WO2002059329A1 - Nouvelles sequences nucleotidiques codant pour le gene de cite - Google Patents

Nouvelles sequences nucleotidiques codant pour le gene de cite Download PDF

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Publication number
WO2002059329A1
WO2002059329A1 PCT/EP2001/000797 EP0100797W WO02059329A1 WO 2002059329 A1 WO2002059329 A1 WO 2002059329A1 EP 0100797 W EP0100797 W EP 0100797W WO 02059329 A1 WO02059329 A1 WO 02059329A1
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Prior art keywords
gene
polynucleotide
coding
sequence
amino acid
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PCT/EP2001/000797
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German (de)
English (en)
Inventor
Mike Farwick
Klaus Huthmacher
Achim Marx
Brigitte Bathe
Walter Pfefferle
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Degussa Ag
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Priority to PCT/EP2001/000797 priority Critical patent/WO2002059329A1/fr
Priority to EP01919248A priority patent/EP1354051A1/fr
Publication of WO2002059329A1 publication Critical patent/WO2002059329A1/fr

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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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the invention relates to nucleotide sequences coding for the citE gene from coryneform bacteria and a method for the fermentative production of amino acids using bacteria in which the citE gene is weakened.
  • L-amino acids in particular L-lysine, are used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.
  • Microorganisms use mutagenesis, selection and mutant selection methods. In this way, strains are obtained which are resistant to antimetabolites or auxotrophic for regulatory-important metabolites and which produce amino acids.
  • the inventors have set themselves the task of providing new measures for improved fermentative production of amino acids.
  • one or more amino acids including their salts are selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L- Cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine are meant. L-lysine is particularly preferred.
  • L-lysine or lysine are mentioned in the following, not only the bases but also the salts such as e.g. Lysine monohydrochloride or lysine sulfate is meant.
  • the invention relates to an isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence coding for the citE gene, selected from the group
  • polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No.
  • polynucleotide which is complementary to the polynucleotides of a) or b),
  • polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c),
  • polypeptide preferably has the activity of citrate lyase E.
  • the invention also relates to the above-mentioned polynucleotide, which is preferably a replicable DNA containing:
  • the invention further relates to polynucleotides selected from the group
  • a replicable polynucleotide in particular DNA, containing the nucleotide sequence as shown in SEQ ID No. 1;
  • a polynucleotide encoding a polypeptide having the amino acid sequence as described in SEQ ID No. 2 includes;
  • the invention also relates to polynucleotides which essentially consist of a polynucleotide sequence which can be obtained by screening by means of hybridization of a corresponding library of a coryneform bacterium which contains the complete gene or parts thereof, with a probe which has the sequence of the invention
  • Polynucleotides containing the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA to nucleic acids respectively
  • Polynucleotides which contain the sequences according to the invention are furthermore suitable as primers, with the aid of which polymerase chain reaction (PCR) can be used to produce DNA from genes which code for citrate lyase E.
  • PCR polymerase chain reaction
  • Such oligonucleotides serving as probes or primers contain at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably at least 15, 16, 17, 18 or 19 successive nucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable , If applicable, too
  • Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides are suitable.
  • Isolated means separated from its natural environment.
  • Polynucleotide generally refers to
  • Polyribonucleotides and polydeoxyribonucleotides which can be unmodified RNA or DNA or modified RNA or DNA.
  • the polynucleotides according to the invention include a polynucleotide according to SEQ ID No. 1 or a fragment produced therefrom and also one which is at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90% and entirely are particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID No. 1 or a fragment made from it.
  • Polypeptides are understood to mean peptides or proteins which contain two or more amino acids linked via peptide bonds.
  • polypeptides according to the invention include a polypeptide according to SEQ ID No. 2, in particular those with the biological activity of citrate lyase E and also those which are at least 70% to 80%, preferably at least 81% to 85%, particularly preferably at least 86% to 90% and very particularly preferably too at least 91%, 93%, 95%, 97% or 99% are identical to the polypeptide according to SEQ ID No. 2 and have said activity.
  • the invention further relates to a process for the fermentative production of amino acids, selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- Methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine, using coryneform bacteria, which in particular already produce amino acids and in which those for the nucleotide sequences encoding the citE gene are weakened, in particular switched off or expressed at a low level.
  • amino acids selected from the group L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- Methionine, L-isole
  • Context the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which is responsible for a corresponding enzyme or protein encodes a low activity or inactivates the corresponding gene or enzyme (protein) and, if appropriate, combines these measures.
  • the microorganisms which are the subject of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative of coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned in particular, which is known in the art for its ability to produce L-amino acids.
  • Suitable strains of the genus Corynebacterium in particular of the species Corynebacterium glutamicum (C. glutamicum), are in particular the known wild-type strains
  • E. coli Escherichia coli
  • the creation of gene banks is recorded in well-known textbooks and manuals. Examples include the textbook by Winnacker: Genes and Clones, An Introduction to Genetic Technology (Verlag Chemie, Weinheim, Germany, 1990), or the manual by Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).
  • a very well-known gene bank is that of the E. coli K-12 strain W3110, which was developed by Kohara et al. (Cell 50, 495-508 (1987)) in ⁇ vectors. Bathe et al.
  • Plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can also be used to produce a C. glutamicum gene bank in E. coli become.
  • Particularly suitable hosts are E. coli strains which are defective in terms of restriction and recombination, such as the strain
  • ⁇ ⁇ ⁇ ⁇ d ⁇ C ⁇ CQ H- DJ 0 2 ⁇ H- O ⁇ ⁇ ⁇ £ EP
  • DNA sequences labeled with SEQ ID No. 1 or parts of SEQ ID No. 1 hybridize part of the invention.
  • DNA sequences are the result part of the invention, which are prepared by the polymerase chain reaction (PCR) using primers (of SEQ ID No. 1.
  • PCR polymerase chain reaction
  • Such oligonucleotides typically have a length of at least 15 nucleotides.
  • the person skilled in the art can find instructions for identifying DNA sequences by means of hybridization in the manual "The DIG System Users Guide for Filter Hybridization” by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)).
  • the hybridization takes place under stringent conditions, ie only hybrids are formed in which the probe and target sequence, ie the polynucleotides treated with the probe, are at least 70% identical. It is known that the stringency of the hybridization, including the washing steps, is influenced or determined by varying the buffer composition, the temperature and the salt concentration.
  • the hybridization reaction is preferably carried out at a relatively low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).
  • a 5x SSC buffer at a temperature of approx. 50 ° C - 68 ° C can be used for the hybridization reaction.
  • Probes can also hybridize with polynucleotides that have less than 70% identity to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be done, for example, by lowering the salt concentration to 2x SSC and possibly subsequently 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim,
  • Insertions and deletions are considered. Depending on the effect of the amino acid exchange on the enzyme activity, one speaks of missense mutations or nonsense mutations. Insertions or deletions of at least one base pair (bp) in a gene lead to
  • a central part of the coding region of the gene of interest is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum.
  • vectors are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pKl ⁇ mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pKl ⁇ mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994).
  • the plasmid vector which contains the central part of the coding region of the gene, is then converted into the desired strain of C. glutamicum by conjugation or transformation.
  • the conjugation method is described, for example, by Schfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al.
  • a mutation such as a deletion, insertion or base exchange in the gene of interest is produced in vitro.
  • the allele produced is in turn cloned into a vector which is not replicative for C. glutamicum and then cloned by Transformation or conjugation transferred to the desired host by C. glutamicum after homologous recombination by means of a first, integration-causing "cross-over” event and a suitable one second excision-causing "cross-over” event in the target gene or in the target sequence is achieved by incorporating the mutation or the alley.
  • This method was, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) used to switch off the pyc gene from C. glutamicum by deletion.
  • L-amino acids in addition to the weakening of the citE gene, one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, the amino acid export and, if appropriate to reinforce regulatory proteins, in particular to overexpress them.
  • the term “amplification” describes the increase in the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA, for example by increasing the copy number of the gene or genes, using a strong promoter or uses a gene or allele which codes for a corresponding enzyme (protein) with a high activity and, if appropriate, combines these measures.
  • the lysC gene coding for a feed-back resistant aspartate kinase (Accession No.P26512; EP-B-0387527; EP-A-0699759; WO 00/63388), • the lysE gene coding for lysine export (DE-A -195 48 222),
  • the gene pgi coding for glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969), The gene poxB coding for the pyruvate oxidase (DE: 1995 1975.7, DSM 13114),
  • the weakening of the homoserine dehydrogenase can also be caused, inter alia, by amino acid exchanges, such as, for example, by exchanging L-valine for L-alanine, L-glycine or L-leucine at position 59 of the enzyme protein, by exchanging L-valine for L- Isoleucine, L-valine or L-leucine at position 104 of the enzyme protein and / or by replacing L-asparagine with L-threonine or L-serine at position 118 of the enzyme protein.
  • amino acid exchanges such as, for example, by exchanging L-valine for L-alanine, L-glycine or L-leucine at position 59 of the enzyme protein, by exchanging L-valine for L- Isoleucine, L-valine or L-leucine at position 104 of the enzyme protein and / or by replacing L-asparagine with L-threonine or L-serine at position 118 of the enzyme protein.
  • the weakening of the homoserine kinase can also be caused, inter alia, by amino acid exchanges, such as, for example, by exchanging L-alanine for L-valine, L-glycine or L-leucine at position 133 of the enzyme protein and / or by exchanging L-proline for L-threonine, L-isoleucine or L-serine can be achieved at position 138 of the enzyme protein.
  • amino acid exchanges such as, for example, by exchanging L-alanine for L-valine, L-glycine or L-leucine at position 133 of the enzyme protein and / or by exchanging L-proline for L-threonine, L-isoleucine or L-serine can be achieved at position 138 of the enzyme protein.
  • the attenuation of the aspartate decarboxylase can also be achieved, inter alia, by amino acid exchanges, such as, for example, by replacing L-alanine with L-glycine, L-valine or L-isoleucine at position 36 of the enzyme protein.
  • amino acid exchanges such as, for example, by replacing L-alanine with L-glycine, L-valine or L-isoleucine at position 36 of the enzyme protein.
  • DJ ⁇ Q 3 DJ co d ⁇ DJ: ⁇ d ⁇ ⁇ ⁇
  • This goal is usually achieved within 10 hours to 160 hours.
  • the method according to the invention serves for the fermentative production of amino acids, in particular L-lysine.
  • composition of common nutrient media such as LB or TY medium can also be found in the manual by Sambrook et al. be removed. example 1
  • Chromosomal DNA from C. glutamicum ATCC 13032 is as described by Tauch et al. (1995, plasmid 33: 168-179) described isolated and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, code no. 27-0913-02).
  • the DNA fragments are dephosphorylated with shrip alkaline phosphatase (Röche Molecular Biochemicals, Mannheim, Germany, product description SAP, code no. 1758250).
  • the DNA of the cosmid vector SuperCosl (Wahl et al.
  • the cosmid DNA is then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04).
  • BamHI Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04.
  • the cosmid DNA treated in this way is mixed with the treated ATCC13032 DNA and the mixture is treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no.27-0870-04) ,
  • T4 DNA ligase Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no.27-0870-04
  • the ligation mixture is then packed in phages using the Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, Code no. 200217).
  • the cells are taken up in 10 mM MgSO 4 and with an aliquot of the Phage suspension mixed. Infection and titering of the cosmid bank are, as in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the cells are plated on LB agar (Lennox, 1955, Virology, 1: 190) + 100 mg / 1 ampicillin. After overnight incubation at 37 ° C., recombinant individual clones are selected.
  • the cosmid DNA of a single colony is isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Product No. 27-0913 -02) partially split.
  • the DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Röche Molecular Biochemicals, Mannheim, Germany, product description SAP, product No. 1758250).
  • the cosmid fragments in the size range from 1500 to 2000 bp are isolated using the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).
  • the DNA of the sequencing vector pZero-1 obtained from Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, Product No. K2500-01) is mixed with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Product No. 27 - 0868-04) split.
  • BamHI Amersham Pharmacia, Freiburg, Germany, product description BamHI, Product No. 27 - 0868-04
  • the ligation of the cosmid fragments in the sequencing vector pZero-1 is carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the DNA mixture is incubated with T4 ligase (Pharmacia Biotech, Freiburg, Germany) overnight. This ligation mixture is then converted into the E.
  • the plasmid preparation of the recombinant clones is carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany).
  • the sequencing is carried out according to the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academys of Sciences, U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990,
  • the raw sequence data obtained are then processed using the Staden program package (1986, Nucleic Acids Research, 14: 217-231) version 97-0.
  • the individual sequences of the pZerol derivatives are blended into a coherent contig asse.
  • the computer-aided coding area analysis is carried out with the program XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231).
  • the nucleotide sequence obtained is shown in SEQ ID No. 1 shown. Analysis of the nucleotide sequence reveals an open reading frame of 821 bp, which is referred to as the citE gene.
  • the citE gene codes for a polypeptide of 273 amino acids.
  • the ATCC 13032 strain is used according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) chromosomal DNA isolated. Based on the sequence of the citE gene known from Example 2 for C. glutamicum, the following oligonucleotides are selected for the polymerase chain reaction (see also SEQ ID No. 3 and SEQ ID No. 4):
  • the primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and after
  • the amplified DNA fragment is inserted into the vector pCR2.1-TOPO (Mead at al. (1991) Bio / Technology 9: 657) using the TOPO TA cloning kit from Invitrogen Corporation (Carlsbad, CA, USA; catalog number K4500-01) -663).
  • the E. coli strain TOP10 is then electroporated using the ligation approach (Hanahan, In: DNA cloning. A practical approach. Vol.I. IRL-Press, Oxford, Washington DC, USA, 1985). Plasmid-bearing cells are selected by plating out the Transformation approach on LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which was supplemented with 50 mg / 1 kanamycin. Plasmid DNA is isolated from a transformant using the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0.8%). The plasmid becomes pCR2. Called lcitEint and is shown in Figure 1.
  • the strain DSM 5715 is an AEC-resistant lysine producer, which is described in EP-B-04351342.
  • the vector pCR2. lcitEint cannot replicate independently in DSM5715 and only remains in the cell if it has integrated into the chromosome of DSM 5715. Selection of clones with pCR2 integrated in the chromosome. lcitEint is carried out by plating the electroporation batch onto LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2nd and Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), which has been supplemented with 15 mg / 1 kanamycin.
  • citEint fragment is marked according to the method "The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) with the Dig Hybridization Kit from Boehringer.
  • Chromosomal DNA of a potential integrant is extracted using the method of Eikmanns et al. (Microbiology 140: 1817 - 1828 (1994)) isolated and each with the
  • the strain is first incubated on agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg / 1) for 24 hours at 33 ° C.
  • a preculture is inoculated (10 ml medium in 100 ml Erlenmeyer flask)
  • the complete medium CgIII is used as the medium for the preculture.
  • the pH is adjusted to pH 7.4 Kanamycin (25 mg / 1) is added to this.
  • the preculture is incubated on the shaker at 33 ° C. at 240 rpm for 48 hours.
  • a main culture is inoculated from this preculture so that the initial OD (660 nm) of the main culture is 0.1 OD.
  • the medium MM is used for the main culture
  • MOPS morpholinopropanesulfonic acid
  • CSL, MOPS and the salt solution are adjusted to pH 7 with ammonia water and autoclaved. Then the sterile substrate and vitamin solutions are added, and the dry autoclaved CaC0 3 is added. The cultivation is carried out in a volume of 10 ml in a 100 ml Erlenmeyer flask with baffles. Kanamycin (25 mg / 1) is added. The cultivation takes place at 33 ° C and 80% humidity.
  • the OD is determined at a measuring wavelength of 660 nm using the Biomek 1000 (Beckmann Instruments GmbH, Kunststoff).
  • the amount of lysine formed is determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
  • Figure 1 Map of plasmid pCR2. lcitEint.
  • KmR Kanamycin resistance gene
  • citEint internal fragment of the citE gene
  • ColEl origin of replication of the plasmid ColEl
  • This international depository accepts the microorganism referred to under I, which it received on 2 001 - 01 - 12 (date of first filing) 1
  • microorganism referred to under I has been received by this International Agency for the filing of the application (date of first filing) and an application for the conversion of this first filing into a deposit under the Bndapester Treaty has been received on (date of receipt of the application for conversion)
  • the microorganism identified under I. above was accompanied by:
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)

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Abstract

L'invention concerne un polynucléotide isolé contenant une séquence polynucléotidique, sélectionné dans le groupe comprenant a) un polynucléotide identique à au moins 70 % à un polynucléotide qui code pour un polypeptide contenant la séquence d'acides aminés SEQ ID No. 2 ; b) un polynucléotide codant pour un polypeptide qui contient une séquence d'acides aminés identique à au moins 70 % à la séquence d'acides aminés SEQ ID No. 2 ; c) un polynucléotide complémentaire des polynucléotides de a) ou b) ; et d) un polynucléotide contenant au moins 15 nucléotides successifs de la séquence polynucléotidique de a), b) ou c). L'invention concerne également un procédé pour préparer, par fermentation, des L-acides aminés au moyen de bactéries coryneformes dans lesquelles au moins le gène de citE est présent sous forme affaiblie. L'invention concerne en outre l'utilisation de polynucléotides, qui contiennent les séquences selon l'invention, comme sondes d'hybridation.
PCT/EP2001/000797 2001-01-25 2001-01-25 Nouvelles sequences nucleotidiques codant pour le gene de cite WO2002059329A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5688799A (en) * 1979-12-21 1981-07-18 Ajinomoto Co Inc Preparation of l-lysine
WO2001000844A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Proteines codant pour les genes corynebacterium glutamicum, intervenant dans le metabolisme du carbone et dans la production d'energie
EP1108790A2 (fr) * 1999-12-16 2001-06-20 Kyowa Hakko Kogyo Co., Ltd. Nouveaux polynuclétides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5688799A (en) * 1979-12-21 1981-07-18 Ajinomoto Co Inc Preparation of l-lysine
WO2001000844A2 (fr) * 1999-06-25 2001-01-04 Basf Aktiengesellschaft Proteines codant pour les genes corynebacterium glutamicum, intervenant dans le metabolisme du carbone et dans la production d'energie
EP1108790A2 (fr) * 1999-12-16 2001-06-20 Kyowa Hakko Kogyo Co., Ltd. Nouveaux polynuclétides

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* Cited by examiner, † Cited by third party
Title
DATABASE EMBL 10 May 2001 (2001-05-10), NAKAGAWA S ET AL: "Sequence 4463 from Patent EP1108790.", XP002176658 *
DATABASE EMBL 10 May 2001 (2001-05-10), NAKAGAWA S ET AL: "Sequence 7061 from Patent EP1108790.", XP002176656 *
DATABASE EMBL 10 May 2001 (2001-05-10), NAKAGAWA S ET AL: "Sequence 962 from Patent EP1108790.", XP002176659 *
DATABASE EMBL 10 May 2001 (2001-05-10), NAKAGAWA S ET AL: "Sequence 963 from Patent EP1108790.", XP002176657 *
DATABASE EMBL 10 May 2001 (2001-05-10), NAKAGAWA S ET AL: "Sequence 964 from Patent EP1108790.", XP002176660 *
DATABASE EMBL 12 August 1999 (1999-08-12), JAKOBY M J: "Corynebacterium glutamicum yjcc gene, amtR gene and citE gene, partial", XP002176655 *
DATABASE WPI Section Ch Week 198136, Derwent World Patents Index; Class B05, AN 1981-64859D, XP002176661 *
JAKOBY MARC ET AL: "AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum.", MOLECULAR MICROBIOLOGY, vol. 37, no. 4, August 2000 (2000-08-01), pages 964 - 977, XP002176654, ISSN: 0950-382X *

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