DE10136987A1 - Nucleotide sequences encoding the clpC gene - Google Patents

Abstract

The invention relates to an isolated polynucleotide comprising a polynucleotide sequence chosen from the group consisting of a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% 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 comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the clpC gene is present in attenuated form, and the use of polynucleotides which comprise the sequences according to the invention as hybridization probes.

Description

The invention relates to coding for the clpC gene Nucleotide sequences from coryneform bacteria and a Process for the fermentative production of amino acids using bacteria in which the clpC gene is weakened.

State of the art

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.

It is known that amino acids by fermentation of Strains coryneformer bacteria, in particular Corynebacterium glutamicum. Because of the Size is constantly improving Manufacturing process worked. process improvements can fermentation measures such as Stirring and supply of oxygen, or the Composition of nutrient media, such as the Sugar concentration during fermentation, or the Work-up to the product form by, for example Ion exchange chromatography or the intrinsic Performance characteristics of the microorganism itself affect.

To improve the performance characteristics of this Microorganisms become methods of mutagenesis, selection and mutant selection applied. In this way you get Strains that are resistant to antimetabolites or auxotrophs for regulatory metabolites and the Produce amino acids.

For some years now also methods of recombinant DNA technology for strain improvement of L-  Amino acid producing strains of Corynebacterium Used by single amino acid biosynthetic genes amplified and the effect on the amino acid Production examined.

Object of the invention

The inventors have set themselves the task of new Measures for the improved fermentative production of To provide amino acids.

Description of the invention

Are mentioned in the following L-amino acids or amino acids, are thus including one or more amino acids their salts selected from the group L-asparagine, L- Threonine, L-serine, L-glutamate, L-glycine, L-alanine, L- Cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L- Tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine. Particularly preferred is lysine.

If in the following L-lysine or lysine are mentioned, are so that not only the bases, but also the salts such. B. Lysine monohydrochloride or lysine sulfate.

The invention relates to an isolated polynucleotide from coryneform bacteria containing a coding for the clpC gene polynucleotide sequence selected from the group

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

wherein the polypeptide preferably the activity of the clpC- Protease has.

The invention likewise provides the abovementioned polynucleotide, which is preferably a replicable DNA comprising:

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) within the range of degeneration of the corresponds to genetic codes, or
• c) at least one sequence identical to the Sequences (i) or (ii) complementary sequences hybridized, and optionally
• d) functionally neutral sense mutations in (i).

Other items are:
a replicable polynucleotide, in particular DNA, containing the nucleotide sequence as shown in SEQ ID no. 1 shown;
a polynucleotide encoding a polypeptide having the amino acid sequence as set forth in SEQ. ID. 2, contains;
a vector containing parts of the polynucleotide according to the invention, but at least 15 consecutive nucleotides of the claimed sequence,
and coryneform bacteria in which the clpC gene is attenuated, in particular by insertion or deletion.

The invention also relates to polynucleotides which are in consist essentially of a polynucleotide sequence, the are available by screening by hybridization a corresponding gene bank of a coryneform bacterium, which contains the complete gene or parts thereof, with a probe containing the sequence of the invention Polynucleotide according to SEQ ID no. 1 or a fragment thereof contains and isolation of said polynucleotide sequence.

Polynucleotides containing the sequences according to the invention are used as hybridization probes for RNA, cDNA and DNA suitable for nucleic acids or Isolate polynucleotides or full length genes encode the clpC protease, or such Nucleic acids or polynucleotides or genes to isolate, which is very similar to the sequence of the clpC gene. You can also use as a probe so-called "arrays", "micro arrays" or "DNA chips" be applied to the appropriate polynucleotides or derived therefrom sequences such. B. RNA or cDNA to detect and determine.

Polynucleotides containing the sequences according to the invention are still suitable as primers, with their Help with the polymerase chain reaction (PCR) DNA of genes can be prepared for the clpC protease encode.

Such as probes or primers serving oligonucleotides contain at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, most preferably at least 15, 16, 17, 18 or 19 consecutive Nucleotides. Also suitable are oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38,  39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. If necessary, too Oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides suitable.

"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.

The polynucleotides according to the invention include Polynucleotide according to SEQ ID no. 1 or one of them produced fragment and also those that belong to at least 70% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% and completely more preferably at least 91%, 93%, 95%, 97% or 99% identical to the polynucleotide according to SEQ ID no. 1 or a fragment made therefrom.

By "polypeptides" is meant peptides or proteins, the two or more linked via peptide bonds Contain amino acids.

The polypeptides of the invention include a polypeptide according to SEQ ID no. 2, in particular those with the biological activity of the clpC protease and also such at least 70% to 80%, preferably at least 81% to 85%, more preferably at least 86% to 90% and most preferably at least 91%, 93%, 95%, 97% or 99% are identical to the polypeptide according to SEQ ID No. 2 and have the said activity.

The invention further relates to a method for fermentative production of amino acids selected from L-asparagine, L-threonine, L-serine, L-glutamate, L- Glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-  Isoleucine, L-Leucine, L-Tyrosine, L-Phenylalanine, L-Histidine, L-lysine, L-tryptophan and L-arginine, using coryneform bacteria, in particular already Produce amino acids and those responsible for the clpC gene attenuated nucleotide sequences encoding, in particular switched off or expressed at a low level.

The term "weakening" describes in this Related to the reduction or elimination of the intracellular activity of one or more enzymes (Proteins) in a microorganism produced by the corresponding DNA are encoded by, for example used a weak promoter or a gene or allele used for a corresponding enzyme with a low activity encodes or the corresponding gene or Inactivated enzyme (protein) and optionally this Measures combined.

By the measures of weakening becomes the activity or concentration of the corresponding protein in the general to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type Protein, or activity or concentration of the protein in the initial microorganism, lowered.

The microorganisms that are the subject of the present Invention, amino acids from glucose, sucrose, Lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be around Representative coryneformer bacteria especially the genus Corynebacterium act. In the genus Corynebacterium is in particular the species Corynebacterium glutamicum too which is known in the art for their ability To produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum (C. glutamicum), are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium molassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-amino acid producing mutants or strains thereof.

The new clpC gene coding for the enzyme clpC protease of C. glutamicum was isolated.

For the isolation of the clpC gene or other genes of C. glutamicum first becomes a gene bank of this Microorganism in Escherichia coli (E. coli) created. The Creating gene libraries is well known Textbooks and manuals written down. As an an example be the textbook of Winnacker: Genes and Clones, One Introduction to genetic engineering (Verlag Chemie, Weinheim, Germany, 1990), or the Handbook of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is that of E. coli K-12 strain W3110, available from Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of 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 the 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 (1992)) again describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (scab and Collins, 1980, Gene 11, 291-298).  

For the preparation of a gene bank of C. glutamicum in E. coli For example, plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268). As hosts are suitable especially those E. coli strains that are restriction and recombination defects are such as the strain DHSamcr, by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) has been. The with the help of cosmids or other X-vectors cloned long DNA fragments can be subsequently again in common for DNA sequencing suitable Vectors are subcloned and subsequently sequenced, as it is z. In Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).

The resulting DNA sequences can then be labeled with known Algorithms or sequence analysis programs such. B. the of Staden (Nucleic Acids Research 14, 217-232 (1986)), of Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or Butler's GCG program (Methods of Biochemical Analysis 39, 74-97 (1998)).

The new coding for the clpC gene DNA sequence of C. glutamicum was found as SEQ. ID. 1 component of the present invention. Furthermore, 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 clpC gene product shown.

Coding DNA sequences consisting of SEQ ID no. 1 through the degeneracy of the genetic code are also part of the invention. In the same way DNA sequences identified by SEQ ID NO. 1 or parts of SEQ ID No. 1 hybridize, part of the invention. In the Experts are still conservative amino acid exchanges such as B. Exchange of glycine for alanine or of  Aspartic acid against glutamic acid in proteins as "Sense mutations" known to none fundamental change in the activity of the protein lead, d. H. are functionally neutral. It is also known that changes at the N- and / or C-terminus of a protein its function does not significantly affect or even can stabilize. Information on this is the expert among others Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), O'Regan et al. (Gene 77: 237-251 (1989)); Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)); Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in known textbooks of Genetics and molecular biology. Amino acid sequences that correspondingly from SEQ ID no. 2, are also part of the invention.

Similarly, DNA sequences SEQ ID NO. 1 or parts of SEQ ID no. 1 hybridize part of the Invention. Finally, DNA sequences are part of Invention produced by the polymerase chain reaction (PCR) be made using primers that are from SEQ ID no. 1 result. Have such oligonucleotides typically at least 15 nucleotides in length.

Instructions for the identification of DNA sequences by means of Hybridization is found among others in the art Manual "The DIG System Users Guide for Filters Hybridization "the company Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). The hybridization takes place under stringent Conditions, that is, only hybrids formed in which probe and target sequence, d. H. with probe treated polynucleotides, at least 70% are identical. It is known that the stringency of Hybridization including washing steps by Varying the buffer composition, the temperature and the  Salt concentration is influenced or determined. The Hybridization reaction is preferably carried out at relative low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).

For the hybridization reaction, for example, a 5 × SSC buffer at a temperature of about 50 ° C-68 ° C be used. It can also probe with Hybridize to polynucleotides that are less than 70% Identity to the sequence of the probe. Such hybrids are less stable and are underwent by washing stringent conditions removed. This can be, for example by lowering the salt concentration to 2X SSC and optionally subsequently 0.5 × SSC (The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) can be achieved, with a Temperature of about 50 ° C-68 ° C is set. It is possibly possible the salt concentration up to 0.1 × Lower SSC. By gradually increasing the Hybridization temperature in steps of about 1-2 ° C of 50 ° C to 68 ° C isolated polynucleotide fragments for example, at least 70% or at least 80% or at least 90% to 95% identity to the sequence of own used probe. Further instructions for the Hybridization are in the form of so-called kits on the market available (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

Instructions for the amplification of DNA sequences using the polymerase chain reaction (PCR) will be apparent to those skilled in the art inter alia in the handbook of Gait: Oligonucleotides Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Academic Spectrum Verlag, Heidelberg, Germany, 1994).  

It was found that coryneform bacteria after Attenuation of the clpC gene in an improved manner Produce amino acids.

To achieve a weakening either the Expression of the clpC gene or the catalytic Properties of the enzyme protein are minimized or turned off. If necessary, both measures be combined.

The reduction of gene expression can be achieved by appropriate Cultural tour or genetic modification (mutation) the signal structures of gene expression take place. Signaling structures of gene expression are, for example Repressor genes, activator genes, operators, promoters, Attenuators, ribosome binding sites, the start codon and Terminators. Information on this is the expert z. In of 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)), in Pátek et al. (Microbiology 142: 1297 (1996)), Vasicova et al. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks of genetics and Molecular biology such. B. the textbook by Knippers ("Molecular Genetics", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or that of Winnacker ("Gene and clones ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations that lead to a change or reduction of the catalytic properties of enzyme proteins are known from the prior 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 ("Threonine dehydratase Corynebacterium glutamicum: abolition of allosteric  Regulation and structure of the enzyme ", reports of the Forschungszentrum Jülichs, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Summary presentations can be known textbooks of genetics and Molecular biology such. B. von Hagemann ("General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986) become.

Mutations are transitions, transversions, Insertions and deletions into consideration. In dependence of the effect of amino acid exchange on the Enzyme activity is mediated by missense mutations ("missense mutations ") or non-nonsense mutations spoken. Insertions or deletions of at least a base pair (bp) in a gene lead to Frame shift mutations, in the consequence of which incorrect amino acids are incorporated or the Translation aborts prematurely. Deletions of several Codons typically result in a complete one Failure of enzyme activity. Instructions for generation Such mutations are prior art and can be known textbooks of genetics and Molecular biology such. B. the textbook by Knippers ("Molecular Genetics", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ("Gene and Klone ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that of Hagemann ("General Genetics", Gustav Fischer Verlag, Stuttgart, 1986).

A common method, genes of C. glutamicum too mutate is that of Schwarzer and Pühler (Bio / Technology 9, 84-97 (1991)) method of gene disruption ("gene disruption") and gene exchange ("gene replacement ").

In the method of gene disruption becomes a central Part of the coding region of the gene of interest in a Plasmid vector cloned in a host (typically  E. coli) but not in C. glutamicum. When For example, vectors come from pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pKlBmob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269: 32678-84; U.S. Patent 5,487,993), pCR® Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the central part of the coding region of the gene contains subsequently by conjugation or transformation into the desired strain of C. glutamicum transferred. The method the conjugation is described by Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)) described. Methods for transformation are for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a cross-over Event becomes the coding region of the gene in question interrupted by the vector sequence and you get two incomplete alleles to which the 3 'and 5' End is missing. For example, this method was used by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.

In the method of gene replacement (gene replacement) is a mutation such. B. a deletion, insertion or Base exchange in the gene of interest in vitro manufactured. The produced allele turns into a cloned for C. glutamicum non-replicative vector and this subsequently by transformation or conjugation  into the desired host of C. glutamicum. To homologous recombination by means of a first, integration effecting a cross-over event and an appropriate second excision-causing cross-over event in the target gene or in the target sequence to reach the installation the mutation or the allele. This method was for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) to the pyc gene of C. glutamicum by a deletion.

In the clpC gene, a deletion, Insertion or a base exchange can be installed.

Additionally, it may be responsible for the production of L-amino acids be advantageous, in addition to the attenuation of the clpC gene one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, of the pentose phosphate cycle, amino acid export and optionally to amplify regulatory proteins, especially overexpress.

The term "reinforcement" describes in this context the increase in intracellular activity of one or several enzymes (proteins) in a microorganism that be encoded by the corresponding DNA by For example, the copy number of the gene or genes increases, uses a strong promoter or a gene or Allele used for a corresponding enzyme (protein) encoded with a high activity and optionally this Measures combined.

By the measures of reinforcement, in particular Overexpression, is the activity or concentration of the corresponding protein generally by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000% of the wild-type Protein or activity or concentration of the protein in the initial microorganism increases.  

Thus, in addition to the attenuation of the clpC gene, one or more of the genes selected from the group may be simultaneously produced for the production of L-amino acids

• The gene coding for the dihydrodipicolinate synthase dapA (EP-B 0 197 335),
• - that for the glyceraldehyde-3-phosphate dehydrogenase coding gene gap (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene tpi coding for the triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - The gene coding for the 3-phosphoglycerate kinase gene pgk (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - The coding for the glucose-6-phosphate dehydrogenase Gen zwf (JP-A-09224661),
• - The gene coding for the pyruvate carboxylase gene pyc (DE-A-198 31 609),
• The gene coding for malate quinone oxidoreductase mqo (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),
• - that for a feed-back-resistant aspartate kinase coding gene lysC (Accession No. P26512; EP-B-0387527; EP-A-0699759),
• - the gene encoding lysine export, lysE (DE-A-195 48 222),
• Homoserine dehydrogenase-encoding gene hom (EP-A 0131171),
• - The gene encoding ilvA for the threonine dehydratase (Möckel et al., Journal of Bacteriology (1992) 8065- 8072)) or that for a "feed-back-resistant" threonine  Dehydratase-encoding allele ilvA (Fbr) (Möckel et al., (1994) Molecular Microbiology 13: 833-842),
• - The gene coding for the acetohydroxy acid synthase gene ilvBN (EP-B 0356739),
• - The gene encoding the Dihydroxysäuredehydratase ilvD (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979),
• The gene coding for the Zwa1 protein zwa1 (DE 199 59 328.0, DSM 13115)

amplified, in particular overexpressed.

Furthermore, it may be advantageous for the production of amino acids, in addition to the attenuation of the clpC gene simultaneously one or more of the genes selected from the group

• - The coding for the phosphoenolpyruvate carboxykinase Gene pck (DE 199 50 409.1, DSM 13047),
• - The gene coding for the glucose-6-phosphate isomerase gene pgi (US 09 / 396,478, DSM 12969),
• - _d as poxB gene which codes for pyruvate oxidase (DE 199 51 975.7, DSM 13114),
• - the zwa2 protein encoding gene zwa2 (DE 199 59 327.2, DSM 13113)

attenuate, in particular to reduce expression.

Furthermore it can be used for the production of amino acids be beneficial, in addition to the attenuation of the clpC gene to eliminate unwanted side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in:  

Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention are also the subject of the invention and can continuous or discontinuous in the batch process (Batch cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) cultured for the purpose of producing L-amino acids become. A summary of known Cultivation methods is in the textbook of Chmiel (Bioprocessing Technology 1. Introduction to the Bioprocess Engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (Bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must suitably Claims of the respective strains suffice. descriptions of culture media of various microorganisms are in Manual of Methods for General Bacteriology American Society for Bacteriology (Washington D.C., USA, 1981).

As a carbon source, sugars and carbohydrates can z. Glucose, sucrose, lactose, fructose, maltose, Molasses, starch and cellulose, oils and fats, such as Example soybean oil, sunflower oil, peanut oil and coconut oil, Fatty acids, such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerine and Ethanol and organic acids, such as acetic acid be used. These substances can be used individually or as Mixture be used.

As nitrogen source, organic nitrogen-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 singly or as a mixture.

As phosphorus source can phosphoric acid, Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used. The culture medium must continue to salts of metals contain, such as magnesium sulfate or Iron sulfate necessary for growth. Finally, essential growth factors such as amino acids and vitamins in addition to the above substances be used. The culture medium can also suitable precursors are added. The mentioned Feedstocks can be used to culture in the form of a one-off Approach added or appropriately during the Cultivation be fed.

For pH control of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as Phosphoric acid or sulfuric acid in a suitable manner used. To control the foaming can Anti-foaming agents, such as fatty acid polyglycol esters be used. To maintain the stability of Plasmids may be selectively selective to the medium Substances such as antibiotics are added. Around maintain aerobic conditions, become oxygen or oxygen-containing gas mixtures, such as Air entered into the culture. The temperature of the culture is usually at 20 ° C to 45 ° C and preferably at 25 ° C to 40 ° C. The culture is continued until a maximum of the desired product has formed. This goal will normally take up to 10 hours Reached 160 hours.

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

The inventive method is used for fermentative Production of amino acids.

The following microorganism was deposited on April 24, 2001 as a pure culture with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• Escherichia coli Top10 / pCR2.1clpCint as DSM 14258.

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

The isolation of plasmid DNA from Escherichia coli as well all techniques for restriction, Klenow and alkaline Phosphatase treatment was performed according to Sambrook et al. (Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) carried out. Methods for the transformation of Escherichia coli are also described in this manual.

The composition of common nutrient media such as LB or TY Medium can also be found in the manual of Sambrook et al. be removed.

example 1 Preparation of a Genomic Cosmid Gene Bank C. glutamicum ATCC 13032

Chromosomal DNA from C. glutamicum ATCC 13032 was as in Tauch et al., (1995, Plasmid 33: 168-179), isolated and with the restriction enzyme Sau3AI (Amersham  Pharmacia, Freiburg, Germany, product description Sau3AI, Code no. 27-0913-02) partially cleaved. The DNA Fragments were extracted with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product description SAP, code no. 1758250) dephosphorylated. The DNA of the cosmid vector SuperCos1 (Wahl et al., 1987, Proceedings of the National Academy of Sciences, USA 84: 2160-2164), obtained from the company Stratagerie (La Jolla, USA, Product description SuperCos1 Cosmid Vector Kit, code no. 251301) was used with the Restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) cleaved and also with shrimp alkaline phosphatase dephosphorylated.

Subsequently, the cosmid DNA 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 labeled with the treated ATCC13032 DNA and the mixture with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04) treated. The ligation mixture was then washed with Help with the Gigapack II XL Packing Extract (Stratagenek La Jolla, USA, Product description Gigapack II XL Packing Extract, Code no. 200217) in phages.

To infect the E. coli strain NM554 (Raleigh et al 1988, Nucleic Acid Res. 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 cosmidbank were performed as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) + 100 μg / ml ampicillin. After incubation overnight at 37 ° C, recombinant single clones were selected.

Example 2 Isolation and sequencing of the c1pC gene

The cosmid DNA of a single colony was treated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) isolated according to the manufacturer and with the Restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27- 0913-02) partially split. The DNA fragments were with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated. To Gelelektrophoretischer separation was carried out the isolation the cosmid fragments in the size range of 1500 to 2000 bp with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1 obtained from the company Invitrogen (Groningen, Netherlands, product description Zero Background Cloning Kit, Product No. K2500-01) with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04). The ligation of the cosmid fragments into the sequencing vector pZero-1 was as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), whereby the DNA Mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) was incubated overnight. This Ligation mixture was then added to the E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences, U.S.A., 87: 4645-4649) (Tauch et al. 1994, FEMS Microbiol. Letters, 123: 343-7) and on LB Agar (Lennox, 1955, Virology, 1: 190) with 50 μg / ml Zeocin plated.

The plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden,  Germany). The sequencing was carried out after the dideoxy Chain termination method of 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 dRhodamine Terminator Cycle Sequencing Kit "by PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) used. Gel electrophoretic separation and Analysis of the sequencing reaction was carried out in one "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 obtained crude sequence data were then under Application of the Staden Program Package (1986, Nucleic Acids Research, 14: 217-231), version 97-0. The Single sequences of pZerol derivatives became one coherent contig assembled. The computer-aided Coding area analysis was carried out with the program 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: 33893402) against the non-redundant database of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).

The nucleotide sequence obtained is shown in SEQ ID no. 1 shown. Analysis of the nucleotide sequence revealed open reading frame of 2778 bp, which is called clpC gene was designated. The clpC gene encodes a polypeptide of 925 amino acids.  

Example 3 Preparation of an integration vector for the Integration mutagenesis of the clpC gene

From strain ATCC 13032, the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) isolated chromosomal DNA. Based on the sequence of the clpC gene known from example 2 for C. glutamicum, the following oligonucleotides were selected for the polymerase chain reaction (see SEQ ID No. 3 and SEQ ID No. 4):
CLPc int1:
5'-GAG ACC CTC AAG GAC AAG C-3 '
CLPc int2:
5'-GAT GTA GCG ATC AGC AAG C-3 '

The primers shown were from MWG Biotech (Ebersberg, Germany) synthesized and after the Standard PCR method of Innis et al. (PCR protocols, A guide to methods and applications, 1990, Academic Press) with the Taq polymerase from Boehringer Mannheim (Germany, Product Description Taq DNA Polymerase, Product No. 1 146 165) carried out the PCR reaction. With Help the polymerase chain reaction allow the primer the amplification of a 453 bp internal fragment of the clpC gene. The thus amplified product was in a 0.8% agarose gel electrophoresed.

The amplified DNA fragment was digested with the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, CA) UNITED STATES; Catalog number K4500-01) into the vector pCR2.1-TOPO (Mead et al. (1991) Bio / Technology 9: 657-663).

Subsequently, the E. coli strain TOP10 was electroporated with the ligation mixture (Hanahan, In: DNA cloning, A practical approach, Vol. I. IRL-Press, Oxford, Washington DC, USA, 1985). The selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al, Molecular cloning. A laboratory manual 2 ^nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989..), With 50 mg / l kanamycin had been supplemented.

Plasmid DNA was isolated from a transformant using the QlAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0, 8%). The plasmid was named pCR2.1clpCint and is shown in FIG .

Example 4 Integration mutagenesis of the clpC gene in strain DSM 5715

The vector pCR2.1clpCint mentioned in Example 3 was after the electroporation method of Tauch et.al. (FEMS Microbiological Letters, 123: 343-347 (1994)) in Corynebacterium glutamicum DSM5715 electroporated. In which Strain DSM 5715 is an AEC resistant Lysine producers. The vector pCR2.1clpCint can be found in DSM5715 do not self-replicate and only stay in the Get cell when it enters the chromosome of DSM5715 integrated. The selection of clones with the chromosome integrated pCR2.1clpCint was made by plating out the Electroporation approach on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), supplemented with 15 mg / l kanamycin.

For proof of integration, the clpCint fragment was according to the method "The DIG System Users Guide for Filters Hybridization "the company Boehringer Mannheim GmbH (Mannheim, Germany, 1993) with the dig- Hybridization kit of the company Boehringer marked. Chromosomal DNA of a potential integrante was reduced the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and each with the Restriction enzymes EcoRI and PstI cut. The resulting fragments were purified by agarose gelation. Separated electrophoresis and with the Dig Hybrisierungskit Boehringer hybridized at 68 ° C. That in example  3 called plasmid pCR2.1clpcint had within the chromosomal clpC gene into the chromosome of DSM5715 inserted. The root was named DSM5715 :: pCR2.1clpCint designated.

Example 5 Production of lysine

The C. glutamicum strain obtained in Example 4 DSM5715 :: pCR2.1clpCint was used in one to produce Lysine suitable nutrient medium cultured and the lysine content determined in culture supernatant.

For this, the strain was first on agar plate with the appropriate antibiotic (brain-heart agar with kanamycin (25 mg / L) for 24 hours at 33 ° C incubated. Starting from This agar plate culture was inoculated into a preculture (10 ml of medium in 100 ml Erlenmeyer flask). As a medium for the Preculture, the complete medium CgIII was used.

Medium Cg III

NaCl 2.5 g / l Bacto-peptone 10 g / l Bacto-yeast extract 10 g / l Glucose (separately autoclaved) 2% (w / v) AL = L <The pH was adjusted to pH 7.4

To this was added kanamycin (25 mg / L). The preculture was 16 hours at 33 ° C at 240 rpm on the shaker incubated. From this Vorkultur became a main culture seeded so that the initial OD (660 nm) of the major culture 0.1 OD. For the main culture, the medium MM used.  

Medium MM

CSL (Corn Steep Liquor) 5 g / l MOPS (morpholinopropanesulfonic acid) 20 g / l Glucose (separately autoclaved) 50 g / l AL = L <salts: (NH ₄ ) ₂ SO ₄ 25 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ .2H ₂ O 10 mg / l FeSO ₄ .7H ₂ O 10 mg / l MnSO ₄ .H ₂ O 5.0 mg / l Biotin (sterile filtered) 0.3 mg / l Thiamine.HCl (sterile-filtered) 0.2 mg / l Leucine (sterile filtered) 0.1 g / l CaCO ₃ 25 g / l

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

The cultivation is carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It became kanamycin (25 mg / l) was added. The cultivation took place at 33 ° C and 80% Humidity.

After 72 hours, the OD became at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH,  Munich). The amount of lysine formed was with 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

Brief description of the figure is attached:

Fig. 1: Map of the plasmid pCR2.1clpCint.

The abbreviations and designations used have the following meaning.
KmR: kanamycin resistance gene
EcoRI: restriction enzyme EcoRI site
PstI: restriction enzyme PstI site
clpCint: internal fragment of the clpC gene
ColE1: origin of replication of the plasmid ColE1

SEQUENCE LISTING

Claims (21)

An isolated polynucleotide of coryneform bacteria containing a polynucleotide sequence encoding the clpC gene selected from the group

• a) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide having 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 consecutive nucleotides of the polynucleotide sequence of a), b) or c),

wherein the polypeptide preferably has the activity of the clpC protease. 2. Polynucleotide according to claim 1, wherein the polynucleotide a replicable in coryneform bacteria, preferred recombinant DNA. 3. Polynucleotide according to 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. A replicatable DNA according to claim 2, comprising

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) within the range of the degeneracy of the genetic code, or
• c) at least one sequence that hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
• d) functionally neutral sense mutations in (i).

6. A replicable DNA according to claim 5, characterized characterized in that the hybridization under a stringency corresponding to at most 2 × SSC is carried out. 7. polynucleotide sequence according to claim 1, which is for a Polypeptide encoded that in SEQ ID. 2 contains shown amino acid sequence. 8. Coryneform bacteria in which the clpC gene attenuated, in particular, is switched off. 9. Integration vector pCR2.1clpCint, the

• 1. 9.1. carries a 453 bp internal fragment of the cplC gene,
• 2. 9.2. the restriction map is reproduced in Fig. 1, and
• 3. 9.3. which is deposited in the E. coli strain Top10 / pCR2.1clpCint under No. DSM 14258 in the German Collection of Microorganisms and Cell Cultures.

10. A process for the fermentative production of L-amino acids, in particular lysine, characterized in that one carries out the following steps:

• a) fermentation of the desired L-amino acid-producing coryneform bacteria, in which at least the clpC gene or nucleotide sequences coding for it are attenuated, in particular switched off;
• b) accumulation of the L-amino acid in the medium or in the cells of the bacteria, and
• c) isolating the L-amino acid.

11. The method according to claim 10, characterized characterized in that you have bacteria used, in which additional genes of the Biosyntheseweges the desired L-amino acid strengthened. 12. The method according to claim 10, characterized characterized in that you have bacteria in which the metabolic pathways at least are partially switched off, which is the formation of the reduce the desired L-amino acid. 13. The method according to claim 10, characterized characterized in that the expression of the the polynucleotide (s) that bind to the clpC gene coded (coded) attenuates, in particular off. 14. The method according to claim 10, characterized characterized in that the catalytic Properties of the polypeptide (enzyme protein) decreases, for which the polynucleotide clpC encodes. 15. The method according to claim 10, characterized in that fermented for the production of L-amino acids coryneform microorganisms, in which one or more of the genes selected from the group

• 1. 15.1 the gene dapA coding for the dihydrodipicolinate synthase,
• 2. 15.2 the gene gap coding for the glyceraldehyde-3-phosphate dehydrogenase,
• 3. 15.3 the gene tpi coding for the triosephosphate isomerase,
• 4. 15.4 the pgk gene coding for the 3-phosphoglycerate kinase,
• 5. 15.5 the gene coding for the glucose-6-phosphate dehydrogenase zwf,
• 6. 15.6 the gene coding for the pyruvate carboxylase pyc,
• 7. 15.7 the mqo gene encoding malate quinone oxidoreductase,
• 8. 15.8 the lysC gene coding for a feedback-resistant aspartate kinase,
• 9. 15.9 the lysine export-encoding gene lysE,
• 10. 15.10 the gene encoding homoserine dehydrogenase hom,
• 11. 15.11 the ilvA gene coding for the threonine dehydratase or the allele ilvA (Fbr) coding for a feedback-resistant threonine dehydratase,
• 12. 15.12 the gene ilvBN coding for the acetohydroxy acid synthase,
• 13. 15.13 the gene ilvD coding for the dihydroxy acid dehydratase,
• 14. 15.14 enhances or overexpresses the Zwa1 gene coding for the Zwa1 protein.

16. The method according to claim 10, characterized in that fermented for the production of L-amino acids coryneform microorganisms, in which one or more of the genes selected from the group

• 1. 16.1 the gene coding for the phosphoenolpyruvate carboxykinase pck,
• 2. 16.2 the gene coding for the glucose-6-phosphate isomerase gene pgi,
• 3. 16.3 the gene poxB coding for the pyruvate oxidase,
• 4. 16.4 the Zwa2 protein encoding gene zwa2,

weakens. 17. Coryneform bacteria containing a vector, the Parts of the polynucleotide according to claim 1, at least but 15 consecutive nucleotides of the claimed sequence, carries. 18. The method according to one or more of the claims 10-16, characterized in that one microorganisms of the species Corynebacterium glutamicum starts. 19. The method according to claim 18, characterized characterized in that the Corynebacterium glutamicum strain DSM5715 :: pCR2.1clpCint is used. 20. Method for Finding RNA, cDNA and DNA to Nucleic acids, or polynucleotides or genes to isolate those encoding the clpC protease or a high similarity with the sequence of the clpC gene  have, characterized that the polynucleotide containing the Polynucleotide sequences according to claims 1, 2, 3 or 4, as hybridization probes. 21. The method according to claim 20, characterized characterized in that arrays, micro arrays or DNA chips.