -lipoic acid produced in the method according to the invention is carried out, for example, by means of a bioassay using a lipoic auxotrophic indicator strain (lipA mutant).
  • lipoic auxotrophic indicator strain lipA mutant
  • This type of turbidimetric quantification of R- ⁇ -lipoic acid is known from the literature (Herbert and Guest, 1970, Meth. Enzymol. 18A, 269-272).
  • the indicator strain W14851ip2 (ATCC 25645) used in the context of the present invention would also grow without supplemented R- ⁇ -lipoic acid if the medium also contained acetate and succinate in addition to glucose.
  • the R- ⁇ -lipoic acid producers prefer with succinate as the only carbon source.
  • This strain is supplemented with the culture supernatant of a cell culture according to the invention; The lipoic acid content in the culture medium can then be determined on the basis of the growth of the indicator strain.
  • the following examples serve to further explain the invention.
  • the bacterial strain Escherichia coli W3110 / pASK-IBA3-lipA which was used for the execution of the examples, was deposited with the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH, D-38142 Braunschweig) under the number DSM 15104 in accordance with the Budapest Treaty.
  • the lipA gene from E. coli was determined by means of the polymerase chain reaction (PCR) using the Pwo DNA polymerase common practice known to those skilled in the art.
  • the chromosomal DNA of the E. coli wild-type strain W3110 (ATCC 27325) served as the template.
  • the phosphorothioate-protected oligonucleotides lipAS1 and lipAS2 with the following sequences were used:
  • lipASl (SEQ ID NO: 3)
  • lipAS2 (SEQ ID NO: 4)
  • the DNA fragment with a length of approx. 1 kb obtained during the PCR was then purified using a DNA adsorption column from the QIAprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions.
  • the vector pASK-IBA3 was cut with the restriction enzyme Eco31I (isoschizomer from Bsal) under the conditions specified by the manufacturer, then dephosphorylated by treatment with alkaline phosphatase at the 5 'ends and then by means of the PCR fragment cleaned using the GENECLEAN method.
  • the ligation of the PCR fragment with the cut and dephosphorylated vector was carried out according to the manufacturer's instructions using the T4 DNA ligase.
  • the transformation of E. coli cells of the DH5 ⁇ strain with the ligation mixture was carried out by means of electroporation in a manner known to the person skilled in the art.
  • the transformation mixture was applied to LB-A picillin agar plates (10 g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 NaCl, 15 g / 1 agar, 100 mg / 1 ampicillin) and incubated at 37 ° C. overnight ,
  • the desired transformants were identified by means of a restriction analysis after plasmid isolation using a QIAprep Spin Miniprep Kit (Qiagen, Hilden).
  • Example 2 Production of a producer of R- ⁇ -lipoic acid
  • the plasmid pASK-IBA3-lipA described in Example 1 was transformed into the E. coli strain W3110 by electroporation and after selection on LB agar plates with 100 mg / 1 ampicillin, this became Plasmid from one of the transformants reiso- lated, cleaved with restriction endonucleases and checked.
  • the control plasmid pASK-IBA3 was experienced in an analogous manner.
  • the strain W3110 / pASK-IBA3-lipA was used for the fermentative production of R- ⁇ -lipoic acid.
  • the strain W3110 was used as a comparison with the plasmid pASK-IBA3, which was cultivated under exactly the same conditions.
  • 5 ml of LB liquid medium containing 100 mg / 1 ampicillin were inoculated with the respective strain and incubated for 16 h at 37 ° C. and 160 rpm on a shaker. The cells were then harvested by centrifugation and washed twice with the appropriate volume of sterile saline (0.9% NaCl).

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Abstract

L'invention concerne des cellules et un procédé permettant de produire de l'acide R-α-lipoïque par fermentation. La souche d'organisme hôte selon l'invention, approprié pour la production d'acide R-α-lipoïque par fermentation, se caractérise en ce qu'il surexprime un gène codant une synthase d'acide lipoïque et sépare l'acide R-α-lipoïque formé, sous forme libre, dans le milieu de culture.
PCT/EP2003/008178 2002-08-01 2003-07-24 Cellules secretant de l'acide r-alpha-lipoique et procede de production par fermentation de l'acide r-alpha-lipoique WO2004013314A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003260329A AU2003260329A1 (en) 2002-08-01 2003-07-24 CELLS, WHICH SECRETE R-Alpha-LIPOIC ACID AND METHOD FOR PRODUCING SAID R-Alpha-LIPOIC ACID BY FERMENTATION

Applications Claiming Priority (2)

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DE10235270A DE10235270A1 (de) 2002-08-01 2002-08-01 Zellen, die R-alpha-Liponsäure sekretieren und Verfahren zur fermentativen Herstellung der R-alpha-Liponsäure
DE10235270.4 2002-08-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053131A1 (fr) * 2002-12-12 2004-06-24 Consortium für elektrochemische Industrie GmbH PROCEDE DE PRODUCTION D'ACIDE R-α-LIPOIQUE PAR FERMENTATION
WO2009091582A1 (fr) * 2008-01-17 2009-07-23 Indigene Pharmaceuticals, Inc. PRODUCTION D'ACIDE R-α-LIPOÏQUE PAR UN PROCÉDÉ DE FERMENTATION UTILISANT DES MICRO-ORGANISMES GÉNÉTIQUEMENT MODIFIÉS

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206035B (zh) * 2020-02-17 2021-12-28 河南科技学院 调节目的植物叶片衰老进程的基因及方法及其在棉花作物上的应用

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002085293A2 (fr) * 2001-04-20 2002-10-31 Cargill, Incorporated Production d'acide alpha-lipoique

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002085293A2 (fr) * 2001-04-20 2002-10-31 Cargill, Incorporated Production d'acide alpha-lipoique

Non-Patent Citations (4)

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Title
MILLER J R ET AL: "Escherichia LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 39, 2000, pages 15166 - 15178, XP002961226, ISSN: 0006-2960 *
REED ET AL.: "Lipoic acid metabolism in Escherichia coli: Sequencing and functional characterization of lipA and lipB genes", JOURNAL OF BACTERIOLOGY, vol. 175, no. 5, March 1993 (1993-03-01), pages 1325 - 1336, XP008025890 *
THOMSEN-ZIEGER ET AL.: "Apicomplexan parasites contain a single lipoic acid synthase located in the plastid", FEBS LETT, vol. 547, 18 June 2003 (2003-06-18), pages 80 - 86, XP004438823 *
YASUNO R ET AL: "Biosynthesis of lipoic acid in arabidopsis: cloning and characterization of the cDNA for lipoic acid synthase", PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 118, 1998, pages 935 - 943, XP002961224, ISSN: 0032-0889 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053131A1 (fr) * 2002-12-12 2004-06-24 Consortium für elektrochemische Industrie GmbH PROCEDE DE PRODUCTION D'ACIDE R-α-LIPOIQUE PAR FERMENTATION
WO2009091582A1 (fr) * 2008-01-17 2009-07-23 Indigene Pharmaceuticals, Inc. PRODUCTION D'ACIDE R-α-LIPOÏQUE PAR UN PROCÉDÉ DE FERMENTATION UTILISANT DES MICRO-ORGANISMES GÉNÉTIQUEMENT MODIFIÉS

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DE10235270A1 (de) 2004-02-12
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