DE10017057A1 - New ribosomal L1 protein gene from coryneform bacteria, useful, when downregulated, for increasing fermentative production of amino acids - 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

The invention relates to the rplK gene coding Nucleotide sequences from coryneform bacteria and a Process for the fermentative production of amino acids, especially L-lysine, weakening the rplK gene.

State of the art

L-amino acids, especially L-lysine, can be found in the Animal nutrition, in human medicine and in pharmaceutical industry application.

It is known that these amino acids by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of the great importance is constantly attached to the improvement of the Manufacturing process worked. To improve the Performance characteristics of these microorganisms Methods of mutagenesis, selection and mutant selection applied. In this way you get tribes that resistant to antimetabolites such as B. the lysine analogue S- (2-aminoethyl) cysteine or auxotrophic for regulatory are important metabolites and produce L-amino acids.

Methods of recombinant DNA technology for strain improvement L- Amino acid producing strains of Corynebacterium used by single amino acid biosynthesis genes amplified and the impact on the L-amino acid Production examined. Review articles on this can be found among others at 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 protein K) is an ingredient first described for Escherichia coli the bacterial ribosome, the translational apparatus of the Cell on which protein synthesis takes place.

Ribosomes are cellular particles made up of three Ribonucleic acid (RNA) molecules and a defined one Assemble number of proteins. Ribosomes are mostly obtained from cell extracts by ultracentrifugation. The further purification from the remaining cell components usually takes place by sedimentation in sucrose Gradients. This technique of preparation has become one of the Common names for the components of the Ribosomes that directly related to the Take sedimentation properties. A functional one For this reason, bacterial ribosome is often called 70S ribosome, which consists of the small subunit 30S subunit and the large subunit 50S Subunit exists. The small 30S subunit from E. coli consists of 21 different polypeptides and one RNA molecule with a length of 1542 nucleotides, the Known to those skilled in the art as 16S rRNA; the large 50S subunit contains 31 more besides the RplK protein Polypeptides, along with two RNA molecules with one Length of 120 or 2904 nucleotides, the so-called 5S- or 23S RNA molecules. In the meantime, has for the ribosomal proteins an alternative nomenclature established. So the polypeptides of the small 30S Subunit by the specialist with S1 to S21, that of the large 50S subunit labeled L1 through L32. The RplK Protein corresponds to the ribosomal L11 protein (Noller and Nomura, In: Neidhardt et al. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington D.C., 167-186, 1996). In recent years, L11-like ones Proteins also in other organisms like Borrelia burgdorferi (Fraser et al., Nature, 390, 580-586, 1997), Helicobacter pylori (Tomb et al., Nature, 388, 539-547, 1997), Serratia marcescens (Sor and Nomura, Molecular and  general Genetics, 210, 52-59, 1987), Haemophilus infuenzae (Fleischmann et al., Science, 269, 496-512, 1995) and in the gram-positive bacterium Bacillus subtilis (Jeong et al., Molecular Microbiology, 10, 133-142, 1993) identified.

The process of translation taking place at the ribosome, that is the Matrix RNA (mRNA) directed biosynthesis of polypeptides, is complex. In addition to the ribosome, there are other proteins for essential to the translation process, which the expert calls Protein synthesis factors are referred to (Noller, Annual Review of Biochemistry, 60, 191-227, 1991). The L11 protein mediates the interaction between the ribosome and some Protein synthesis factors; as an example here Elongation factor G (EF-G) and the termination factor 1 (RF-1). The absence of the L11 protein in the Ribosomes of E. coli L11 mutants therefore lead to one Reduction of the translation rate (Xing and Draper, Biochemistry, 35, 1581-1588, 1996).

The L11 protein is also essential for binding and activity of the RelA protein on the ribosome. RelA catalyzes synthesis under amino acid deficiency conditions of guanosine tetraphosphate (ppGpp) by transfer a pyrophosphate group from ATP to GDP. ppGpp affected in E. coli the expression of many genes, either negative or positive. In general, the expression of Gene products that are effective in biosynthetic pathways, stimulates. Gene products that are catabolically active, are usually negatively regulated accordingly. By ppGpp have a large number of genes and Operons regulates one in amino acid biosynthesis play a central role. To the previously known in E. coli positively influenced genes include a. argF, argI, argECBH (arginine biosynthesis), gltB, glnA, gdh (Glutamine / glutamate biosynthesis), ilvB, IlvA (isoleucine Biosynthesis), metC, metF, metK (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: Cellular and molecular biology. American Society for Microbiology, Washington D.C., 1458-1496, 1996). The function of ppGpp as a positive regulator of amino acid biosynthesis meanwhile also in other bacteria like 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, 26, 65-79, 1997).

It is clear from the prior art that there is an interest insists on finding out if knowledge of the Nucleotide sequence of the rplK gene of coryneform bacteria Improve the amino acid production of these bacteria contributes.  

Object of the invention

The inventors set themselves new measures for the improved fermentative production of amino acids, especially L-lysine.  

Description of the invention

L-amino acids, especially lysine, can be found in the Human medicine and in the pharmaceutical industry, in the Food industry and especially in the Animal nutrition application. There is therefore a general one Interested in new approaches for improved processes for Production of amino acids, especially L-lysine, to provide.

The invention relates to an isolated polynucleotide containing a polynucleotide sequence selected from the group

• a) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide that the amino acid sequence of SEQ ID No. 2 contains
• b) polynucleotide encoding a polypeptide that a Contains amino acid sequence that is at least 70% is identical to the amino acid sequence of SEQ ID No. 2,
• c) polynucleotide that is complementary to the Polynucleotides of a) or b), and
• d) polynucleotide containing at least 15 successive bases of the polynucleotide sequence of a), b) or c).

The invention also relates to the polynucleotide according to claim 1, which is preferably a replicable DNA comprising:

• a) the nucleotide sequence shown in SEQ ID No. 1, or
• b) at least one sequence that corresponds to sequence (i) within the range of degeneration of the corresponds to genetic codes, or
• c) at least one sequence that matches the sequence (i) or (ii) complementary sequence hybridized, and optionally
• d) functionally neutral meaning mutations in (i).

Other items are
a polynucleotide according to claim 4, containing the nucleotide sequence as in SEQ ID No. 1 shown
a polynucleotide according to claim 2, which codes for a polypeptide having the amino acid sequence as in SEQ ID No. 2 contains,
a polynucleotide according to claim 1, in particular point d, containing the nucleotide sequence as in SEQ ID No. 3 shown
a polynucleotide according to claim 1, point d, which codes for a polypeptide having the amino acid sequence as described in SEQ ID No. 4 contains,
a vector containing a mutant polynucleotide according to claim 1, point d shown in SEQ ID No. 3 and Fig. 1 (Δ = deHa) and deposited according to the Budapest Treaty in E. coli DH5α / pΔrplK as DSM 13 158,
and coryneform bacteria serving as the host cell which contain an insertion or deletion in the rplk gene.

The invention also relates to polynucleotides which are in the essentially consist of a polynucleotide sequence which are available through hybridization screening a corresponding gene bank that contains the complete gene the polynucleotide sequence corresponding to SEQ ID No. 1 contains with a probe that the sequence of the said Polynucleotides according to SEQ ID No. 1 or a fragment thereof contains and isolation of said DNA sequence.

Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA to cDNA  isolate in full length for the ribosomal Encode protein L11 and such cDNA or genes isolate that have a high similarity of the sequence to that of the rplK gene.

Polynucleotide sequences according to the invention are still suitable as a primer, with the help of the polymerase Chain reaction (PCR) DNA can be produced from genes those for the RplK gene product or ribosomal protein L11 encode.

Such oligonucleotides serving as probes or primers contain at least 30, preferably at least 20, whole particularly preferably at least 15 consecutive Nucleotides. Oligonucleotides are also suitable at least 40 or 50 base pairs in length.

"Isolated" means from its natural environment separated out.

"Polynucleotide" generally refers to Polyribonucleotides and polydeoxyribonucleotides, wherein it unmodified RNA or DNA or modified RNA or DNA can act.

"Polypeptides" means peptides or proteins, the two or more linked via peptide bonds Contain amino acids.

The polypeptides according to the invention include the polypeptide according to SEQ ID No. 2, especially those with the biological activity of the RplK gene product and also those that are at least 70% identical to the Polypeptide according to SEQ ID No. 2, preferably at least 80% and especially at least 90% to 95% identity with the Polypeptide according to SEQ ID No. 2 and the activity mentioned exhibit.

The invention further relates to a method for fermentative production of amino acids, in particular Lysine, using coryneform bacteria that in particular already produce and in the amino acids which the nucleotide sequences coding for the rplK gene are weakened.

The term "weakening" describes in this Related to reducing or eliminating the intracellular activity or function of one or more Enzymes or proteins in a microorganism that are caused by the corresponding DNA can be encoded by for example, uses a weak promoter or a Gene or allele used that for a corresponding enzyme or encodes protein with a low activity or that corresponding gene or enzyme or protein inactivated and where appropriate, combined these measures.

The microorganisms that are the subject of the present Invention are, amino acids, especially lysine, from Glucose, sucrose, lactose, fructose, maltose, molasses, Manufacture starch, cellulose or from glycerin and ethanol. It can be representative of coryneform bacteria act in particular of the genus Corynebacterium. In the The genus Corynebacterium is especially the species Corynebacterium glutamicum to be called in the professional world is known for its ability to produce L-amino acids to produce.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium melassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and mutants or strains produced therefrom which produce L-amino acids,
such as the L-lysine producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464
Corynebacterium glutamicum DSM 5715
Corynebacterium glutamicum DSM 12866 and
Corynebacterium glutamicum DM58-1

The inventors succeeded in the new rplK gene from Isolate Corynebacterium glutamicum.

To isolate the rplK gene from C. glutamicum initially a gene bank of Corynebacterium glutamicum created. The creation of gene banks is general known textbooks and manuals described. As An example is the textbook by Winnacker: genes and clones, An introduction to genetic engineering (Verlag Chemie, Weinheim, Germany, 1990) or the Sambrook manual et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well known Genbank is that of E. coli K-12 strain W3110, which is from Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors was created. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank from C. glutamicum ATCC13032 using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in E. coli K-12 Strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Börmann et al. (Molecular Microbiology 6 (3), 317-326)) in turn describe one C. glutamicum ATCC13032 library using the Cosmids pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

For the production of a C. glutamicum gene bank in E. coli plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can be used. Are suitable as hosts  especially those E. coli strains that are restricted and recombination defect. An example of this is the Strain DH5αmcr, which was described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) has been described.

The long DNA fragments cloned with the help of cosmids can then turn into common for which Suitable sequencing vectors are subcloned.

Methods for DNA sequencing are among others Sanger et al. (Proceedings of the National of Sciences of the United States of America USA, 74: 5463-5467, 1977) described.

In this way, the new one coding for the rplK gene DNA sequence of C. glutamicum obtained as SEQ ID NO. 1 Is part of the present invention. Furthermore was from the present DNA sequence with those described above Methods the amino acid sequence of the corresponding protein derived. In SEQ ID NO. 2 is the resulting Amino acid sequence of the RplK gene product or the L-11 Protein represented.

Coding DNA sequences which result from SEQ ID NO. 1 through which result in degeneracy of the genetic code also part of the invention. Are in the same way DNA sequences that start with SEQ ID NO 1 or parts of SEQ ID NO. 1 hybridize, part of the invention.

Amino acid sequences which are made up in a corresponding manner SEQ ID NO. 2 result are also part of the Invention.

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

To achieve a weakening, either Expression of the rplK gene or, preferably, the functional properties of the protein decreased will. If necessary, both measures can be combined will.

The reduction in gene expression can be reduced by appropriate Culture management or genetic modification (mutation) the signal structures of gene expression. Signal structures of gene expression are, for example Repressor genes, activator genes, operators, promoters, Attenuators, ribosome binding sites, the start codon and Terminators. The expert will find information on this. B. in patent application WO 96/15246, to Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), at Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), by Patek et al. (Microbiology 142: 1297 (1996) and in well-known textbooks of genetics and Molecular biology such as B. Knippers' textbook ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that of Winnacker ("Gene and clones ", VCH publishing company, Weinheim, Germany, 1990).

Mutations that lead to a change or reduction in the functional properties of proteins are out known in the art. As examples are the Works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel ("Die Threonindehydratase aus Corynebacterium glutamicum: cancellation of allosteric Regulation and structure of the enzyme ", reports of the Research Center Jülichs, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Summary presentations can be known textbooks of genetics and Molecular biology such as B. that of Hagemann ("General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986) will.

Transitions, transversions, Insertions and deletions are considered. In dependence of the effect of amino acid exchange on activity is caused by missense mutations or Nonsense mutations spoken. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations (frame shift mutations) resulting in incorrect amino acids be installed or the translation is terminated prematurely. Instructions for creating such mutations are part of the State of the art and can be known textbooks of Genetics and molecular biology such as B. the textbook of Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ("Genes and Clones", VCH Publishing Company, Weinheim, Germany, 1990) or that of Hagemann ("Allgemeine Genetics ", Gustav Fischer Verlag, Stuttgart, 1986) will. Methods of making mutations with the help the polymerase chain reaction (PCR) are available from Newton C. R. Graham A. "PCR" 2nd edition, Spektrum Akademischer Verlag, Heidelberg, 1997.

An example of a plasmid with the aid of which deletion mutagenesis of the rplK gene can be carried out is the plasmid pΔrplK shown in FIG. 1.

Plasmid pΔrplK consists of the one described by Jäger et al. (Journal of Bacteriology, 1992, 174: 5462-65) pK18mobsacB, into which an allele of the rplK gene is shown in SEQ ID No. 3 was installed. That called ΔrplK Allele carries a 12 bp long deletion in the 5 'range of Gene. The variant of the encoded by the ΔrplK allele Proteins L11 is listed as SEQ ID No. 4 shown. The shown variant of the protein L 11 differs of the wild type form of the protein L 11 due to the absence of the Tetrapeptides "Proline-Alanine-Leucine-Glycine" accordingly the amino acids from positions 30 to 33 of SEQ ID No. 2nd  

After homologous recombination, the plasmid pΔrplK leads to Exchange of the chromosomal rplK gene for the ΔrplK Allele. Instructions and explanations for Insertion mutagenesis can be found, for example, in Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)) or Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)).

In addition, it can be used for the production of L-amino acids, L-lysine in particular, may be advantageous in addition to Attenuation of the rplK gene of one or more enzymes of the to strengthen respective biosynthetic pathways, in particular to overexpress.

For example, especially for the production of L-lysine, one or more of the genes selected from the group

• The dapA coding for the dihydrodipicolinate synthase Gene (EP-B 0 197 335),
• - one for a feed back resistant aspartate kinase encoding lysC gene (Kalinowski et al. (1990) Molecular and General Genetics 224: 317-324),
• - The pyc gene coding for the pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - the mqo coding for the malate quinone oxidoreductase Gen (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),
• - the lysE gene coding for lysine export (DE-A-195 48 222),
• The zwa1 gene (DE 199 59 328.0; DSM 13115),

amplified at the same time, especially overexpressed will.

Furthermore, it can be advantageous for the production of L-amino acids, in addition to the attenuation of the rplK gene, at the same time one or more of the genes selected from the group:

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

weaken.

It can also be used for the production of L-amino acids be beneficial in addition to overexpression of the 6- Phosphogluconate dehydrogenase undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The mutant polynucleotide according to claim 1 point d containing microorganisms are also the subject of Invention and can be continuous or discontinuous in a batch process (set cultivation) or in a fed batch (Feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing L-amino acids in particular L-lysine can be cultivated. A Summary of known cultivation methods are in the Textbook by Chmiel (Bioprocess Engineering 1. Introduction to Bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and  peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must be in a suitable manner The requirements of the respective tribes meet. Descriptions of culture media of various microorganisms are in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981) included. Sugar and Carbohydrates such as B. glucose, sucrose, lactose, Fructose, maltose, molasses, starch and cellulose, oils and Fats such as B. soybean oil, sunflower oil, peanut oil and Coconut fat, fatty acids such as B. palmitic acid, stearic acid and linoleic acid, alcohols such as e.g. B. glycerin and ethanol and organic acids such as B. acetic acid can be used. These substances can be used individually or as a mixture will. Organic nitrogen can be used as the nitrogen source containing compounds such as peptones, yeast extract, Meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate and ammonium nitrate can be used. The Nitrogen sources can be used individually or as a mixture be used. Phosphoric acid, Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts are used. The culture medium must also contain salts of metals such as B. magnesium sulfate or iron sulfate, for the Growth are necessary. After all, essential Growth substances such as amino acids and vitamins in addition to above substances are used. The culture medium Suitable precursors can also be added. The The feedstocks mentioned can be used for culture in the form of a one-off approach or appropriately to be fed during cultivation.

Basic compounds are used to control the pH of the culture such as sodium hydroxide, potassium hydroxide, ammonia or Ammonia water or acidic compounds such as phosphoric acid  or sulfuric acid used in a suitable manner. For Antifoam agents such as e.g. B. fatty acid polyglycol esters. For Maintaining the stability of plasmids can do that Medium suitable selectively acting substances such. B. Antibiotics are added. To aerobic conditions oxygen or oxygen are maintained containing gas mixtures such. B. Air into culture registered. The temperature of the culture is usually at 20 ° C to 45 ° C and preferably at 25 ° C to 40 ° C. The Culture continues until a maximum of desired product. That goal will usually within 10 hours to 160 hours reached.

Methods for determining L-amino acids are from the State of the art known. The analysis can be done like Spackman et al. (Analytical Chemistry, 30, (1958), 1190) described by anion exchange chromatography with subsequent ninhydrin derivatization, or they can be done by reversed phase HPLC, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174) described.

The following microorganism was deposited with the German Collection for Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:

• - Escherichia coli strain DH5α / pΔrplK as DSM 13158

Examples

The present invention will hereinafter be described with reference to Embodiments explained in more detail.

example 1 Manufacture from a genomic cosmid library Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was as described by Tauch et al. (1995, plasmid 33: 168-179), isolated and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164), obtained from Stratagene (La Jolla, USA, product description SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04). The cosmid DNA treated in this way was mixed with the treated ATCC13032 DNA and the mixture was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no. 27-0870-04) . The ligation mixture was 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). To infect the E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16: 1563-1575), the cells were taken up in 10 mM MgSO ₄ and mixed with an aliquot of the phage suspension. Infection and titering of the cosmid bank were carried out as in Sambrook et al. (1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor), wherein the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) with 100 mg / l ampicillin. After overnight incubation at 37 ° C., recombinant individual clones were selected.

Example 2 Isolation and sequencing of the rplK gene

The cosmid DNA of a single colony was obtained with 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 were made with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, Product No. 1758250) dephosphorylated. To Isolation was carried out by gel electrophoresis the cosmid fragments in the size range from 1500 to 2000 bp with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1 purchased from Invitrogen (Groningen, Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product description BamHI, Product No. 27-0868-04) split. The ligation of the cosmid fragments in the Sequencing vector pZero-1 was as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) performed, the DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) via Was incubated overnight. This ligation mixture was subsequently into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A.,  87: 4645-4649) electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-7) and on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l / Zeocin. The plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Sequencing was done after the dideoxy Chain termination method by Sanger et al. (1977, Proceedings of the National Academies of Sciences U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). It became the "RR dRhodamin Terminator Cycle Sequencing Kit "from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany). The gel electrophoretic separation and analysis of the Sequencing reaction was carried out in a "rotiphoresis NF Acrylamide / Bisacrylamide "Gel (29: 1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377" Sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then under Use of the Staden program package (1986, Nucleic Acids Research, 14: 217-231) version 97-0. The Individual sequences of the pZero1 derivatives became one connected contig assembled. The computerized Coding area analysis was carried out with the XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were carried out with the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402), against the non-redundant database of the "National Center for Biotechnology Information "(NCBI, Bethesda, MD, USA).

The nucleotide sequence obtained is in SEQ ID NO. 1 shown. Analysis of the nucleotide sequence revealed a open reading frame of 438 base pairs, which as rplK- Gen was called. The rplK gene codes for a 145 amino acid polypeptide shown in SEQ ID No. 2nd

Example 3 Generation of a vector with a copy of the rplK gene

A chromosomal 1200 bp DNA fragment that contains the rplK C. glutamicum gene was determined by means of PCR cloned.

For this, chromosomal DNA from Corynebacterium was used glutamicum ATCC 13032 as described by Tauch et al. (1995, plasmid 33: 168-179). With the help of Polymerase chain reaction was a DNA fragment with a Size of 1200 bp, which contains the rplK gene, amplified. For this purpose, based on SEQ ID No. 1 derived the following primers.

P1-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 AGC TAC GTC GAT GG-3 '

The oligonucleotides shown were from ARK Scientific (ARK Scientific GmbH Biosystems, Darmstadt, Germany) and synthesized the PCR reaction Use of the Pfu DNA polymerase (Stratagene, La Jolla, USA) and the PTC 100 thermal cycler (MJ Research Inc., Waltham, USA).

During the PCR, a cycle consisting of thermal Denaturation (94 ° C, 90 seconds), Annealing (58 ° C, 90 seconds) and polymerase reaction (72 ° C, 90 seconds) 35 go through times. The 1200 bp DNA fragment thus obtained was then carried out using Qiagen PCR Purification Spin kits (Qiagen, Hilden, Germany) cleaned up.

For cloning the rplK gene-containing DNA Amplificate on a plasmid which is in C. glutamicum can replicate, the plasmid pECM3, a Deletion derivative of that described by Tauch et al. (FEMS Microbiological Letters, 123, 343-347 (1994)) described plasmid pECM2, produced. For this, the plasmid pECM2 with the Restriction enzymes BamHI (Amersham-Pharmacia, Freiburg, Germany) and BglII (Amersham-Pharmacia, Freiburg, Germany) restricted and with T4 ligase (Amersham Pharmacia, Freiburg, Germany), as in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Habor Laboratory (1989)), which caused the plasmid pECM3 was obtained. The transformation of the Grant et al. (Proceedings of the National Academy of Science USA, 87, 4645-4649 (1990)) described E. coli strain DH5αMCR the plasmid pECM3 was carried out as in Tauch et al. (FEMS Microbiological Letters, 123, 343-347 (1994)). The transformants were placed on LBG agar (10 g trypton, 5 g Yeast extract, 5 g NaCl, 2 g glucose, 15 g agar per liter), chloramphenicol (Merck, Darmstadt, Germany) (50 mg / l) was added, selected.

The 1200 bp DNA containing rplK gene was then Amplificate with the plasmid pECM3, which was previously with the Restriction enzyme SmaI (Amersham-Pharmacia, Freiburg, Germany) was linearized, mixed and with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) treated, whereby the plasmid prplK was obtained. The transformation of the E. coli strain DH5αMCR with the plasmid prplK was carried out as in Tauch et al. (FEMS Microbiological Letters, 123, 343-347 (1994)) and the transformants were analyzed on LBG Agar, the chloramphenicol (Merck, Darmstadt, Germany) (50 mg / l) was added, selected. The plasmid prplK thus carries the complete rplK gene of C. glutamicum and can be found in both E. coli and C. Replicate glutamicum autonomously.  

Example 4 Incorporation of a deletion into the rplK gene

An allele called ΔrplK was identified by means of PCR technology of the rplK gene, which is a 12 bp long deletion wearing. The 12 bp deletion generated in the rplK gene leads to Loss of the tetrapeptide "proline-alanine-leucine-glycine" in the N-terminal region of the L11 protein from C. glutamicum.

As primers for the generation of the rplK deletion all in addition to the primers P1-up and P2- described in Example 3 down the oligonucleotides described below used by the company ARK Scientific (ARK Scientific GmbH Biosystems, Darmstadt, Germany).

P1-down (see also SEQ ID No. 7):
5'-extension-CGC CGT GAG C-5'-side-GCC AAC TGG AGG AGC AGG GT-3 '

P2-up: (See also SEQ ID No. 8):
5'-Extension-TCC AGT TGG C-5'-side-GCT CAC GGC GTC AAC ATC AG-3 '

The primers used for the PCR were from the known DNA sequence derived. These each have a 10 bp 5 'extension, which is exactly complementary to the 5' sides of the Primer P1-up and P2-down is. From C. glutamicum ATCC 13032 the chromosomal DNA was extracted and with it as Matrix using the specified oligonucleotides P1- up, P1-down, P2-up and P2-down, the Pfu DNA polymerase (Stratagene, La Jolla, USA), and the PTC 100 thermal cycler (MJ Research Inc., Waltham, USA) initially in separate PCR reactions generated two 600 bp DNA fragments. At A cycle was established for each of these PCR reactions from thermal denaturation (94 ° C, 90 seconds), Annealing (58 ° C, 90 seconds) and polymerase reaction (72 ° C, 90 seconds) run 35 times.

The first 600 bp DNA amplificate, as rplK part 1 with the oligonucleotides P1-up and P1- get down. It contains the 5 'region of the rplK gene (Nucleotide 1-87) and additionally that of oligonucleotide P1- down-coming 10 bp extension, which the nucleotides 100-109 of the rplK gene (SEQ ID No. 1). Thus, this points amplified rplK gene region compared a 12 bp gap on the chromosomal DNA template used. The second 600 bp DNA fragment, rplK part 2, was with the Obtain and include oligonucleotides P2-down and P2-up the 3 'region of the rplK gene (nucleotide 78-435) and carries the identical gap (nucleotide 88-99) within the amplified rplK gene region. These two 600 bp DNA Accordingly, amplificates have a 20 bp overlap DNA area. The two 600 bp PCR products rplK part 1 and rplK part 2 were now in another PCR reaction used together as a DNA template, whereby due to of the overlapping, complementary DNA region of the "Rear strand" of rplK part 1 with the "back strand" of rplK- Part 2 could connect. The extension of this overlapping DNA area or the addition of oligonucleotides P1-up and P2-down, resulted in the generation of a 1200 bp PCR Amplificate, which is a 12 bp deletion derivative of C. glutamicum rplK gene contains. In these PCR reactions was a cycle consisting of thermal Denaturation (94 ° C, 90 seconds), Annealing (58 ° C, 90 Seconds) and polymerase reaction (72 ° C, 90 seconds) 35 times run through.

The nucleotide sequence of the ΔrplK allele is shown in SEQ ID No. 3rd and the variant of the L11 protein encoded by this allele in SEQ ID No. 4 shown. This L11 protein variant the tetrapeptide "proline-alanine-leucine-glycine" is missing corresponding to amino acid positions 30 to 33 of Wild type form of the L11 protein shown in SEQ ID No. 2nd  

Example 5 Incorporation of the ΔrplK allele into the chromosome

The ΔrplK allele, which has a 12 bp deletion in the rplK gene was included using integration mutagenesis With the aid of Schäfer et al., Gene, 14, 69-73 (1994) described sacB system in the chromosome of C. glutamicum incorporated. This system allows the specialist the identification or selection of allele exchanges, that occur through homologous recombination.

1. Construction of the exchange vector pΔrplK

The 1200 bp rplK deletion allele obtained in Example 4 ΔrplK was determined using the Qiagen PCR Purification Spin Kit (Qiagen, Hilden, Germany) cleaned and with the Schaefer et al., Gene, 14, 69-73 (1994) mobilizable cloning vector pK18mobsacB for ligation used. This was previously with the restriction enzyme SmaI (Amersham-Pharmacia, Freiburg, Germany) linearized, mixed with the rplK deletion allele and and with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) treated.

The plasmid pΔrplK resulted.

The transformation of the E. coli strain DH5α with the plasmid pΔrplK was carried out as in Tauch et al., FEMS Microbiological Letters, 123, 343-347 (1994). The Transformants were on LBG agar, the kanamycin (Merck, Darmstadt, Germany) (50 mg / l) had been added, selected. In this way the trunk was created DH5α / pΔrplK.

One clone was selected and named ATCC13032ΔrplK designated.

2. Implementation of the allele exchange

A 12 bp chromosomal deletion in the rplK gene of C. glutamicum was found using integration mutagenesis With the help of Schäfer et al., Gene, 14, 69-73 (1994) described the sacB system. This System allows the specialist to identify or Selection of allele exchanges that are characterized by homologous Do recombination.

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

On the subsequent excision of the plasmid pΔrplK with Aid of the sacB system was only possible in the presence of a Wild type alleles can be selected by rplK. That was what in Example 3, plasmid prplK constituted the complete rplK gene carries, by electroporation according to the method of Liebl et al., FEMS Microbiology Letters 65, 299-304 (1989) transferred to the integrant tribe. The selection of the Strain was carried out on LBG agar, the kanamycin (25 mg / l) (Merck, Darmstadt, Germany) and chloramphenicol (10 mg / l) (Merck, Darmstadt, Germany) had been added.

A selected, transformed colony was inoculated into 100 ml LBG liquid medium (in a 250 ml Erlenmeyer flask with baffles) and incubated for 24 hours at 30 ° C. and 300 rpm. Then 2 × 10 ⁶ cells / ml of this liquid culture were applied to LBG agar containing 10% sucrose (Merck, Darmstadt, Germany) and incubated at 30 ° C. for 48 hours. C. glutamicum cells, which were able to grow on this medium, had lost the integrated plasmid pΔrplK due to a second recombination event between the rplK deletion allele and the natural rplK region. This second recombination event either leads to the restoration of the natural, chromosomal rplK gene arrangement, or it results in the generation of a C. glutamicum ΔrplK mutant that has lost the 12 bp N-terminal DNA fragment. The chromosomal DNA was extracted from selected, "sucrose resistant" and potential pΔrplK-bearing C. glutamicum cells. This served as a matrix with which the olidonucleotides Pdel-up and Pdel-down2 (ARK Scientific GmbH Biosystems, Darmstadt, Germany) were derived using the rplK sequence. The PCR experiments were carried out with the primers, the Pfu DNA polymerase (Stratagene, La Jolla, USA) and the PTC 100 thermal cycler (MJ Research Inc., Waltham, USA). In the PCR, a cycle consisting of thermal denaturation (94 ° C., 90 seconds), annealing (58 ° C., 90 seconds) and polymerase reaction (72 ° C., 90 seconds) was carried out 35 times. The DNA amplificates obtained in this way were then purified using the Qiagen PCR Purification Spin Kit (Qiagen, Hilden, Germany). Analysis of the nucleotide sequences of the purified DNA amplificates, which was carried out as described above, showed that in 43% of the cases a DNA amplificate was generated which lacked the 12 bp DNA region. As a result, the second recombination event in the associated pΔrplK-carrying transconjugants led to the generation of the chromosomal 12 bp deletion in the rplK gene.

Then a selected, deletion-carrying transconjugant through the plasmid prplK the Schäfer et al., Journal of Bacteriology, 176, 7309-7319 (1994) described method of "plasmid curing" away. The C. glutamicum ATCC13032 strain thus obtained ΔrplK thus carries a chromosomal 12 bp deletion within the rplK gene, resulting in the loss of the "Proline-alanine-leucine-glycine" tetrapeptide of the L11 protein leads.

Example 6 Production of lysine

The strain C. glutamicum ATCC13032 obtained in Example 5 ΔrplK was used in a suitable for the production of lysine Culture medium cultivated and the lysine content in Culture supernatant determined.

For this purpose, the strain was first placed on an agar plate (brain heart Agar) incubated for 24 hours at 33 ° C. Starting from a preculture was inoculated into this agar plate culture (10 ml Medium in 100 ml Erlenmeyer flask). As a medium for The complete medium CgIII (10 g / l Bacto-peptone, 10 g / l yeast extract, 2.5 g / l NaCl, 20 g / l glucose, pH 7.4) used. The preculture was carried out for 24 hours at 33 ° C. at 240 rpm incubated on the shaker. From this preculture inoculated a main culture so that the initial OD (660 nm) the main culture was 0.1 OD. For the main culture the medium mm used.  

Medium MM

CSL (Corn Steep Liquor) 5.0 g / l MOPS (morpholinopropanesulfonic acid) 20.0 g / l Glucose (autoclaved separately) 50.0 g / l (NH ₄ ) ₂ SO ₄ ) 25.0 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7 H ₂ O 1.0 g / l CaCl ₂ .2 H ₂ O 10.0 mg / l FeSO ₄ .7 H ₂ O 10.0 mg / l MnSO ₄ .H ₂ O 5.0 mg / l Biotin (sterile filtered) 0.3 mg / l Thiamin.HCl (sterile filtered) 0.2 mg / l CaCO ₃ 25.0 g / l

CSL, MOPS and the salt solution are adjusted to pH 7 with ammonia water and autoclaved. The sterile substrate and vitamin solutions are then added, and the dry autoclaved CaCO _{3 is} added.

The cultivation takes place in a volume of 10 ml in a 100 ml Erlenmeyer flasks with baffles. The cultivation took place at 33 ° C and 80% humidity.

After 48 hours the OD was measured at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Munich) determined. The amount of lysine formed was included an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection certainly.

Table 1 shows the result of the experiment.

Table 1

The following figures are attached:

Fig. 1: Map of the plasmid pΔrplK.

The abbreviations and designations used have following meaning.

pdeltarplK: pΔrplK
sacB gene: SacB gene from Bacillus suptilis, encoded for the enzyme levansucrase
lacZ-alpha ': part of the 5' end of the β-galactosidase gene
oriV: origin of replication
KmR: Kanamycin resistance
RP4 mob: mob region of the plasmid RP4
BamHI: interface of the restriction enzyme BamHI
EcoRI: Interface of the restriction enzyme EcoRI
ΔrplK rplK allele with a deletion of 12 bp in the N-terminal region

SEQUENCE LOG

Claims (21)

1. Isolated polynucleotide from coryneform bacteria, containing a polynucleotide sequence selected from the group

• a) Polynucleotide that is at least 70% identical to a polynucleotide that codes for a polypeptide that contains the amino acid sequence of SEQ ID No. 2 contains
• b) 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. 2,
• c) polynucleotide which is complementary to the polynucleotides of a) or b), and
• d) polynucleotide containing at least 15 successive bases of the polynucleotide sequence of a), b) or c).

2. polynucleotide according to claim 1, wherein the polynucleotide is a replicable, preferred is recombinant DNA. 3. The polynucleotide of claim 1, wherein the polynucleotide is an RNA. 4. polynucleotide according to claim 2, containing the nucleic acid sequence as in SEQ ID No. 1 shown. 5. The polynucleotide sequence according to claim 2, which encodes a polypeptide that contains the amino acid sequence as in SEQ ID No. 2 contains. 6. polynucleotide according to claim 1, in particular point d, containing the nucleotide sequence as in SEQ ID No. 3rd shown.   7. polynucleotide according to claim 1, in particular point d, which codes for a polypeptide that the in SEQ ID No. 4 contains amino acid sequence shown. 8. Replicable DNA according to claim 2, containing

• a) the nucleotide sequence shown in SEQ ID No. I, or
• b) at least one sequence which speaks to sequence (i) within the range of the degeneration of the genetic code, or
• c) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
• d) Functional mutants of meaning in (i)

9. Vector containing a polynucleotide according to claim 1, deposited in E. coli, DH5α / pΔrpk as DSM 13158, shown in SEQ ID No. 3 and Fig. 1. 10. Coryneform bacteria serving as host cells, the a deletion or an insertion in the rplk gene contain. 11. A process for the preparation of L-amino acids, in particular L-lysine, characterized in that the following steps are carried out,

• a) fermentation of the bacteria producing the desired L-amino acid, in which at least the rplk gene is weakened,
• b) accumulation of the desired L-amino acid in the medium or in the cells of the bacteria, and
• c) isolating the L-amino acid.

12. The method according to claim 11, characterized, that you use bacteria in which you additionally further genes of the desired biosynthetic pathway L-amino acid enhanced. 13. The method according to claim 11, characterized, that you use bacteria in which the Metabolic pathways at least partially switched off which are the formation of the desired L-amino acid reduce. 14. The method according to claim 11, characterized, that one according to the expression of the polynucleotide Claim 1, in particular 1a to 1d reduced. 15. The method according to claim 11, characterized, that the catalytic properties of the Polypeptide (enzyme protein) for which the Polynucleotide according to claim 1, in particular 1a to 1d coded. 16. The method according to claim 11, characterized in that bacteria are used in which an insertion mutagenesis using the plasmid pΔrpk, shown in Fig. 1 and deposited as DSM 13158 is generated to attenuate. 17. The method according to claim 11, characterized in that for the production of L-amino acids, in particular L-lysine, bacteria are fermented, in which one overexpresses one or more of the genes selected from the group

• 1. the dapA gene coding for the dihydrodipicolinate synthase,
• 2. an aspartate kinase resistant to a feed back,
• 3. the DNA fragment which mediates resistance to S- (2-aminoethyl) cysteine,
• 4. the pyc gene encoding the pyruvate carboxylase,
• 5. pyc gene (Eikmanns (1992) Journal of Bacteriology)
• 6. the mqo gene coding for the malate: quinone oxidoreductase
• 7. the lysE gene coding for lysine export,
• 8. the zwa1 gene.

18. The method according to claim 11, characterized in that for the production of amino acids, in particular L-lysine, bacteria are fermented, in which one or more of the genes selected simultaneously from the group

• 1. the pck gene coding for the phosphoenolpyruvate carboxykinase,
• 2. the pgi gene coding for the glucose-6-phosphate isomerase,
• 3. the poxB gene coding for the pyruvate oxidase,
• 4. the zwa2 gene or
• 5. the rela gene coding for PPGPP synthetase I.

weakened. 19. The method according to one or more of the preceding Expectations, characterized, that microorganisms of the genus Corynebacterium glutamicum. 20. Use of polynucleotide sequences according to claim 1 as a primer for the production of the DNA of genes that a develop an effect corresponding to the rplK gene by the polymerase chain reaction. 21. Use of polynucleotide sequences according to claim 1 as hybridization probes.