DE10108463A1 - New nucleotide sequences coding for the citA gene - Google Patents

Abstract

The invention relates to isolated polynucleotides 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 citA 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 the citA gene coding Nucleotide sequences from coryneform bacteria and a Process for the fermentative production of amino acids, in particular L-lysine, by attenuation of the citA gene. The citA gene encodes the sensor kinase Cit A of one Two-component system.

State of the art

L-amino acids, in particular L-lysine, can be found in US Pat Human medicine and in the pharmaceutical industry, in the Food industry and especially in the Animal nutrition application.

It is known that amino acids are produced by fermentation of Strains coryneformer bacteria, in particular Corynebacterium glutamicum. Because of the Great importance is constantly attached to the improvement of 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.

Object of the invention

The inventors have set themselves the task of new Measures for the improved fermentative production of Amino acids, especially L-lysine, provide.

Description of the invention

Are mentioned below L-lysine or lysine, are with it also the salts such. B. lysine monohydrochloride or Lysine sulfate meant.

The invention relates to an isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence coding for the citA gene 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 comprises the activity sensor kinase CitA 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 citA 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 inventive  Contains polynucleotide or a fragment thereof and Isolation of said polynucleotide sequence.

Polynucleotides containing the sequences according to the Invention are as hybridization probes for RNA, cDNA and DNA suitable for nucleic acids, respectively Isolate polynucleotides or full length genes code for the CitA protein or such nucleic acids or to isolate polynucleotides or genes that a high similarity to the sequence of the citA gene respectively.

Polynucleotides containing the sequences according to the Invention are also suitable as primers, with their Help with the polymerase chain reaction (PCR) DNA of genes can be produced which code for the CitA protein.

Such as probes or primers serving oligonucleotides contain at least 30, preferably at least 20, completely more preferably at least 15 consecutive Nucleotides. Also suitable are oligonucleotides with a length of at least 40 or 50 nucleotides.

"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%, preferably at least 80% and especially at least 90% to 95% are identical to the Polynucleotide according to SEQ ID no. 1 or one of them prepared fragment.  

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

The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, in particular those with the biological activity of the CitA protein and also such at least 70%, preferably at least 80% and especially at least 90% to 95% identical with the polypeptide according to SEQ ID no. 2 and the named Have activity.

The invention further relates to a method for fermentative production of amino acids, in particular L-lysine, using coryneform bacteria that especially already produce amino acids and in which the nucleotide sequences encoding the citA gene toned down, especially off or on low Level are expressed.

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.

The microorganisms that are the subject of the present Invention are amino acids, in particular L-lysine from glucose, sucrose, lactose, fructose, maltose, Molasses, starch, cellulose or glycerol and ethanol produce. It may be representative coryneformer Bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art  Corynebacterium glutamicum to call in the professional world known for their ability to use L-amino acids to produce.

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
or L-amino acid producing mutants or strains thereof, 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 DM58-1
Corynebacterium glutamicum DG52-5
Corynebacterium glutamicum DSM 5715 and
Corynebacterium glutamicum DSM 12866

The inventors succeeded, the new, for the CitA protein encoding citA gene of C. glutamicum, which is a sensor Kinase of a two-component system is to isolate.

For the isolation of the citA 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 DH5α (Jeffrey H. Miller: "A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and related bacteria ", Cold Spring Harbor Laboratory Press, 1992).

The with the help of cosmids or other λ vectors cloned long DNA fragments can subsequently again in common for DNA sequencing suitable Subcloned vectors.

Methods for DNA sequencing are among others Sanger et al. (Proceedings of the National Academy of  Sciences of the United States of America USA, 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 Researoh 16, 1829-1836 (1988)) or Butler's GCG program (Methods of Biochemical Analysis 39, 74-97 (1998)).

In this way, the new coding for the citA gene DNA sequence of C. glutamicum obtained as SEQ ID no. 1 is part of the present invention. Furthermore, from the present DNA sequence with the methods described above, the amino acid sequence of corresponding protein derived. In SEQ ID no. 2 is the resulting amino acid sequence of the citA 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. B. Replacement of glycine Alanine or of aspartic acid against glutamic acid in Proteins known as "sense mutations", which does not lead to a fundamental change in the activity of the Lead protein, d. H. are functionally neutral. Furthermore is known that changes at the N and / or C terminus of a Proteins whose function does not significantly affect or even stabilize. Details can be found on the Expert among others in 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.

Finally, DNA sequences are part of the invention, by the polymerase chain reaction (PCR) under Use of primers are made up of 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 the washing steps through 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 can for example, a 5x SSC buffer at a temperature of about 50-68 ° C are used. This probes can also hybridize with polynucleotides containing 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 followed by 0.5x SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim,  Mannheim, Germany, 1995) can be achieved, with a Temperature of about 50-68 ° C is set. By Gradually increase the hybridization temperature in Steps of about 1-2 ° C from 50 to 68 ° C can Polynucleotide fragments are isolated, for example at least 70% or at least 80% or at least 90% to 95% identity to the sequence of the probe used have. Further instructions for hybridization are in Form of so-called kits available on the market (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany 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 Gait Handbook: Oligonucleotides A: a Practical Approach (IRL Press, Oxford, UK, 1984) Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994)

In working on the present invention could be noted that coryneform bacteria after Attenuation of the citA gene in an improved manner Amino acids, especially L-lysine, produce.

To achieve a weakening either the Expression of the citA gene or the catalytic Properties of the enzyme protein are minimized or turned off. If necessary, both can Measures are 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)) Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek 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, ISSNO9442952, 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 caused by missense mutations (missense mutations) or 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, 84-87 (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)), pK18mob 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 then 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 used.

In the method of gene replacement is (gene replacement) a mutation such as 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 then 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. to eliminate glutamicum by a deletion.

In the citA gene can in this way a deletion, Insertion or a base exchange can be installed.

In addition, it can be used for the production of L-amino acids, especially L-lysine, be beneficial in addition to Attenuation of the citA gene of one or more enzymes of the biosynthesis pathway, glycolysis,  Anaplerotics, the pentose phosphate cycle or the Amino acid exports and optionally regulatory To amplify proteins, in particular 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.

Thus, for example, for the production of L-lysine one or more of the genes selected from the group can be used simultaneously

• 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 coding for the glucose-6-phosphate dehydrogenase Gen zwf (JP-A-09224661),
• The gene coding for the pyruvate carboxylase pyc (DE-A-198 31 609),
• - the gene encoding lysine export, lysE (DE-A-195 48 222)
• - that for a feed back resistant aspartate kinase encoding gene lysC (EP-B-0387527; EP-A-0699759; WO 00/63388), or  
• The gene coding for the Zwa1 protein zwa1 (DE: 199 59 328.0, DSM 13115)

amplified, in particular overexpressed.

In addition, it may be advantageous for the production of amino acids, in particular L-lysine, in addition to the attenuation of the citA 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),
• - The gene encoding the pyruvate oxidase poxB (DE: 1995 1975.7, DSM 13114)
• - the zwa2 protein encoding gene zwa2 (DE: 199 59 327.2, DSM 13113)

mitigate.

Furthermore, it can be used for the production of amino acids, In particular, L-lysine be beneficial, in addition to the Attenuation of the citA gene 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 microorganisms produced according to the invention are also the subject of the invention and can continuous or discontinuous in a batch process (Batch cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) for the purpose of producing L-amino acids, in particular L-lysine to be cultured. A summary about  well-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 be suitable meet the requirements of the respective strains. Descriptions of culture media of various Microorganisms are described in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981). When Carbon source can be sugars and carbohydrates such. B. Glucose, sucrose, lactose, fructose, maltose, molasses, Starch and cellulose, oils and fats, such as 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 a 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 well in Lindroth et al. (Analytical Chemistry (1979) 51: 1167- 1174).

The invention further relates to a method for fermentative production of an amino acid 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, in particular L-lysine, using coryneform bacteria that in particular already one or more of the mentioned Produce amino acids.

The following microorganism was deposited on 23.01.2001 as pure culture with the German collection for microorganisms and cell cultures (DSMZ, Braunschweig, Germany) according to Budapest contract:

• Escherichia coli strain Top10 / pCR2.1citAint as DSM 13,998th

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 restriction techniques, cleric 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.  

Examples 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 digested 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 Stratagene (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) split. The cosmid DNA treated in this way was mixed with the treated ATCC13032 DNA and the T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27- 0870-04). The ligation mixture was afterwards with the help of the Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, product description Gigapack II XL Packing Extract, Code no. 200217) in phage.  

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 gene citA

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 Cosmidfragmente 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), wherein the  DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) was incubated overnight. This Ligation mixture was then in 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 done 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 raw 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 1653 bp, which is called citA gene was designated. The citA gene encodes a polypeptide of 551 amino acids.

Example 3 Preparation of an integration vector for the Integration mutagenesis of the citA 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 citA gene known from Example 2 for C. glutamicum, the following oligonucleotides were selected for the polymerase chain reaction (see SEQ ID No. 4 and SEQ ID No. 5):
citA int1:
5 'TTC CAG TCG GTG AGG TCA GT 3'
citA int2:
5 'GTA CGA TCG CGG ATG GTT AC 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 480 bp internal fragment of the citA gene. The thus amplified product was in a 0.8% agarose gel electrophoresed.

The amplified DNA fragment (see SEQ ID No. 3) was with the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, CA, USA; Catalog Number K4500-01) in  the vector pCR2.1-TOPO (Mead et al. (1991) Bio / Technology 9: 657-663).

Subsequently, 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 QIAprep 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 pCR2.1citAint and is shown in FIG .

The following microorganism was deposited as pure culture on 23. 01. 2001 at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• Escherichia coli Top10 / pCR2.1citAint as DSM 13998

Example 4 Integration mutagenesis of the citA gene in the strains DSM 5715 and FERM-BP 1763

The vector pCR2.1citAint mentioned in Example 3 was prepared according to the electroporation method of Tauch et. al. (FEMS Microbiological Letters, 123: 343-347 (1994)) into the strains Corynebacterium glutamicum DSM 5715 and Brevibacterium lactofermentum FERM-BP 1763 electroporated. The strain DSM 5715 is an AEC-resistant lysine producer (EP-B-0435132), the strain FERM-BP 1763 is a mycophenolic acid-resistant valine producer (US Pat. No. 5,188,948). The vector pCR2.1citAint can not self-replicate in DSM5715 and FERM-BP 1763 and only remains in the cell if it has integrated into the chromosome of DSM 5715 or FERM-BP 1763. The selection of clones with integrated into the chromosome pCR2.1citAint made by plating out the electroporation batch on LB agar (Sambrook et al, Molecular cloning:... A laboratory manual 2 ^nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), the supplemented with 15 mg / l kanamycin.

For proof of integration, the citAint fragment was used 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 ever became a potential integrants according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and each with the Restriction enzymes EcoRI, BamHI and HindIII 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 pCR2.1citAint had within the chromosomal citA gene in the chromosome of DSM5715 and ins Chromosome of FERM-BP 1763 inserted. The tribes were called DSM5715:: pCR2.1citAint and FERM-BP 1763:: pCR2.1citAint designated.

Example 5 Production of lysine

The C.glutamicum strain obtained in Example 4 DSM5715:: pCR2.1citAint was used in a production of 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 of 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) The pH is 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 were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added, and the dry autoclaved CaCO _{3 was} added.

The cultivation was carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It was kanamycin (25 mg / l). 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 insulin derivatization with ninhydrin detection certainly.

Table 1 shows the result of the experiment.  

Table 1

Example 6 Production of valine

The B. lactofermentum strain FERM-BP obtained in Example 4 1763:: pCR2.1citAint was added to the production of valine cultivated suitable nutrient medium and the valine content in Culture supernatant determined.

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 of 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. this Kanamycin (25 mg / L) was added. The preculture became 16 Incubated for hours at 33 ° C at 240 rpm on the shaker. From this preculture a main culture was inoculated, so the initial OD (660 nm) of the main culture was 0.1 OD. The medium MM was used for the main culture.  

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 Isoleucine (sterile filtered) 0.1 g / l Methionine (sterile filtered) 0.1 g / l Thiamine. HCl (sterile filtered) 0.2 mg / l Leucine (sterile filtered) 0.1 g / l CaCO ₃ 25 g / l

CSL (Corn Steep Liquor), MOPS (morpholinopropanesulfonic acid) and the saline solution were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added, and the dry autoclaved CaCO _{3 was} added.

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

After 48 hours, the OD at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of valine 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 2 shows the result of the experiment.

Table 2

SEQUENCE LISTING

The following figure is attached:

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

The abbreviations and designations used have the following meaning.
KmR: kanamycin resistance gene
EcoRI: restriction enzyme EcoRI site
HindIII: restriction enzyme HindIII site
BamHI: Interface of the restriction enzyme BamHI
citAint: internal fragment of the citA gene
ColE1: origin of replication of the plasmid ColE1

Claims (19)

An isolated polynucleotide from coryneform bacteria containing a polynucleotide sequence encoding the citA 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), said polypeptide preferably having the activity of the sensor kinase CitA.

2. polynucleotides according to claim 1, wherein the Polynucleotide one in coryneform bacteria is replicable, preferably recombinant DNA. 3. polynucleotides 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. Replicatable DNA according to claim 2 containing

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

6. Process according to claim 5, characterized; that hybridization under a stringency according to at most 2x SSC is performed. 7. polynucleotide sequence according to claim 2, which is for a Polypeptide encoded that in SEQ ID. 2 contains shown amino acid sequence. 8. Vector pCR2.1citAint, the

• 1. 8.1 carries a 480 bp internal fragment of the citA gene shown in SEQ ID. No. 3,
• 2. 8.2 whose restriction map is reproduced in Fig. 1, and
• 3. 8.3 which is deposited in the E. coli strain Topl0 / pCR2.lcitAint under No. DSM 13998 in the German Collection of Microorganisms and Cell Cultures

9. Internal fragment of the citA gene with a length of 480 bp, shown in SEQ ID no. Third 10. Coryneform bacteria in which the citA gene attenuated, preferably switched off, especially by deletion. 11. 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 desired L-amino acid-producing coryneform bacteria, in which at least the citA gene is attenuated,
• b) enrichment of the desired product 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 one uses bacteria in which one additionally other genes of the biosynthetic pathway of the desired L-amino acid amplified. 13. The method according to claim 11, characterized, that you use bacteria in which the Metabolic pathways are at least partially eliminated which are the formation of the desired L-amino acid to decrease. 14. The method according to claim 11, characterized, that the expression of the polynucleotide (s), which encodes (encodes) the citA gene reduced, in particular turns off. 15. The method according to claim 11, characterized,  that the regulatory (or catalytic) Properties of the polypeptide for which the Polynucleotide citA encoded. 16. The method according to claim 11, characterized in that one fermented for the production of L-amino acids, in particular L-lysine, bacteria, in which simultaneously one or more of the genes selected from the group

• 1. 16.1 the gene dapA coding for the dihydrodipicolinate synthase,
• 2. 16.2 the gene coding for the glyceraldehyde-3-phosphate dehydrogenase gap
• 3. 16.3 the gene coding for the glucose-6-phosphate dehydrogenase zwf,
• 4. 16.4 the gene pyc coding for pyruvate carboxylase,
• 5. 16.5 the lysine export-encoding gene lysE,
• 6. 16.6 the lysC gene coding for a feedback-resistant aspartate kinase,
• 7. 16.7 enhances the Zwa1 gene coding for the Zwa1 protein, preferably overexpressing.

17. The method according to claim 11, characterized in that one or more of the genes selected from the group:

• 1. 17.1 the gene coding for the phosphoenolpyruvate carboxykinase pck,
• 2. 17.2 the gene pgi coding for the glucose-6-phosphate isomerase,
• 3. 17.3 the pyruvate oxidase-encoding gene poxB
• 4. 17.4 attenuates the Zwa2 gene coding for the Zwa2 protein.

18. Method according to one or more of previous claims, characterized, that one microorganisms of the genus Corynebacterium starts. 19. Method for Finding RNA, cDNA and DNA to Nucleic acids, or polynucleotides or Isolate genes encoding sensor kinase CitA or a high similarity with the sequence of the citA Have genes, characterized, that one of the polynucleotides, the sequences according to the claims 1 to 4, as Hybridization probes used.