EP1546350A1 - Cellules surexprimant un gene de ligase b de proteine lipoyle permettant de produire par fermentation de l'acide lipoique r-alpha - Google Patents
Cellules surexprimant un gene de ligase b de proteine lipoyle permettant de produire par fermentation de l'acide lipoique r-alphaInfo
- Publication number
- EP1546350A1 EP1546350A1 EP03810946A EP03810946A EP1546350A1 EP 1546350 A1 EP1546350 A1 EP 1546350A1 EP 03810946 A EP03810946 A EP 03810946A EP 03810946 A EP03810946 A EP 03810946A EP 1546350 A1 EP1546350 A1 EP 1546350A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- gene
- lipoic acid
- cell
- lipb
- protein ligase
- 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.)
- Withdrawn
Links
Classifications
-
- 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
- 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
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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
- C12P11/00—Preparation of sulfur-containing organic compounds
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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 C6.
- 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 such as. B. to understand the calcium, potassium, magnesium, sodium or ammonium salt.
- 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 is carried out by the sulfur transferase lipoic acid synthase [EC 2.8.1.-], the -ZipA gene product, catalyzed.
- the amino acid L-cysteine ultimately serves as the sulfur donor.
- the subsequent transfer of the R- ⁇ -lipoic acid from R- ⁇ -lipoyl-ACP to the E2 subunit of the ⁇ -keto acid dehydrogenases is carried out by the lipoyl protein ligase B [EC 6.-.-.-], the lipB gene product, catalyzed without the occurrence of R- ⁇ -lipoyl-ACP or R- ⁇ -lipoic acid as free intermediates (Miller et al., 2000, Biochemistry 39: 15166-15178).
- ⁇ -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, due to its property as a strong reducing agent, is able to regenerate other oxidized natural antioxidants in the body, such as ascorbic acid or ⁇ -tocopherol, directly or indirectly, or if these are deficient also to 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 mitochondrial NADH-dependent lipoamide reductase of mammals shows an almost 20-fold higher activity with the R enantiomer than with the S form. Furthermore, compared to the S enantiomer, R- ⁇ -lipoic acid has a significantly stronger effect on the insulin-mediated glucose uptake and the glucose metabolism of skeletal muscle cells in insulin-resistant rats. In animal experiments, 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 is carried out 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 for obtaining 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. Che. Soc. 76: 4748; DE 4137773) or enzymatically (Adger et al., 1995, J. Chem.
- lipoyl protein ligase B gene lipB gene
- the enzyme activity encoded by the ÜpB gene is to be understood as the lipoyl protein ligase activity of a cell which has a strict preference for R- ⁇ -lipoyl-ACP over free R- ⁇ -lipoic acid as a substrate (see. Fig. 1).
- Overexpression in the sense of the present invention is preferably to be understood to mean that the lipoyl protein ligase B gene is at least a factor of 2 in comparison to the respective wild-type cell from which the lipoyl protein ligase B gene was obtained , preferably expressed at least by a factor of 5.
- the lipoyl protein ligase B 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 Lipoyl protein ligase B is retained.
- the copy number of the lipB gene in a cell can be increased and / or the expression of the lipB gene can be increased, preferably by suitable promoters.
- Overexpression of a lipB gene increases the cell's lipoyl protein ligase B activity by at least the same factor.
- a cell according to the invention preferably overexpresses a lipoyl protein ligase B 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%.
- the 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%.
- the number of copies of a lipB gene in a cell can be increased by methods known to the person skilled in the art.
- a lipB gene can be cloned into a plasmid vector with multiple copy numbers per cell (e.g. pUC19, pBR322, pACYC184 for Escherichia coli) and inserted into the cell.
- a lipB gene can be integrated several times into the chromosome of a cell.
- the known systems with temperate bacteriophages, integrative plasmids or integration via homologous recombination can be used as an integration method (e.g. Hamilton et al., 1989, J. Bacteriol. 171: 4617-4622).
- the invention thus also relates to a plasmid characterized in that it contains a lipB gene under the functional control of a promoter.
- the natural promoter and operator region of the lipB gene can serve as the control region for the expression of a plasmid-encoded lipB gene, but the increased expression of a lipB gene can, in particular, also be achieved by means of other promoters.
- Corresponding promoter systems that enable either continuous or controlled, inducible expression of the lipoyl protein ligase B gene such as, for example, the constitutive GAPDH promoter of the grapscher gene in Escherichia coli or the inducible lac, tac, t rc ', lambda, ara and tet-promoters are known to the skilled person (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 the inducible arabinose 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 frequently occurring codons.
- Cells according to the invention preferably contain a plasmid with a lipB gene and the aforementioned modifications of the regulation signals.
- the native weak start codon of the lipB gene (TTG) ' is replaced by the strong start codon ATG.
- a lipB gene is cloned into a plasmid vector, for example, by specifically amplifying a lipB gene by means of the polymerase chain reaction using specific primers which capture the complete lipB gene, and then ligation with vector DNA fragments ,
- Cells according to the invention which have an increased expression of a lipB gene compared to a starting cell and, in connection therewith, an increased lipoyl protein ligase B activity, can be generated from a starting cell using standard techniques of molecular biology. Lipoyl protein ligase B 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.
- Also particularly suitable as starting cells are those cells which already have an increased lipoic acid synthase activity due to an increased expression of the lipA gene.
- the lipB-containing plasmids are introduced into a starting cell using a common transformation method (e.g. electroporation) and selected for plasmid-bearing clones, for example by means of antibiotic resistance.
- a common transformation method e.g. 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 separating enantiomerically pure R- ⁇ -lipoic acid into the culture medium in free form. det and the enantiomerically pure R- ⁇ -lipoic acid is separated from the culture medium.
- 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 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 can be used as carbon sources.
- 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 process according to the invention is carried out, for example, by means of a bioassay using a lipoic auxotrophic indicator strain (1 ipA 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, however, also grow without supplemented R- ⁇ -lipoic acid if the medium also contains acetate and succinate in addition to glucose.
- the lipB gene from E. coli was amplified by means of the polymerase chain reaction (PCR) using the Pwo DNA polymerase according to common practice known to the person skilled in the art.
- the chromosomal DNA of the E. coli wild-type strain W3110 (ATCC 27325) served as the template.
- the 5 '-phosphorylated oligonucleotides lipB-fwd and lipB-rev were used as primers with the following sequences:
- lipB-fwd (SEQ ID NO: 3)
- lipB-rev (SEQ ID NO: 4)
- the DNA fragment obtained in the PCR with a length of approximately 0.68 kb was then purified using a DNA adsorption column from the QIAprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions.
- the cloning and expression vector pKP477 was obtained as follows from the vector pBAD-GFP (Crameri et al., 1996, ⁇ at. Biotechol. 14: 315-319), a derivative of the vector pBADl ⁇ : First, the GFP gene removed by restriction of the vector pBAD-GFP with the endonucleases Nhel and EcoRI. The 5'- The overhanging ends of the remaining approximately 4.66 kb vector fragment were then filled in with the Klenow enzyme and the vector was finally religated using the T4 ligase. E. coli cells of the DH5 ⁇ strain were transformed with the ligation mixture by means of electroporation in a manner known to the person skilled in the art.
- the transformation mixture was applied to LB ampicillin agar plates (10 g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 NaCl, 15 g / 1 agar, 100 mg / 1 ampicilline) and overnight at 37 ° C. incubated. After plasmid isolation using a QIAprep Spin Miniprep Kit (Qiagen, Hilden), the desired transformants were identified by a restriction analysis. The vector obtained in this way is called pKP476. In order to remove the second Ndel interface of the vector pKP476, which is located in the vicinity of the origin of replication, the vector pKP476 was first partially restricted with Ndel in a manner known to the person skilled in the art.
- the plasmid pKP477 contains various genetic elements that allow a controlled expression of any gene. It is a vector with an origin of replication derived from the pBR plasmid family. The expression of the cloned gene is suppressed by the AraC repressor and can be induced by arabinose.
- the vector pKP477 was cut with the restriction enzymes Ndel and Smal under the conditions specified by the manufacturer, then dephosphorylated by treatment with alkaline phosphatase at the 5 'ends and then like the lipB-PCR fragment cleaned using the GE ⁇ E-CLEA ⁇ method. The ligation of the PCR fragment with the cut and dephosphorylated vector pKP477, the transformation and the verification of the transformants was carried out as described above. The resulting plasmid is called pBAD-lipB (Fig. 2).
- the plasmid pBAD-lipB 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, the plasmid was reisolated from one of the transformants, cleaved with restriction endonucleases and checked.
- the control plasmid pKP477 was used in an analogous manner.
- the strain W3110 / pBAD-lipB was used for the fermentative production of R- ⁇ -lipoic acid.
- the strain W3110 was used as a comparison with the "empty" control plasmid pKP477, 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
La présente invention concerne des cellules et un procédé pour produire de l'acide R-α-lipoïque par fermentation. La souche d'organisme hôte selon cette invention, adaptée à la production d'acide R-α-lipoïque par fermentation, est caractérisée en ce qu'elle surexprime un gène codant une ligase B de protéine lipoyle et en ce qu'elle rejette l'acide R-α-lipoïque formé, sous forme libre, dans le milieu de culture.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10245993A DE10245993A1 (de) | 2002-10-02 | 2002-10-02 | Zellen zur fermentativen Herstellung von R-alpha-Liponsäure |
DE10245993 | 2002-10-02 | ||
PCT/EP2003/010687 WO2004044211A1 (fr) | 2002-10-02 | 2003-09-25 | Cellules surexprimant un gene de ligase b de proteine lipoyle permettant de produire par fermentation de l'acide lipoique r-alpha |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1546350A1 true EP1546350A1 (fr) | 2005-06-29 |
Family
ID=32086841
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03810946A Withdrawn EP1546350A1 (fr) | 2002-10-02 | 2003-09-25 | Cellules surexprimant un gene de ligase b de proteine lipoyle permettant de produire par fermentation de l'acide lipoique r-alpha |
Country Status (6)
Country | Link |
---|---|
US (1) | US20060211098A1 (fr) |
EP (1) | EP1546350A1 (fr) |
AU (1) | AU2003301990A1 (fr) |
DE (1) | DE10245993A1 (fr) |
TW (1) | TW200416288A (fr) |
WO (1) | WO2004044211A1 (fr) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10332623A1 (de) * | 2003-07-17 | 2005-02-03 | Consortium für elektrochemische Industrie GmbH | Zellen und Verfahren zur fermentativen Herstellung von R-alpha-Liponsäure |
US20110262976A1 (en) * | 2008-01-17 | 2011-10-27 | Indigene Pharmaceuticals, Inc. | PRODUCTION OF R-a-LIPOIC ACID BY FERMENTATION USING GENETICALLY ENGINEERED MICROORGANISMS |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10042739A1 (de) * | 2000-08-31 | 2002-03-14 | Degussa | Neue für das lipB-Gen kodierende Nukleotidsequenzen |
WO2002085293A2 (fr) * | 2001-04-20 | 2002-10-31 | Cargill, Incorporated | Production d'acide alpha-lipoique |
-
2002
- 2002-10-02 DE DE10245993A patent/DE10245993A1/de not_active Ceased
-
2003
- 2003-09-25 AU AU2003301990A patent/AU2003301990A1/en not_active Abandoned
- 2003-09-25 EP EP03810946A patent/EP1546350A1/fr not_active Withdrawn
- 2003-09-25 WO PCT/EP2003/010687 patent/WO2004044211A1/fr not_active Application Discontinuation
- 2003-09-25 US US10/529,590 patent/US20060211098A1/en not_active Abandoned
- 2003-09-29 TW TW092126913A patent/TW200416288A/zh unknown
Non-Patent Citations (1)
Title |
---|
See references of WO2004044211A1 * |
Also Published As
Publication number | Publication date |
---|---|
TW200416288A (en) | 2004-09-01 |
DE10245993A1 (de) | 2004-05-06 |
AU2003301990A1 (en) | 2004-06-03 |
WO2004044211A1 (fr) | 2004-05-27 |
US20060211098A1 (en) | 2006-09-21 |
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