SK4252001A3 - Nucleotide sequences which code for the rp1k gene - Google Patents

Abstract

Isolated nucleic acid (I) from coryneform bacteria which is at least 70% identical with a sequence that encodes a 145 amino acid (aa) polypeptide (II) fully defined in the specification, encodes a polypeptide at least 70% identical with (II), its complement or contains at least 15 consecutive bases of (I). Independent claims are also included for the following: (1) vector containing (I), deposited in Escherichia coli DH5 alpha /p DELTA rpk, as DSM 13158 (map reproduced in specification); (2) as host cells, coryneform bacteria that have a deletion or insertion in the rplk gene; and (3) method (M1) for producing L-aa, especially lysine, by fermenting bacteria in which at least the rplk gene has been weakened.

Description

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Nucleotide sequences encoding the rplK gene

Technical field

The invention relates to nucleotide sequences encoding the rplK gene from coryneform bacteria and to a method for fermentative preparation of amino acids, in particular L-lysine, to attenuate the rplK gene.

BACKGROUND OF THE INVENTION

L-amino acids, especially L-lysine, find use in animal nutrition, in human medicine and in the pharmaceutical industry.

It is known that these amino acids are prepared by fermenting strains of coryneform bacteria, in particular CoryneJbacterium glutamicum. Due to the great importance, work is still ongoing to improve the preparation process. Mutagenesis, selection and mutant selection methods are used to improve the performance properties of these microorganisms. In this way strains are obtained which are resistant to antimetabolites, such as lysine analogs of S- (2-aminoethyl) -cysteine, or auxotrophic for regulatory important metabolites and produce L-amino acids.

Recombinant DNA techniques have also been used for years to improve L-lysine-producing strains in Corynebacterium glutamicum strains in which individual amino acid biosynthesis genes are amplified and the effects on L-amino acid production are examined. Review articles can be found, inter alia, in Kinoshita ("Glutamic Acid Bacteria, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40 -44 (1991)), Eggeling (Amino Acids 6: 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15: 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).

The rplK protein (ribosomal large subunit of protein K) is first described for Escherichia coli as a part of the bacterial ribosome, the translational machinery of the cell on which protein synthesis takes place.

Ribosomes are cellular particles that consist of three ribonucleic acid (RNA) molecules and a defined number of proteins. Ribosomes are obtained mostly from cell extract by ultracentrifugation. Further purification of the remaining cellular components is usually performed by sedimentation in sucrose gradients. This preparation technique has led to the labels used for ribosome components that are directly related to sedimentation properties. The functional bacterial ribosome is therefore often referred to as the 70S-ribosome, which consists of small subunits of 30S subunits and large subunits of 50S subunits. The small 30S-subunit of E. coli consists of 21 different polypeptides and a single 1542 nucleotide RNA molecule known to those skilled in the art as 16S-rRNA; the large 50S-subunit contains, in addition to the rplK protein, another 31 different polypeptides, along with two RNA molecules of 120 and 2904 nucleotides, respectively, the so-called 5S-23S-rRNA molecule molecules. In the meantime, an alternative nomenclature has been established for ribosomal proteins. This refers to polypeptides of the small 30S-subunit S1 to S21, the large 50S-subunit L1 to L32. The Rp1K protein corresponds to the ribosomal L11 protein (Noller and Nomura, in: Neidhardt et al., Escherichia coli and Salmonella typhimurium from Cellular and molecular biology. American Society for Microbiology, Washington D. C., 167-186, 1996). In recent years, Lll-like proteins have also been identified in other organisms such as Borrelia burgdorferi (Fraser et al., Nature, 390, 580-586, 1997), Helicobacter pylori (Tomb et al., Nature, rrrrrr · r · rr

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388, 539-547 (1997), Serratia marcescens (Sor and Nomura, Molecular and General Genetics, 210, 52-59, 1987), Haemophilus influenzae (Fleischmann et al., Science, 269, 496-512, 1995) and in gram a positive bacterium of Bacillus subtilis (Jeong et al., Molecular Microbiology, 10, 133-142, 1993).

The translation process taking place on the ribosome, i.e. biosynthesis of polypeptides driven by RNA matrices (mRNA), is complex. In addition to the ribosome, other proteins are essential for the translational process, which the skilled artisan refers to as protein synthesis factors (Noller, Annual Rewiew of Biochemistry, 60, 191-227, 1991). The L11-protein mediates the interaction between the ribosome and some factors of protein synthesis; for example, elongation factor G (EF-G) and termination factor 1 (RF-1) may be mentioned. The absence of the Lll protein in the ribosomes of E. coli Lll mutants therefore leads to a decrease in translation rate (Xing and Draper, Biochemistry, 35, 1581-1588, 1996).

The L11 protein is also essential for the binding and activity of the RelA protein on the ribosome. RelA catalyzes the synthesis of guanosine tetraphosphate (ppGpp) under conditions of amino acid deficiency by transferring the pyrophosphate group from ATP to GDP. ppGpp affects the expression of many genes, either negative or positive, in E. coli. Generally, the expression of gene products that are effective in the biosynthetic process is stimulated. Generally, gene products that act catabolically are negatively regulated accordingly. A large number of genes and operons, which play a central role in amino acid biosynthesis, are regulated in E.coli by ppGpp. Among the previously known genes positively influenced in E. coli include argF, arg1, argECBH (arginine biosynthesis), gltB, glnA, gdh (glutamine / glutamate biosynthesis), ilvB, ilvA (isoleucine biosynthesis), metK, metF (methionine biosynthesis), thrA, thrB, thrC (threonine biosynthesis), lysA, lysC, dapB, asd (lysine biosynthesis) (Cashel et al., in: Neidhardt et al., Escherichia coli and Salmonella typhimurium.). American Society for Microbiology, Washington, DC, 14581496, 1996). Meanwhile, the function of ppGpp as a positive regulator of amino acid biosynthesis has also been shown in other bacteria such as Salmonella typhimurium (Rudd et al., Journal of Bacteriology, 163, 534-542, 1985), Vibrio sp. (Flärdh et al., Journal of Bacteriology, 176: 5949-5957, 1994) and B. subtilis (Wendrich and Marahiel, Molecular Microbiology, 1997, 26, 65-79).

It is clear from the prior art that it is of interest to determine whether knowledge of the nucleotide sequence of the rplK gene of coryneform bacteria contributes to improving the amino acid reproduction of these bacteria.

OBJECT OF THE INVENTION

SUMMARY OF THE INVENTION The object of the present invention is to provide new measures for improving the fermentation preparation of amino acids, in particular L-lysine.

Description of the invention

L-amino acids, especially lysine, find use in human medicine and in the pharmaceutical industry, in the food industry and in particular in animal feed. There is therefore a general interest in preparing new formulations for improving the process for preparing amino acids, especially L-lysine.

SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide comprising a sequence of polynucleotides selected from the group consisting of

a) a polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 2

b) a polynucleotide that encodes a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO. 2

c) a polynucleotide that is complementary to polynucleotides a) or b), and

d) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide sequence a), b) or c.

The present invention also provides a polynucleotide according to claim 1, preferably a replicable DNA comprising:

(i) the nucleotide sequence shown in SEQ-ID-No. Or (ii) at least one sequence that corresponds to sequence (i) within the degeneracy region of the genetic code, or (iii) at least one sequence that hybridizes to a sequence complementary to sequence (i) or (ii), and optionally (iv) functionally neutral meaningful mutations in (i).

Further objects of the invention are a polynucleotide according to claim 4 comprising a nucleotide sequence as shown in SEQ ID NO. The polynucleotide of claim 2, encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 2, a polynucleotide according to claim 1, in particular point d, comprising a nucleotide sequence as shown in SEQ ID NO. The polynucleotide of claim 1, item d, which encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 4, a vector comprising the mutated polynucleotide of claim 1, point d, shown in SEQ ID NO. 3 and in Figure 1 (Δ = deHa) and under the Budapest Treaty deposited in E. coli DH5α / pArplk as DSM 13158 and as host cells serving coryneform bacteria which contain insertion or deletion in the rplK gene.

The present invention also relates to polynucleotides which consist mainly of a sequence of polynucleotides which are obtainable by screening by hybridization of the respective gene bank containing the complete gene with the sequence of the polynucleotides as corresponding to SEQ ID NO. 1 with a probe comprising the sequence of said polynucleotide of SEQ ID NO. 1 or a fragment thereof and isolating said DNA sequence.

The polynucleotide sequences of the invention are useful as hybridization probes for RNA, cDNA and DNA to isolate the full-length cDNA encoding the ribosomal L11 protein and those cDNA or genes that show high similarity to the rplK gene sequence.

The polynucleotide sequences of the invention are further suitable as a primer by means of which DNA genes that encode the rplK gene product and ribosomal L11 protein, respectively, can be prepared by polymerase chain reaction (PCR).

Such oligonucleotides serving as probes or primers comprise at least 30, preferably at least 20, most preferably at least 15 consecutive nucleotides. Oligonucleotides of at least 40 or 50 base pairs in length are also suitable.

“Isolated means naturally separate.

and " polynucleotide refers generally to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.

The term "polypeptides" refers to peptides or proteins that contain two or more amino acids linked by a peptide bond.

The polypeptides of the invention terminate the polypeptide of SEQ ID NO. 2, in particular those with the biological activity of the rp1 gene product, as well as those which are at least 70% identical to the polypeptide of SEQ ID NO. 2, preferably at least 80%, and in particular at least 90% to 95% identity to the polypeptide of SEQ ID NO. 2, and exhibit said activity.

The invention further relates to a process for the fermentative preparation of amino acids, in particular lysine, using coryneform bacteria which in particular produce amino acids and in which the nucleotide sequences encoding the rplK gene are attenuated.

The term "attenuation" in this context describes the diminution or elimination of intracellular activity or function of one or more enzymes or proteins in a microorganism, which is encoded by the corresponding DNA using, for example, a weak promoter or gene, an allele that encodes the corresponding enzyme, lower activity or inactivate the corresponding gene or enzyme respectively protein, and also combine these measures.

The microorganisms of the present invention can prepare amino acids, in particular lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerin and ethanol. It may be a representative of coryneform bacteria, especially of the genus

Corynebacterium. In the genus Corynebacterium, mention should be made in particular of the species Corynebacterium glutamicum, 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 glutainicum, are especially known types of wild strains.

Corynebacterium glutamicum ATCC13032

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870

Corynebacterium melassecola ATCC17965

Corynebacterium thermoaminogenes FERM BP-1539

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 a

Brevibacterium flavum divaricatum ATCC14020 and L-amino acids producing mutants and strains thereof, such as L-lysine-producing strains, respectively.

Corynebacterium glutamicum FERM-P 1709

Brevibacterium flavum FERM-P 1708

Brevibacterium lactofermentum FERM-P1712

Corynebacterium glutamicum FERM-P 6463

Corynebacterium glutamicum FERM-P6464

Corynebacterium glutamicum DSM 5715

Corynebacterium glutamicum DSM 12866 a

Corynebacterium glutamicum DM58-1

The inventors have succeeded in isolating a new rplK gene from Corynebacterium glutamicum.

To isolate the rplK gene from C. glutamicum, a gene bank from Corynebacterium glutamicum is first established. The establishment of the gene bank is in the well-known textbooks and rp cosmid

Proceedings manuals described. As an example, the textbook Winnacker: Gene und Klone, Eine Einfuhrung in die

Gentechnology (Verlag Chemie, Weinheim, Germany, 1990) or Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well known gene bank is the E. coli K-12 strain W3110, which was established by Kohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) described gene bank C.

glutamicum ATCC13032, which was established using the SuperCos I vector (Wahl et al., 1987, of the National Academy of Sciences of the United States,

84: 2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575) Bormann et al. (Molecular Microbiology 6 (3), 317-326)), on the contrary, described the C. glutamicum gene bank ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

Plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979) or pUC9 (Vieira et al., 1982, Gene 19: 259-268) may also be used to prepare the C. glutamicum gene bank in E. coli). Particularly suitable as hosts are those E. coli strains that are restriction or recombinantly defective.

An example is the DH5amcr strain described by Grant et al. (Proceedings of the National Academy of Sciences, USA, 87 (1990) 4645-4649).

Long DNA fragments cloned by cosmids can then be subcloned into conventional vectors suitable for sequencing.

DNA sequencing methods are described, among others, by Sagner et al. (Proceedings of the National Academy of Sciences, USA, 74: 5463-5467 (1977)).

In this way, new DNA sequences coding for the C. glutamicum rplK gene were obtained, which are as SEQ ID NO. 1 of the present invention. Furthermore, they were from the present sequence

DNA derived from the above amino acid sequence of the corresponding protein. In SEQ ID NO. 2 shows the resulting amino acid sequence of the rplK gene product and the L-11 protein, respectively.

The DNA coding sequences that result from SEQ ID NO. 1 by degenerating the genetic code are also part of the invention. Similarly, there are DNA sequences that hybridize to SEQ ID NO. 1 or parts of SEQ ID NO. 1, part of the invention. Amino acid sequences that will be correspondingly derived from SEQ ID NO. 2 are also part of the invention.

The inventors have found that coryneform bacteria produce L-amino acids, especially L-lysine, in an improved manner after attenuation of the rplK gene.

To achieve attenuation, either the expression of the rplK gene or, preferably, the functional property of the protein can be reduced. It is also possible to combine both measures.

Reduction of gene expression can be accomplished by appropriate culture guidance or by genetic alteration (mutation) of the gene expression signaling structure. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, start codons and terminators. See, for example, WO 96/15246, Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988), Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1988)), Jensen and Hammer (Biotechnology and Bioengenering 58: 191). (1998)), Patek et al (Microbiology 142: 1297 (1996)) and in well-known textbooks of Genetics and Molecular Biology, such as the textbook by Knippers ("Molecular Genetics)", 6th ed., Georg Thieme Verlag, Stuttgart, Germany, 1995 ) or from Winnacker ("Gene und Klone, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations resulting in a change or decrease in the functional properties of proteins are known in the art. Examples include Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mückel (Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der Allosterischen Regulation and Structure of Enzymes, Berichte des Forschungszentrum Jlichlich, JU1-2906, ISSN09442952, 1994). Summarizing representations may be taken from well-known textbooks of genetics and molecular biology such as Hagemann ("Allgemeine Genetik, Gustav Fischer Verlag, Stuttgart, 1986).

Mutations include transitions, transversions, insertions, and deletions. Depending on the effect of amino acid exchange on activity, it is referred to as "missense" or "nonsense" mutations. Insertions or deletions of at least one base pair in the gene result in a frame mutation (frame mutation) which results in incorrect amino acids or premature translation being incorporated. Instructions for producing such mutations are well within the state of the art and can be taken from known genetic and molecular biology textbooks such as e.g. textbooks: Knippers ("Molecular Genetic, 6th ed., Georg Thieme Verlag, Stuttgart, Germany, 1995), Winnacker:" Gene und Klone, VCH Verlagsgeselschaft, Weiheim, Germany, 1990) or Hagemann ("Allgemeine Genetik, Gustav Fischer, Stuttgart, 1986). Methods for preparing polymerase chain reaction (PCR) mutations are described in: Newton C.R., Graham A., " PCR, 2nd Ed., Spektrum Akademischer Verlag, Heidelberg, 1997.

An example of a plasmid by which deletion mutagenesis of the rplK gene can be performed is the plasmid pÄrplK shown in Figure 1.

Plasmid pArplK consists of the plasmid pK18mobsacB described by Jäger et al. (Journal of Bacteriology, 1992, 174).

5462-65), into which the allele of the rplK gene, as shown in SEQ ID NOs. 3. The allele designated Arp1K carries a 12 bp deletion in the 5 'region of the gene. The ArplK allele encoded by the variant L11 protein is shown as SEQ ID NO. The L11 protein variant depicted differs from the wild-type L11 protein by the lack of the proline-alanine-leucine-glycine tetrapeptide, which corresponds to amino acids at positions 30-33 of SEQ ID NO. Second

Plasmid pArplK results in homologous recombination leading to the replacement of the chromosomal rplK gene for the ArplK allele. For guidance and explanations on insert mutagenesis, see, for example, Schwarzer and Ptihler (Bio / Technology 9, 84-87 (1991)) or Fitzpatrick et al. (Applied Microbiology and Biotechnology 42: 575-580 (1994)).

Furthermore, it may be advantageous for the production of L-amino acids, especially L-lysine, in addition to attenuating the rplK gene, one or more enzymes of each biosynthesis process to potentiate, in particular overexpress.

Thus, for example, in particular for the preparation of L-lysine, one or more genes selected from the group of the dapA gene encoding dihydropicolinate synthase (EP-B 0 197 335), the lysC gene encoding feed back resistant aspartate kinase (Kalinowski et al. (1990) Molecular and General Genetics 224: 317-324), the pyc gene encoding pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), the mqo gene encoding malate: quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), the lysis gene encoding lysine export (DE-A-195 48 222), the zwal gene (DE 199 59 328.0; DSM 13115), simultaneously amplifying and overexpressing, respectively.

Furthermore, it may be advantageous for L-amino acid production that, in addition to attenuating the rplK gene, one or more genes selected from the group of:

• pck gene encoding phosphoenolpyruvate carboxykinase (DE 199 50 409.1; DSM 13047) • pgi gene encoding glucose-6-phosphate isomerase (US 09/396, 478, DSM 12969) • poxB gene encoding pyruvate oxidase (DE 199 51 975.7; DSM 13114), zwa2 gene (DE 199 59 327.2; DSM 13113), relA gene encoding PPGPP synthetase I (EC 2.7.6.5).

In addition, it may be advantageous for L-amino acid production to avoid, in addition to overexpressing 6-phosphogluconate dehydrogenase, unwanted side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms," in: Overproduction of Microbial Products , Academic Press, London, UK, 1982).

The microorganisms containing the mutated polynucleotide obtained according to claim 1 point d are also subject of the invention and can be cultured continuously or discontinuously by batch method (batch culture) or fed batch method by batch method) or repeated fed batch method by repeated batch method) to produce L- amino acids, especially L-lysine. A summary of known cultivation methods is described in the Chmiel textbook (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the Storhas textbook (Bioreactor and Periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of the relevant micro-organism strains. Descriptions of the culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology, American Society for Bacteriology (Washington, D.C., USA, 1981). As the carbon source, sugar and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids such as acid can be used. palmitic acid, stearic acid and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These may be used singly or as a mixture. As nitrogen source, organic nitrogen containing compounds such as peptones, yeast extract, meat extract, malt extract, corn aqueous extract, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be used. The nitrogen sources may be used singly or as a mixture. As the phosphorus source, potassium dihydrogen phosphate or potassium monohydrogen phosphate or the corresponding sodium-containing salts may be used. The culture medium must further contain metal salts such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth agents such as amino acids and vitamins may be used in addition to the above. In addition, suitable precursors may be added to the culture medium. Said substances used may be added to the culture in the form of a disposable batch or suitably added during the cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used to control the pH of the culture. Antifoams such as polyglycol fatty acid esters can be used to control foam development. In order to maintain the stability of the plasmids, suitable selective agents such as antibiotics may be added to the environment. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as air are introduced into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The cultivation is allowed to continue until the desired product is formed. This goal is usually achieved within 10 to 160 hours.

Methods for determining L-amino acids are known in the art. The analysis may be performed as described by Spackmann et al. (Analytical Chemistry 30, 1190 (1958)) followed by ninhydrin derivatization, or may be performed by reverse phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

The following microorganism was deposited under the Budapest Treaty at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany):

Escherichia coli strain DH5α / pArplK as DSM 13158

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be elucidated in the following with reference to Examples:

Example 1

Preparation of the genomic cosmid bank from Corynebaeterium glutamicum ATCC 13032

Chromosomal DNA was isolated from Corynebacterium glutamicum ATCC 13032 as described by Tauch et al. (1995, Plasmid 33: 168-179) and partially digested with a restriction enzyme

Sau3AI (Amersham

Pharmacia,

Freiburg,

Germany

Product code Sau3AI, Code no. 27-0913-02). fragments

The DNAs were dephosphorylated by Molecular Biochemicals, beschreibung SAP, Code no. Cosmid vector DNA with crab alkaline phosphatase (Roche Mannheim, Germany, Produkt1758250).

SuperCosl (Wahl et al. (1987)

Proceedings of the National Academy of Sciences, USA 84: 21602164), from Stratagene (La Jolla, USA, Productbeschreibung SuperCosl Cosmid Vector Kit, Code no. 251301) was digested with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, Produktbeschreibung Xbal, Code). The cosmid DNA was then digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Produktbeschreibung BamHI, Code no. 27-0868-04) and treated with cosmid DNA in this manner. treated with ATCC13032-DNA and a T4-DNA ligase-treated batch (Amersham Pharmacia, Freiburg, Germany, Produktbeschreibung T4-DNA ligase, Code no. 27-0870-04). La Jolla, USA, Gigapack II XL Packing Extract, Code no. 200217) packaged in phages for E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16: 156) 3-1575) cells were captured in 10 mM MgSO ₄ and mixed with an aliquot of phage suspension. Infection and cosmid bank titer were performed as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), and cells were spread on LB-agar (Lennox, 1955, Virology, 1: 190). ) with 100 mg / l ampicillin. After incubation overnight at 37 ° C, individual recombinant clones were selected.

r r

Example 2

Isolation and sequencing of the rplK gene

Single colony cosmid DNA was isolated with Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to manufacturer's data and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Produktbeschreibung Sau3AI2, Product No. 27-091) . DNA fragments were dephosphorylated with crab alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Produktbeschreibung SAP, Product no. 1758250). After separation by gel electrophoresis, cosmid fragments were isolated in the size range from 1500 to 2000 bp with the QiaExII_ Gel Extraction Kit. (Product No. 20021; Qiagen, Hilden, Germany). The pZero-1 DNA sequencing vector obtained from Invitrogen (Groningen, Niederland, Zero Background Cloning Kit, Product No. K250001) was digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Produktbeschreibung BamHI, Product No. 27-0868-04). ). The ligation of the cosmid fragment into the Zero-1 sequencing vector was performed as described by Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor), and the DNA mixture was incubated overnight with T4 ligase (Pharmacia Biotech, Freiburg) This ligation mixture was then electroporated (Tauch et al. 1994, FEMS Microbiol. Letters, 123: 343-7) into E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences, USA, 87). : 4645-4649) and plated on LB-agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l zeocin The preparation of plasmid recombinant clones was performed with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) Sequencing was performed by the dideoxy chain breaking method of Sanger et al.

Proceedings of the National Academy of Sciences (U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acid Research, 18: 1067). The RR dRhodamine Terminator Cycle Sequencing Kit from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. Gel electrophoresis separation and sequencing reaction analysis was performed in a Rotiphorese NF Acrylamide / Bisacrylamide (29: 1) gel (Product No. A124.1, Roth, Karlsruhe, Germany) with a sequencing apparatus "ABI Prism 377 from PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained was then processed using the Staden-Program package (1986, Nucleic Acid Research, 14: 217-231) version 97-0. Individual sequences of pZerol derivatives were pooled in one contiguous contig. Computer-aided. analysis of the coding region was performed with the XNIP program (Staden, 1986, Nucleic Acid Research, 14: 217-231). Further analyzes were performed with the BLAST search Programs (Altschul et al., 1997, Nucleic Acid Research, 25: 3389-3402, against the non-redundant National Center for Biotechnology Information (NCBI, Betheseda, MD, USA).

The nucleotide sequence obtained is shown in SEQ ID NO. 1. Nucleotide sequence analysis gave a free reading frame of 438 base pairs, which was designated as the rplK gene. The rplK gene encodes a polypeptide of 145 amino acids in SEQ ID NO. Second

Example 3

Preparation of rplK gene copy vector

The chromosomal 1200 bp DNA fragment containing the rplK gene from C. Glutamicum was cloned by PCR.

To this end, chromosomal DNA was isolated from Corynebacterium glutamicum ATCC 13032 as described by Tauch et.

Al r al. (1995, Plasmid 33: 168-179). A 1200 bp DNA fragment containing the rplK gene was amplified by the polymerase chain reaction. Then, based on SEQ ID NO. 1 derived the following primers:

Pl-up (see also SEQ ID No. 5):

5 'AGG AGC AGG CTG TTG TCA CC -3'

P2-down (see also SEQ ID No. 6):

5'-GCG GAT TAC GTC GAT GG -3 '

The oligonucleotides shown were synthesized by ARK Scientific (ARK Scientific GmbH, Biosystems, Darmstadt, Germany) and PCR was performed using Pfu DNA polymerase (Stratagene, La Jolla, USA) and PTC-100 thermal cycler (MJ Research'Inc., Waltham, USA).

The PCR was run 35 times, consisting of thermal denaturation (94 ° C, 90 seconds), cooling (58 ° C, 90 seconds), and polymerase reaction (72 ° C, 90 seconds). The 1200 bp DNA fragment thus obtained was then purified by Qiagen PCR Purification Spin Kits (Qiagen, Hilden, Germany).

A plasmid pECM3, a derivative with the deletion of the described plasmid pECM2 (Tauch et al., FEMS Microbiological Letters, 123, 343-347 (1994)) was prepared for cloning a DNA amplifier containing the rplK gene into a plasmid that can replicate in C. glutamicum. ). For this, plasmid pECM2 was restricted with BamHI restriction enzymes (Amersham Pharmacia, Freiburg, Germany) and BglII (Amersham Pharmacia, Freiburg, Germany) and treated with T4-ligase (Amersham Pharmacia, Freiburg, Germany), as in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) to obtain plasmid pECM3. Transformation by Grant et al., Described (Proceedings of the National Academy of Sciences USA 87: 4645-4649 (1990)) of E. coli strain The DH5aMCR plasmid pECM3 was performed as described by Tauch et al (FEMS

Microbiological Letters, 123, 343-347 (1994)). Transformants were selected on LBG-agar (10 g Trypton, 5 g yeast extract, 5 g NaCl, 2 g glucose, 15 g agar per liter) to which chloramphenicol (Merck, Darmstadt, Germany) was added (50 mg / l) .

Then, a 1200 bp DNA amplification containing the rplK gene was mixed with plasmid pECM3, which had been pre-linearized with the restriction enzyme SmaI (Amersham Pharmacia, Freiburg, Germany), and treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany) to obtain the plasmid. prplK. Transformation of the E. coli strain DH5αMCR with the plasmid prplK was performed as described by Tauch et al. (FEMS Microbiological Letters, 123, 343-347 (1994)), and transformants were selected on LBG-agar to which chloramphenicol (Merck, Darmstadt, Germany) was added (50 mg / L). The plasmid prplK thereby carries the complete rplK gene from C. glutamicum and can autonomously replicate in both E. coli and C. glutamicum.

Example 4

Incorporation of the deletion into the rplK gene

Using the PCR technique, an allele of the rplK gene, designated ArplK, was prepared which carries a 12 bp deletion. The 12 bp deletion produced in the rplK gene results in the loss of the proline-alanine-leucine-glycine tetrapeptide in the N-terminal region of the C. glutamicum Lll protein.

In addition to the Pl-up and P2-down primers described in Example 3, the oligonucleotides described below, prepared by ARK Scientific (ARK Scientific GmbH Biosystems, Darmstadt, Germany), were used as a primer for the production of the rplK deletion allele.

Pl-down (see also SEQ ID No. 7):

5'-extension-CGC CGT GAG C-5'-side-GCC AAC

P2-up (see also SEQ ID No. 8):

'-extension-TCC AGT TGG C-5'-side-GCC CAC GGC GTC AAC ATC AG-3'

The primers used for PCR were derived from a known DNA sequence. They each contain a 10 bp 5'-extension that is exactly complementary to the 5'-sides of the P1-up and P2down primers. Chromosomal DNA was extracted from C. glutamicum ATCC 13032 and with it as a matrix using the indicated nucleotide Pl-up, Pl-down, P2-up and P2-down, Pfu-DNA polymerase (Stratagene, La Jolla, USA), and PTC thermal cycler. -100 (MJ. Research. Inc., Waltham, USA) first produced two 600 bp DNA fragments in separate PCR reactions. In these PCR reactions, a cycle consisting of thermal denaturation (94 ° C, 90 seconds), cooling (58 ° C, 90 seconds) and polymerase reaction (72 ° C, 90 seconds) was run 35 times.

The first 600 bp DNA amplification, designated rplK-Part 1, was obtained with Pl-up and Pl-down oligonucleotides. It contains the 5 'region of the rplK gene (nucleotide 1-87) and additionally a 10 bp extension derived from the Pl-down oligonucleotide, which corresponds to nucleotides 100-109 of the rplK gene (SEQ ID No. 1). Thus, this amplified region of the gene shows a gap of 12 bp compared to the chromosomal DNA template used. The second 600 bp DNA fragment, rplK-part 2, was obtained with P2-down and P2-up oligonucleotides and comprises the 3'-region of the rplK gene (nucleotide 78435) and carries an identical gap (nucleotide 88-99) within the amplified region of the rplK gene. Both of these 600 bp DNA amplifiers consequently have a 20 bp DNA overlap. Both the 600 bp PCR products rplK-part 1 and rplK-part 2 were used as DNA templates in the next PCR reaction, whereby a "strand of rplK-part 1 could be ligated there with a strand back of rplK-part 2 based on the overlapping complementary DNA" The extension of this DNA overlap, respectively, the addition of Pl-up and P2-down oligonucleotides, resulted in the production of a 1200 PCR amplification, which includes a 12 bp derivative with a deletion of the C. glutamicum rplK gene. In these PCR reactions, a cycle consisting of thermal denaturation (94 ° C, 90 seconds), cooling (58 ° C, 90 seconds) and polymerase reaction (72 ° C, 90 seconds) was run 35 times.

The nucleotide sequence of the ArplK allele is shown in SEQ ID NO. 3 and a variant of the L11 protein encoded by this allele in SEQ ID NO. This Lll-protein variant lacks the tetrapeptide proline-alanine-leucine-glycine corresponding to amino acid positions 30-33 of the wild-type Lll-protein shown in SEQ ID NO. Second

Example 5

Incorporation of the ArplK allele into the chromosome

The ArplK allele, which contains a 12 bp deletion in the rplK gene, was incorporated into the C. glutamicum chromosome by integration mutagenesis using the SacB helper system described by Schäfer et al., Gene, 14, 69-73 (1994). This system allows one skilled in the art to identify or select allele exchanges that are performed by homologous recombination.

1. Construction of the ArplK exchange vector

In Example 4, the 1200 bp ArplK allele with rplK deletion was obtained by Qiagen PCR Purification Spin Kits (Qiagen,

Hilden, Germany) purified and used for the ligation described (Schäfer et al., Gene, 14, 69-73 (1994)) of the mobilizable cloning vector pK18mobsacB. This vector was pre-linearized with the restriction enzyme SmaI (Amersham Pharmacia, Freiburg, Germany), mixed with the rplK deletion allele and treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany).

This resulted in plasmid pArplK.

Transformation of E. coli strain DH5a with plasmid pArplK was performed as described by Tauch et al. (FEMS Microbiological Letters, 123, 343-347 (1994). Transformants were selected on LBG-agar to which kanamycin (Merck) was added. (Darmstadt, Germany) (50 mg / L) to produce the DH5α / pArplK strain.

One clone was selected and designated as ATCC13032ArplK.

2. Perform allele exchange

The chromosomal 12 bp deletion in the C. glutamicum rplK gene was obtained by integration mutagenesis using the SacB helper system described by Schäfer et al., Gene, 14, 69-73 (1994). This system allows one skilled in the art to identify or select allelic exchanges that are performed by homologous recombination.

The mobilizable plasmid pArplK was - starting from Simon et al., Bio / Technology, 1, 784-794 (1993) of the described donor strain, E. coli S17-1 - using the conjugation method described by Schäfer et al., Journal of Bacteriology, 172, 1663-1666 (1990) introduced as a recipient in C. glutamicum ATCC 13032. Since the plasmid pArplK cannot replicate in C. glutamicum, its establishment is only possible by integration into the C. glutamicum chromosome via homologous recombination between the fragment with the rplK deletion. encoding the plasmid and the identical chromosomal region of rplK. Transconjugants were selected on LBG-agar to which kanamycin (25 mg / l) (Merck, Darmstadt, Germany) and nalidixic acid (Merck, Darmstadt, Germany) (50 mg / l) were added.

For the subsequent excision of plasmid pArplK by the sacBsystem, it could only be selected from rplK in the presence of the wild-type allele. To this end, the plasmid prplK constituted in Example 3, which carries the complete rplK gene, is transferred to the integrant strain by electroporation according to Liebl et al., FEMS Microbiological Letters 65, 299-304 (1989). Strain selection was performed on LBG-agar to which kanamycin (25 mg / l) (Merck, Darmstadt, Germany) and chloramphenicol (10 mg / l) (Merck, Darmstadt, Germany) were added.

The selected, transformed colony was re-seeded in 100-ml - LBG - liquid media - (in an Erl-enmeyer-250 ml bully) and incubated for 24 hours at 30 ° C and 300 rpm. Then, 2x10 ⁶ cells / ml of this liquid culture were spread on LBG-agar containing 10% sucrose (Merck, Darmstadt, Germany) and incubated for 48 hours at 30 ° C. C. glutamine cells that were able to grow in this environment lost the integrated plasmid pArplK due to the second recombination event between the allele with the rplK deletion and the natural region of rplK. This second recombination event either results in the restoration of the natural, chromosomal alignment of the rplK gene, or results in the production of an ArplK mutant that lost the 12 bp N-terminal DNA fragment. Chromosomal DNA was extracted from selected, sucrose-resistant and potential C. glutamicum cells bearing pArplK. This served as the matrix with which the Pdel-up and P-del-down2 oligonucleotides were derived using the rplK sequence (ARK Scientific GmbH Biosystems, Darmstadt, Germany). PCR-experiments were performed with primers, Pfu-DNA polymerase (Stratagene, La Jolla, USA) and PTC 100 thermal cycler (MJ Research Inc., Waltham, USA). The PCR was run 35 times, consisting of thermal denaturation (94 ° C, 90 seconds), cooling (58 ° C, 90 seconds) and polymerase reaction (72 ° C, 90 seconds). The DNA amplifications thus obtained were then purified by Qiagen PCR Purification Spin Kits (Qiagen, Hilden, Germany). Analysis of the nucleotide sequences of the purified amplitudes, which were prepared as described previously, indicated that in 43% of the cases, a DNA amplification lacking a 12 bp stretch of DNA was produced. As a result, in the respective pArplK-bearing transconjugants, the second recombination event resulted in the production of a chromosomal 12 bp deletion in the rplK gene.

Finally, the selection of transconjugants harboring a deletion-plasmid-deleted--prplK-metódou-, _R plazmid.-curings described by Schafer et al., Journal of Bacteriology, 176, 7309-7319 (1990). The C. glutamicum ATCC13032 ArplK strain thus obtained carries such a chromosomal 12 bp deletion within the rplK gene, resulting in the loss of the proline-alanine-leucine-glycine L11 protein tetrapeptide.

Example 6

Preparation of lysine

The C. glutamicum strain ATCC13032 ArplK obtained in Example 5 was cultured in a culture medium suitable for lysine production and the lysine content of the remainder of the culture was determined.

For this, the strain was first incubated on an agar plate (brain-heart agar) for 24 hours at 33 ° C. A preculture (10 ml of medium in 100 ml) was inoculated from this culture on an agar plate

100 ml Erlenmeyer flask). Pre-culture medium was complete CglII medium (10 g / L Bacto-Pepton, g / L yeast extract, 2.5 g / L NaCl, 20 g / L glucose, pH 7.4). The preculture was incubated for 24 hours at 33 ° C at 240 rpm on a shaker. The main culture was inoculated from this preculture so that the initial OD (660 nm) of the main culture was 0.1 OD. MM medium was used for the main culture.

MM Medium

CSL (Corn Steep Liquor) 5 g / L

MOPS (morpholinopropanesulfonic acid) 20 g / l glucose (separately autoclaved) 50 g / l (NH ₄ ) ₂ SO ₄ 25 g / l

KH ₂ PO ₄ 0.1 g / l

MgSO ₄ * 7H ₂ O 1.0 g / 1 JCaCla * -2.HaO --------------------------..... ._ .. .......... lCLmg / 1. .

FeSO ₄ * 7 H ₂ O 10 mg / L

MnSO ₄ * H ₂ O 5.0 mg / 1 biotin (sterile filtered) 0.3 mg / 1 thiamine * HCl (sterile filtered) 0.2 mg / 1

CaCO ₃ 25 g / L

The CSL, MOPS and brine were adjusted to pH 7 with ammonia water and autoclaved. Sterile substrate and vitamin solutions as well as dry autoclaved CaCO _{3 are then added} .

Cultivation is carried out in a volume of 10 ml in 100 ml bullying Erlenmeyer flasks. Cultivation was performed at 33 ° C and 80% air humidity.

After 48 hours, OD was measured at 660 nm with Biomek 1000 (Beckmann Instruments GmbH, Miinchen). The amounts of lysine produced were determined by an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.

r β r r p r

Table I shows the result of the experiment.

Table 1

tribe OD (660) Lysine-HCl g / l ATCC13032 ArplK 13.0 0.98 ATCC 13032 13.8 0.0

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 PÄrplK plasmid card

Abbreviations and designations used have the following meanings:

pdeltarplK pArplK sacB-gene sacB gene from Bacillus suptilis, encoding the enzyme levansaccharase lacZ-alpha 'part of the 5'-end of the β-galactosidase gene oriV origin of replication

KmR: kanamycin resistance

RP4 mob region of mob plasmid RP4

BamHI: BamHI restriction enzyme cut site

EcoR1: EcoR1 restriction enzyme cut site

ArplK: 12 bp deletion rplK allele in the N-terminal region 'f

P *

SEQUENCE PROTOCOL <110> Degussa-Hiils Ag <120> New nucleotide sequences encoding the rplK gene <130> 990178 BT <140>

<141>

<160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 835 '<212> DNA <213> Corynebacterium glutamicum <220>

<221> CDS <222> (201) .. (635) <223> rplK gene <4G0> 1 ttgcgtgtag ggtagacaat cgcgtgtttt ttaagcatgc tcaaaatcat tcatccccgg 60 tggcccggtt acgtaaagat cagcaaagat gatcaactaa agcgatcatc tgaagttgta 120 gegggaccga gcatccggac ggttactagt ggggtttcat cgtcccagtt gtggccggta 180 atg get acaaggaagc aggtttaacg cct aag aag aag aag aag aag gtc act ggc 233 Met Ala Pro Lys Lys

10

ctc atag ac ctc cag atc cag gca gc aac cct get Ala 25 CCT for ca. for 261 Leu Ile Lys Leu gin Ile gin Ala Gly 20 Gin Ala same time for 15 gtt GGC ca. GCA CTT GGT get CAC GGC GTC aac ATC atg gaa ttc TGC 329 wall Gly for Ala Leu Gly Ala His Gly wall same time Ile Met Glu Phe Cys 30 35 40 aag get tac aac get GCG act gaa aac cag ege GGC aac gtt gtt CCT 377 Lys Ala Tyr same time Ala Ala Thr Glu same time gin Arg Gly same time wall wall for 45 50 55 gtt gag ATC acc gtt tac gaa GAC CGT tCA ttc GAC ttc aag CTG aag 425 wall Glu Ile Thr wall Tyr Glu Asp Arg Ser Phe Asp Phe Lys Leu Lys 60 65 ⁷⁰ 75 act CCT ca. get GCA aag CTT CTT CTG aag get get GGC CTG cag aag 473 Thr for for Ala Ala Lys Leu Leu Leu Lys Ala Ala Gly Leu gin Lys 80 85 90 GGC TCC GGC gtt CCT CAC acc cag aag GTC GGC aag gtt TCC atg get 521 Gly Ser Gly wall for His Thr gin Lys wall Gly Lys wall Ser Met Ala 95 100 105 cag gtt CGT gag ATC get gag acc aag aag gaa GAC CTG aac get ege 569 gin wall Arg Glu Ile Ala Glu Thr Lys Lys Glu Asp Leu same time Ala Arg 110 115 120

gin Arg 50 Gly same time wall wall for 55 wall Glu Ile Thr Val Tyr Glu Asp Arg Ser 65 Phe Asp Phe Lys Leu 70 Lys Thr for for Ala Ala Lys Leu Leu Leu 80 Lys Ala Ala Gly Leu 85 gin Lys Gly Ser Gly 90 Val.Pro. His Thr gin 95 Lys wall Gly Lys wall 100 Ser Met Ala gin wall 105 Arg Glu'íle Ala Glu 110 Thr Lys Lys Glu Asp 115 Leu same time Ala Arg Asp 120 Ile Asp Ala Ala Ala 125 Lys Ile Ile Ala Gly 130 Thr Ala Arg. Ser Met 135 Gly Ile Thr Val Glu Gly

<210> 5 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> Pl-up <220>

<223> -Opi.S — artificial — sequence: primer-: <400> 5 aggagcaggc tgttgtcacc <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> P2 down <220>

<223> Artificial sequence description: primer <400> 6 gcggatagct acgtčgatgg 20 <210> 7 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Pl-down <220>

<223> Artificial sequence description: diameter <400> 7 cgccgtgagc gccaactgga ggagcagggt <210> 8 gat atc gac get gcg aag atc getc ggt acc. get cgt tcc atg

Asp Ala Asp Ala Ala Lys Ala Ala Gly Thr Ala Arg Ser Met

125 130 135 ggc atc acc gtc gaa ggc taaaagcttt cacaccggtt agtggctcat 665

Gly clay Thr Val Glu Gly 140 145. <<<> <<> <<

Met Ala For Lys Lys Lys Lys Lys Val Thr Gly Leu Clay Lys Leu Gin

5 10 15.

Gin Ala Gly Gin - Ala Asn Pro Ala Pro Pro Val Gly Pro Ala Leu

25 30

-Gly — Al-a — H-i-s — € -l-y — Va 1 — Asn-il-e · 35

Ala Thr 50 Glu same time gin Arg Gly 55 Tyr 65 Glu Asp Arg Ser Phe 70 Asp Lys Leu Leu Leu Lys 85 Ala Ala His Thr gin Lys 100 wall Gly Lys Ala Glu Thr 115 Lys Lys Glu Asp Ala Lys 130 Ile Ile Ala Gly Thr 135

40 45 -ÄiTr same time wall wall for wall 60 Glu Ile Thr wall Phe Lys Leu Lys 75 Thr for for Ala Ala 80 Gly Leu gin 90 Lys Gly Ser Gly wall 95 for wall Ser 105 Met Ala gin wall Arg 110 Glu Ile Leu 120 same time Ala Arg Asp Ile 125 Asp Ala Ala Ala Arg Ser Met Gly 140 Ile Thr wall Glu

Gly ^r ? 145 <210> 3 <211> 825 <212> DNA <213> Corynebacterium glutamicum <220>

<221> CDS <222> (200) .. (622) <223> Delta rplK <400> 3 tgcgtgtagg gtagacaatc gcgtgttttt taagcatgct caaaatcatt catccccggt 60 ggcccggtta cgggaccgag caaggaagca cgtaaagatc agcaaagatg catccggacg gttactagtg ggtttaacg get atg cct Met Ala Pro atcaactaaa gcgatcatct gaagttgtag gggtttcatc gtcccagttg tggccggtaa aag aag aag aag aag gtc act ggc Lys Lys

120

180

232

CTC Leu atc aag ctc cag gin atc cag gca. Gin Ala gca cac gca aac cct get cct approx 280 Lys Leu 15 Gly 20 gin Ala same time For Ala 25 for for gtt GGC get CAC GGC GTC aac ATC atg gaa ttc TGC aag get tac aac 328 wall Gly Ala His Gly wall same time Ile Met Glu Phe Cys Lys Ala Tyr same time 30 35 40 get GCG act gaa aac cag ege GGC aac gtt gtt CCT gtt gag ATC acc 376 Ala Ala Thr Glu same time gin Arg Gly same time wall wall for wall Glu Ile Thr 45 50 55 gtt tac gaa GAC CGT tCA ttc GAC ttc aag CTG aag act CCT ca. get 424 wall Tyr Glu Asp Arg Ser Phe Asp Phe Lys Leu Lys Thr for for Ala 60 65 70 ⁷⁵ GCA aag CTT CTT CTG aag get get GGC CTG cag aag GGC TCC GGC gtt 472 Ala Lys Leu Leu Leu Lys Ala Ala Gly Leu gin Lys Gly Ser Gly wall 80 85 90

cct cac acc cag aag gtc ttt atg get cag gtt CGT Arg gag Glu 520 for His Thr gin 95 Lys wall Gly Lys wall 100 Ser Met Ala gin wall 105 ATC get gag acc aag aag gaa GAC CTG aac get ege gat ATC GAC get 568 Ile Ala Glu Thr Lys Lys Glu Asp Leu same time Ala Arg Asp Ile Asp Ala 110 115 120 get GCG aag ATC ATC get GGT acc get CGT TCC atg GGC ATC ACC GTC 616 Ala Ala Lys Ile Ile Ala Gly Thr Ala Arg Ser Met Gly Ile Thr wall 125 130 135

gaa ggc taaaagcttt cacaccggtt agtggctcat tcaaaatgaa tggccaccaa 672

Glu Gly

140 ca.attttca ccatgaaagg gatcgacgct ca.aagtttt gaatttctaa ggtcgcatct atgtggcagg gccagctccg gcccgttaaa ccacagaatt 732 tgagcaagaa ctctaaggcg taccgcgagg ccgctg2a2a <cg> <cg> <cg> <cg> <cg> <cg>

<400> 4 Met Ala 1 for Lys Lys 5 Lys Lys Lys wall Thr 10 Gly Leu Ile Lys Leu 15 gin Ile gin Ala Gly 20 gin Ala same time for Ala 25 for for wall Gly Ala 30 His Gly wall ASN Ile 35 Met Glu Phe Cys Lys 40 Ala Tyr same time Ala Ala 45 Thr Glu same time

<211> 30 <212> DNA <213> Artificial sequence <220>

<223> P2-up <220>

<223> Artificial sequence description: primer <400> 8

Iccagtlggc gclcacggcg Icaacalcag

Claims (19)

Patent Claims • 1. An isolated polynucleotide from coryneform bacteria comprising a polynucleotide sequence selected from the group consisting of: a) a polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO. 2 b) a polynucleotide that encodes a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO. 2 c) a polynucleotide that is complementary to polynucleotides a) or b), and ______d) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide sequence a), b) or c. The polynucleotide of claim 1, wherein the polynucleotide is replicable, preferably recombinant DNA. The polynucleotide of claim 1, wherein the polynucleotide is RNA. The polynucleotide of claim 2, comprising the nucleic acid sequence as shown in SEQ ID NO. First The polynucleotide sequences of claim 2, encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. Second A polynucleotide according to claim 1, in particular point d, comprising a nucleotide sequence as shown in SEQ ID NO. Third r r A polynucleotide according to claim 1, in particular point d, encoding a polypeptide comprising the nucleotide sequence shown in SEQ ID NO. 4th The replicable DNA of claim 2, comprising (i) the nucleotide sequence shown in SEQ-ID-No. Or (ii) at least one sequence that corresponds to sequence (i) within the degeneracy region of the genetic code, or (iii) at least one sequence that hybridizes a sequence complementary to sequence (i) or (ii), and optionally (iv) functionally faithful meaningful mutants in (i). A vector comprising the polynucleotide of claim 1, deposited in E. coli, DH5α / βArpk as DSM 13158, shown in SEQ ID NO. 3 and 1. 1 10. As a host cell serving coryneform bacteria that contain a deletion or insertion in the rplK gene. A process for the preparation of L-amino acids, in particular L-lysine, characterized in that the following steps are performed: a) fermentation of the bacteria producing the desired L-amino acid in which at least the rplK gene is attenuated, (b) enrichment with the desired amino acid in the medium or in the cells of the bacteria; and c) isolating the L-amino acid. Method according to claim 11, characterized in that bacteria are used in which additional genes are additionally amplified in the process of biosynthesis of the desired L-amino acid. * · * E e P · n p P P p The method according to claim 11, characterized in that bacteria are used in which at least part of the metabolism processes which attenuate the formation of the desired amino acid are used. Method according to claim 11, characterized in that the expression of the polynucleotide according to claim 1, in particular 1 to 1, is reduced. d. The method according to claim 11, characterized in that the catalytic properties of the polypeptide (enzyme protein) encoding the polynucleotide according to claim 1, in particular 1 to 1 d, are reduced. Method according to claim 11, characterized in that bacteria are used in which insertion mutagenesis is produced to attenuate using the plasmid pArpk shown in FIG. 1 and deposited as DSM 13158. Method according to claim 11, characterized in that for the preparation of L-amino acids, in particular L-lysine, bacteria are fermented in which one or more genes selected from the group are simultaneously expressed. 17.1 dapA gene encoding a dihydrodipicolinate synthase 17.2 lysC gene encoding feed back resistant aspartate kinase 17.3 DNA fragment mediating S- (2-aminoethyl) -cysteine resistance 17.4 pyc gene encoding pyruvate carboxylase, 17.5 pyc gene (Eikmanns (1992) Journal of Bacteriology) 17.6 mqo gene coding for malate: quinone oxidoreductase 17.7 lysE gene encoding lysine export 17.8 zwal gene. Method according to claim 11, characterized in that for the preparation of amino acids, in particular L-lysine, bacteria are fermented in which one or more genes selected from the group are simultaneously attenuated. 18.1 pck gene encoding phosphoenolpyruvate carboxykinase, 18.2 pgi gene encoding glucose-6-phosphate isomerase 18.3 poxB gene encoding pyruvate oxidase 18.4 zwa2 gene or 18.5 Rela gene encoding PPGPP-synthetase I. Method according to one or more of the preceding claims, characterized in that microorganisms of the genus Corynebacterium glutamicum are used. Use of the polynucleotide sequences of claim 1 as primers for the preparation of DNA genes that develop an effect corresponding to the rplK gene by a polymerase chain reaction. Use of the polynucleotide sequences of claim 1 as hybridization probes.