DE102005013676A1 - Alleles of the zwf gene from coryneform bacteria - Google Patents

Abstract

The invention relates to mutants and alleles of the zwf gene of coryneform bacteria, coding for variants of the Zwf subunit of glucose-6-phosphate dehydrogenase (EC: 1.1.1.49), and to processes for the preparation of amino acids, in particular L-lysine and L-lysine. Tryptophan using bacteria containing these alleles.

Description

object In the invention, mutants and alleles of the zwf gene of coryneform bacteria are coding for variants the Zwf subunit of glucose-6-phosphate dehydrogenase (EC: 1.1.1.49) and method for producing amino acids, in particular L-lysine and L-tryptophan using bacteria containing these alleles contain.

Find amino acids in human medicine, in the pharmaceutical industry, in the food industry and especially in animal nutrition application.

It is known to be amino acids by fermentation of stems coryneform bacteria, in particular Corynebacterium glutamicum, getting produced. Because of the great importance is constantly on the improvement of the manufacturing process worked. process improvements can fermentation measures such as emotion and supply of oxygen, or the composition of the 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 concern yourself.

to Improve the performance characteristics of these microorganisms Methods of mutagenesis, selection and mutant selection applied. In this way receives man tribes, which is resistant to antimetabolite or auxotrophic for regulatory are significant metabolites and produce the amino acids. A friend Antimetabolite is the lysine analog S- (2-aminoethyl) -L-cysteine (AEC).

since Several years ago, methods of recombinant DNA technology were also used for strain improvement of L-amino acid producing strains of Corynebacterium, by using single amino acid biosynthetic genes amplified and investigated the effect on amino acid production. A summary Presentation about various aspects of genetics, metabolism and biotechnology of Corynebacterium glutamicum can be found in Pühler (chief ed.) in the Journal of Biotechnology 104 (1-3), 1-338, Of 2003.

The Nucleotide sequence of for the glucose-6-phosphate dehydrogenase Corynebacterium glutamicum encoding gene is among others in the database of the National Center for Biotechnology Information (NCBI) available from the National Library of Medicine (Bethesda, MD, USA). she can still the patent application WO 01/00844 as sequence no. 243 (= AX065117).

In WO 01/70995 is an improvement in fermentative production of L-amino acids by coryneform bacteria by enhancing the gene zwf.

In WO 01/98472, WO 03/042389 and US 2003/0175911 A1 is of new Mutations in the zwf gene reported.

Moritz et al. (European Journal of Biochemistry 267, 3442-3452 (2000)) report on physiological and biochemical studies on glucose-6-phosphate dehydrogenase of Corynebacterium glutamicum. After investigations by Moritz et al. Glucose-6-phosphate dehydrogenase consists of a Zwf subunit and an OpcA subunit.

The Microbial biosynthesis of L-amino acids in coryneform bacteria is a complex system and complex with various others Metabolic pathways networked in the cell. Therefore, no prediction can be made which mutation is the catalytic activity of glucose-6-phosphate dehydrogenase changed in this way, that the production of L-amino acids is improved. It is therefore desirable other variants of glucose-6-phosphate dehydrogenase available to have.

Of the better clarity half is the nucleotide sequence of the glucose-6-phosphate dehydrogenase or of for the Zwf subunit of glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum encoding zwf gene ("wild-type gene") as indicated the NCBI database in SEQ ID NO: 1 and the resulting amino acid sequence coded glucose-6-phosphate dehydrogenase shown in SEQ ID NO: 2 and 4. In SEQ ID NO: 3 are upstream and downstream additional nucleotide sequences specified.

Task of invention

The Inventors have set themselves the task of new measures for improved production of amino acids, in particular L-lysine and L-tryptophan.

description the invention

object of the invention are generated or isolated mutants coryneformer Bacteria that prefer amino acids excrete, and which contain a gene or allele, that for a polypeptide with glucose-6-phosphate dehydrogenase activity encoded, characterized in that the polypeptide is an amino acid sequence comprising, at the position 321 or a corresponding or comparable position of the amino acid sequence any proteinogenic amino acid except glycine is included. The exchange of glycine against L-serine is preferred.

The Polypeptide contained in the mutants of the invention, may also be a Zwf polypeptide or Zwf subunit of glucose-6-phosphate dehydrogenase be designated.

For the coryneform bacteria, the genus Corynebacterium is preferred. Particularly preferred are amino acid-secreting strains which are based on the following types:
Corynebacterium efficiens, such as the strain DSM44549,
Corynebacterium glutamicum, such as strain ATCC13032,
Corynebacterium thermoaminogenes such as strain FERM BP-1539, and
Corynebacterium ammoniagenes, such as strain ATCC6871,
the species Corynbacterium glutamicum being most preferred.

Some representatives of the species Corynebacterium glutamicum are also known in the art under other species. These include, for example:
Corynebacterium acetoacidophilum ATCC13870,
Corynebacterium lilium DSM20137,
Corynebacterium molassecola ATCC17965,
Brevibacterium flavum ATCC14067,
Brevibacterium lactofermentum ATCC13869, and
Brevibacterium divaricatum ATCC14020.

Known representatives of amino acid leaving strains of coryneform bacteria are, for example
the L-lysine producing strains
Corynebacterium glutamicum DM58-1 / pDM6 (= DSM4697) described in EP 0 358 940 .
Corynebacterium glutamicum MH20-22B (= DSM16835) described in Menkel et al. (Applied and
Environmental Microbiology 55 (3), 684-688 (1989)),
Corynebacterium glutamicum AHP-3 (= FermBP-7382) described in EP 1 108 790 .
Corynebacterium thermoaminogenes AJ12521 (= FERM BP-3304) described in US 5,250,423 .
or the L-tryptophan producing strains
Corynebacterium glutamicum K76 (= FermBP-1847) described in US 5,563,052 .
Corynebacterium glutamicum BPS13 (= FermBP-1777) described in US 5,605,818 , and
Corynebacterium glutamicum FermBP-3055 described in US 5,235,940 ,

information for the taxonomic classification of strains of this group of bacteria can be found, inter alia, Seiler (Journal of General Microbiology 129, 1433-1477 (1983)), fighter and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)), Liebl et al (International Journal of Systematic Bacteriology 41, 255-260 (1991)) and in US-A-5,250,434.

With tribes the designation "ATCC" can of of the American Type Culture Collection (Manassas, Va, USA) become. strains labeled "DSM" can by the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Strains called "FERM" can be obtained from National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) be obtained. The mentioned strains of Corynebacterium thermoaminogenes (FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5,250,434.

Under proteinogenic amino acids one understands the amino acids, in natural Proteins, that is in proteins of microorganisms, plants, animals and humans occurrence. These include in particular L-amino acids selected from the group L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine.

The mutants according to the invention preferably shed the said proteinogenic amino acids, in particular L-lysine and L-tryptophan. The term amino acids also includes their salts such as the lysine monohydrochloride or lysine sulfate in the case of the amino acid L-lysine.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, the the amino acid sequence of SEQ ID NO: 2, wherein at position 321 any proteinogenic amino acid except glycine is included. Replacing glycine with L-serine is preferred. Optionally, it contains the amino acid sequence the polypeptide beyond at position 8 an amino acid exchange L-serine against another proteinogenic amino acid, preferably L-threonine.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, at position 321 of the amino acid sequence of SEQ ID NO: 2 corresponding position any proteinogenic amino acid except glycine, preferred Contains L-serine, wherein the gene comprises a nucleotide sequence linked to the nucleotide sequence is identical to a polynucleotide produced by a polymerase chain reaction (PCR) is obtainable using a primer pair whose nucleotide sequences each comprise at least 15 consecutive nucleotides, the from the nucleotide sequence between position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11 and from the complementary nucleotide sequence between Position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11 are selected. examples for Such suitable primer pairs are shown in SEQ ID NO: 17 and SEQ ID NO: 18 and shown in SEQ ID NO: 19 and SEQ ID NO: 20. As starting material ("Template" DNA) becomes chromosomal DNA coryneformer bacteria, in particular with a Mutagen have been treated. Particularly preferred is the chromosomal DNA of the genus Corynebacterium, and most preferably the of the species Corynebacterium glutamicum.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, the one amino acid sequence with a length corresponding to 514 L-amino acids at position 321 except for any proteinogenic amino acid Glycine, preferably L-serine, is included. Optionally, it contains the amino acid sequence the polypeptide beyond an amino acid exchange L-serine against another proteinogenic amino acid, preferably L-threonine, at position 8.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, at position 312 to 330 of the amino acid sequence, the amino acid sequence according to position 312 to 330 of SEQ ID NO: 6 or 8. Prefers contains the amino acid sequence of the encoded polypeptide has an amino acid sequence corresponding to position 307 to 335 of SEQ ID NO: 6 or 8 or positions 292 to 350 of SEQ ID NO: 6 or 8 or positions 277 to 365 of SEQ ID NO: 6 or 8 or positions 262 to 380 of SEQ ID NO: 6 or 8 or position 247 to 395 of SEQ ID NO: 6 or 8 or positions 232 to 410 of SEQ ID NO: 6 or 8 or positions 202 to 440 of SEQ ID NO: 6 or 8 or positions 172 to 470 of SEQ ID NO: 6 or 8 or position 82 to 500 of SEQ ID NO: 6 or 8 or positions 2 to 512 of SEQ ID NO: 6 or 8 or positions 2 to 513 of SEQ ID NO: 6 or 8 or Position 2 to 514 of SEQ ID NO: 6 or B. Most preferably the length of the encoded polypeptide 514 amino acids.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, which at position 321 or at the corresponding position the amino acid sequence every amino acid contains glycine, the preference is given to the exchange for L-serine and its amino acid sequence Furthermore at least 90%, preferably at least 92% or at least 94% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 6. An example for an amino acid sequence, the least 99% identity with the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 8 and 10. The Polypeptide of this glucose-6-phosphate dehydrogenase has in addition to to the amino acid exchange Position 321 the amino acid exchange L-serine against L-threonine at position 8.

object The invention further mutants coryneformer bacteria, the contain a zwf allele for that encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, which at position 321 or at the corresponding position the amino acid sequence every amino acid contains glycine, the preference is given to the exchange for L-serine and its Nucleotide sequence as well at least 90%, preferably at least 92% or at least 94% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% is identical to the nucleotide sequence of SEQ ID NO: 5. An example for a nucleotide sequence of a zwf allele having at least 99% identity with the Nucleotide sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 7. The nucleotide sequence of this zwf allele has in addition to the nucleotide substitution guanine for adenine at position 961 (See SEQ ID NO: 5) the nucleotide exchange thymine to adenine at the position 22 (See SEQ ID NO: 7). Another example of a nucleotide sequence of a zwf alleles having at least 99% identity with the nucleotide sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 9. The nucleotide sequence this zwf allele has in addition to the nucleotide exchange guanine to adenine at position 961 (See SEQ ID NO: 5) and the nucleotide exchange thymine against adenine at the Position 22 (See SEQ ID NO: 7) the nucleotide exchanges cytosine against thymine at position 138, cytosine against thymine at position 279, Thymine against cytosine at position 738, cytosine against thymine at position 777 and guanine against adenine at position 906 (See SEQ ID NO: 9).

It It is known that conservative amino acid substitutions the enzyme activity only insignificantly change. Accordingly, the zwf allele present in the mutants of the present invention may be a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity coded, in addition to SEQ ID NO: 6 and SEQ ID NO: 8 and SEQ ID NO: 10, respectively shown amino acid sequence contain one (1) or more conservative amino acid substitutions. Preferably contains the polypeptide at most two (2), at most three (3), at most four (4) or at most five (5) conservative amino acid substitutions.

at the aromatic amino acids one speaks of conservative exchanges when phenylalanine, tryptophan and tyrosine are interchangeable. For the hydrophobic amino acids One speaks of conservative exchanges when leucine, isoleucine and valine are exchanged against each other. Speaking of the polar amino acids one from conservative exchange, when glutamine and asparagine against each other be replaced. The basic amino acids are called conservative ones Swap when arginine, lysine and histidine are exchanged become. At the acidic amino acids one speaks of conservative exchanges when aspartic acid and glutamic acid be exchanged for each other. In the hydroxyl-containing amino acids one speaks of conservative exchanges when serine and threonine be exchanged for each other.

One example for a conservative amino acid exchange is the replacement serine for threonine at position 8 of SEQ ID NO: 6, leading to the amino acid sequence according to SEQ ID NO: 8 or SEQ ID NO: 10 leads.

at The work on the present invention was compared by comparing amino acid sequence with the Clustal program (Thompson et al., Nucleic Acids Research 22, 4637-4680 (1994)) found that the amino acid sequences of glucose-6-phosphate dehydrogenase various bacteria such as Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium bovis, Streptomyces coeliclor, Streptomyces avermitilis, Corynebacterium efficiens and Corynebacterium glutamicum a sequence motif consisting of the sequence Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, a sequence motif consisting of the sequence Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys and also a sequence motif consisting of the sequence Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg contain. The term "Ali" stands for the amino acids Ala or Val, the term "Afa" for the amino acids Lys or Thr and the term "Bra" for the amino acids Ile or Leu.

Accordingly such mutants are preferred to coryneform bacteria that have a zwf allele contain that for encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, which is at least one amino acid sequence selected from the group Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys and Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg and at position 321 or equivalent or comparable Position of the amino acid sequence each amino acid excluding glycine, preferably L-serine. Optionally, it contains the amino acid sequence Furthermore an amino acid exchange L-serine against another proteinogenic amino acid, preferably L-threonine, at position 8 according to SEQ ID NO. 2

The amino acid sequence motif Val-Ile-Phe-Gly-Val-Thr-Gly-Asp-Leu is, for example, in SEQ ID NO: 6, 8 or 10 from position 32 to 40 included. The amino acid sequence motif Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys is included, for example, in SEQ ID NOS: 6, 8 or 10 from position 203 to 210, respectively. The amino acid sequence motif Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Leu-Arg-Thr-Gly-Lys-Arg is included, for example, in SEQ ID NO: 6, 8 or 10 from position 354 to 367.

object Finally, the invention is Mutant coryneform bacteria containing a zwf allele, the for a Polypeptide encoded with glucose-6-phosphate dehydrogenase enzyme activity, which is the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10.

It it is known that by intrinsic enzymes, so-called aminopeptidases, the terminal one Methionine is removed during protein synthesis.

Under the term "one to position 321 of the amino acid sequence corresponding position "or" one to position 321 of the amino acid sequence comparable position " the fact that by insertion or deletion of one coding for an amino acid Codons in the N-terminal Range (relative to position 321 of SEQ ID NO: 6, 8 or 10) of the encoded polypeptide, the position indication and length specification formally increased by one unit in the case of an insertion or in the case of a deletion is reduced by one unit. For example moves through Deletion of for the amino acid L-asparagine-encoding AAC codons at position 4 of SEQ ID NO: 6, 8 or 10 the L-serine from position 321 to position 320. The length specification would then be: 513 amino acids. Similarly, by insertion or deletion of one coding for an amino acid Codons in the C-terminal region (relative to position 321) of the coded polypeptide the length specification formally increased by one unit in the case of an insertion or in the case of a deletion reduced by one unit. Such comparable Positions can be determined by comparing the amino acid sequences in the form of an "alignment", for example with Easily identify help from the Clustal program.

By Such insertions and deletions will essentially alter the enzymatic activity not touched. "Essentially not touched "means that yourself the enzymatic activity of the mentioned variants by a maximum of 10%, a maximum of 7.5%, a maximum of 5%, at most 2.5% or at most 1% of the activity of the polypeptide having the amino acid sequence of SEQ ID NO: 6 or 8 or 10, respectively.

object Accordingly, the invention also includes zwf alleles which, for polypeptide variants of SEQ ID NO: 6 or 8 or 10 encode one or more Insertion (s) or deletion (s) included. Preferably, the polypeptide contains maximum 5, maximum 4, maximum 3 or maximum 2 insertions or deletions of amino acids.

The Sequence motifs Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, and Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys and Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg are not likely to be affected by such insertions / deletions torn.

to Preparation of the mutants of the invention can classical in vivo mutagenesis method with cell populations coryneformer Bacteria using mutagenic substances such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), 5-bromouracil, or ultraviolet Light to be used. Mutagenesis methods are for example in Manual of Methods for General Bacteriology (Gerhard et al. American Society for Microbiology, Washington, DC, USA, 1981) or Tosaka et al. (Agricultural and Biological Chemistry 42 (4), 745-752 (1978)) or Konicek et al (Folia Microbiologica 33, 337-343 (1988)) described. Typical mutagenesis using MNNG include Concentrations of 50 to 500 mg / l or even higher concentrations up to a maximum of 1 g / l, an incubation period of 1 to 30 minutes at one pH of 5.5 to 7.5. Under these conditions, the number of viable cells is reversed a share of approx. 50% to 90% or approx. 50% to 99% or approx. 50% reduced to 99.9% or more.

From the mutagenized cell population mutants or cells are removed and propagated. Preferably, in a further step, their ability to eliminate amino acids, preferably L-lysine or L-tryptophan, in a batch culture using a suitable nutrient medium is investigated. Suitable nutrient media and test conditions are, inter alia, in US 6,221,636 , in the US 5,840,551 , in the US 5,770,409 , in the US 5,605,818 , in the US 5,275,940 and in the US 4,224,409 described. In this way, mutants are identified which, in comparison to the parent strain or non-mutagenized parent strain, excrete more amino acids into the nutrient medium or into the cell interior. These include, for example, those mutants whose amino acid secretion is increased by at least 0.5%.

Subsequently, will provided from the mutants DNA, or isolated and with the help of the polymerase chain reaction using primer pairs that amplify the zwf gene or the zwf allele according to the invention or the mutation according to the invention at position 321, the corresponding polynucleotide synthesized. Preferably, the DNA is isolated from such mutants which are described in U.S. Pat increased Way amino acids excrete.

For this can any primer pairs from the upstream and downstream of the mutation according to the invention located nucleotide sequence and the complementary nucleotide sequence selected become. A primer of a primer pair in this case preferably comprises at least 15, at least 18, at least 20, at least 21 or at least 24 consecutive nucleotides selected from the nucleotide sequence between position 1 and 1267 of SEQ ID NO: 3 or SEQ ID NO: 11. The associated second primer of a primer pair comprises at least 15, at least 18, at least 20, at least 21 or at least 24 consecutive Nucleotides selected from the complementary Nucleotide sequence of position 2100 and 1271 of SEQ ID NO: 3 or SEQ ID NO: 11. If the amplification of the coding region is desired, then the primer pair is preferably selected from the nucleotide sequence Position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11 and of complementary Nucleotide sequence between position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11 selected. Is the amplification of a part of the coding region, such as as shown in SEQ ID NOS: 14 and 15, the primer pair becomes preferably from the nucleotide sequence between position 309 and 1267 of SEQ ID NO: 3 or SEQ ID NO: 11 and of the complementary nucleotide sequence between position 1848 and 1271 of SEQ ID NO: 3 or SEQ ID NO: 11 selected. Suitable primer pairs are, for example, under SEQ ID NO: 17 and SEQ ID NO: 18 represented primer pair zwf-K1 and zwf-K2 or the primer pair represented by SEQ ID NO: 19 and SEQ ID NO: 20 zwf-L1 and zwf-L2. The primer can also be labeled with recognition sites for restriction enzymes, with a biotin group or other accessories, as described in the prior art are equipped be. The total length of the primer is generally not more than 30, 40, 50 or 60 nucleotides.

For the production of polynucleotides by amplification of selected sequences, such as the zwf allele according to the invention, from presented, for example, chromosomal DNA ("template DNA") by amplification By means of PCR, generally thermostable DNA polymerases used. Examples of such DNA polymerases are the Taq polymerase from Thermus aquaticus, which was produced by Qiagen (Hilden, Germany) the Vent polymerase from Thermococcus litoralis, among others by the company New England Biolabs (Frankfurt, Germany) or the Pfu polymerase from Pyrococcus furiosus, which was produced by Stratagene (La Jolla, USA). Preference is given to polymerases with "proof-reading" activity. "Proof-reading" activity means that these polymerases are capable of defective incorporation of nucleotides to recognize and remedy the error by re-polymerization (Lottspeich and Zorbas, Bioanalytics, Spektrum Akademischer Verlag, Heidelberg, Germany (1998)). Examples of polymerases with "proof-reading" activity are the Vent polymerase and the Pfu polymerase.

The conditions in the reaction mixture are set according to the manufacturer. The polymerases are generally supplied by the manufacturer together with the usual buffer, which usually has concentrations of 10-100 mM Tris / HCl and 6-55 mM KCl at pH 7.5-9.3. Magnesium chloride is added at a concentration of 0.5-10 mM unless it is included in the manufacturer's supplied buffer. Deoxynucleoside triphosphates are also added to the reaction mixture in a concentration of 0.1-16.6 mM. The primers are presented in the reaction mixture with a final concentration of 0.1-3 μM and the template DNA in the optimal case with 10 ² to 10 ⁵ copies. The corresponding polymerase is added to the reaction mixture in an amount of 2 to 5 units. A typical reaction mixture has a volume of 20-100 μl.

When further additives can the reaction bovine serum albumin, Tween-20, gelatin, glycerol, formamide or DMSO (Dieffenbach and Dveksler, PCR Primer - A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA 1995).

A typical PCR course consists of three different, consecutively repeating temperature steps. In advance, the reaction is started with a temperature increase to 92 ° C - 98 ° C for 4 to 10 minutes to denature the submitted DNA. Then, a step of denaturing the initial DNA for 10-60 seconds at about 92-98 ° C is repeated, followed by a step of binding the primers to the subject DNA for 10-60 seconds at a certain primer-dependent temperature ("Annealing temperature"), which according to experience lies at 50 ° C. to 60 ° C. and can be calculated individually for each primer pair .The skilled person is given detailed information on this by Rychlik et al. (Nucleic Acids Research 18 (21): 6409-6412). This is followed by a synthesis step for extending the primers presented ("extension") at the respective optimum activity for the polymerase, usually depending on the polymerase in the range from 73 ° C. to 67 ° C., preferably 72 ° C. to 68 ° C. The duration This extension step depends on the performance of the polymerase and the length of the PCR product to be amplified In a typical PCR, this step lasts 0.5-8 minutes, preferably 2-4 minutes These three steps will take 30-35 times, optionally up to Repeated 50 times A final "extension" step of 4 - 10 minutes ends the reaction. The polynucleotides prepared in this way are also referred to as amplicons; the term nucleic acid fragment is also common.

Further Details and information on the PCR can be found in the manual PCR strategies (Innis, Felfand and Sninsky, Academic Press, Inc., 1995), in PCR primers - a laboratory manual, in the Gait manual: Oligonucleotides synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

The Nucleotide sequence is subsequently for example, according to the chain termination method of Sanger et al. (Proceedings of the National Academies of Sciences, U.S.A., 74, 5463-5467 (1977)) with those of Zimmermann et al. (Nucleic Acids Research 18, 1067pp (1990)) and that of this Nucleotide sequence encoded polypeptide in particular with respect to amino acid sequence analyzed. For this, the nucleotide sequence is converted into a program for translation from DNA sequence to an amino acid sequence entered. Suitable programs are, for example, the program "Patentin", which at patent offices, for example available from the US Patent Office (USPTO) is, or the "Translate Tool "on the ExPASy Proteomics Server on the World Wide Web (Gasteiger et al., Nucleic Acids Research 31, 3784-3788 (2003)) is.

On In this way, mutants are identified whose zwf alleles are polypeptides encode enzyme activity with glucose-6-phosphate dehydrogenase, which at position 321 of the amino acid sequence, respectively the corresponding or comparable position, any proteinogenic amino acid excluding glycine. Preferably, the exchange is against L-serine. Optionally contains the amino acid sequence Furthermore an amino acid exchange L-serine against another proteinogenic amino acid, preferably L-threonine, at position 8 or at the corresponding or comparable Position.

• a) Behandlung eines coryneformen Bakteriums, das die Fähigkeit besitzt Aminosäuren auszuscheiden, mit einem mutagenen Agenz,
• b) Isolierung und Vermehrung der in a) erzeugten Mutante,
• c) vorzugsweise Bestimmung der Fähigkeit der Mutante in einem Medium mindestens 0,5% mehr Aminosäure auszuscheiden oder im Zellinneren anzureichern als das in a) eingesetzte coryneforme Bakterium,
• d) Bereitstellung von Nukleinsäure aus der in b) erhaltenen Mutante,
• e) Herstellung eines Nukleinsäuremoleküls/Amplifikates/Nukleinsäurefragments unter Verwendung der Polymerasekettenreaktion, der Nukleinsäure aus d), und eines Primerpaares bestehend aus einem ersten Primer umfassend mindestens 15 aufeinanderfolgenden Nukleotide ausgewählt aus der Nukleotidsequenz zwischen Position 1 und 1267 von SEQ ID NO:3 oder SEQ ID NO:11 und einem zweitem Primer umfassend mindestens 15 aufeinanderfolgenden Nukleotide ausgewählt aus der komplementären Nukleotidsequenz zwischen Position 2100 und 1271 von SEQ ID NO:3 oder 11.
• f) Bestimmung der Nukleotidsequenz des in e) erhaltenen Nukleinsäuremoleküls, und Bestimmung der kodierten Aminosäuresequenz,
• g) gegebenfalls Vergleich der in f) bestimmten Aminosäuresesequenz mit SEQ ID NO:6, 8 oder 10, und
• h) Identifizierung einer Mutante, die ein Polynukleotid enthält, das für ein Polypeptid kodiert, welches an Position 321 oder an vergleichbarer Position jede proteinogene Aminosäure ausgenommen Glycin, bevorzugt L-Serin, und welche gegebenenfalls an Position 8 oder an vergleichbarer Position jede proteinogene Aminosäure ausgenommen L-Serin, bevorzugt L-Threonin, enthält.

The invention accordingly provides a mutant of a coryneform bacterium, characterized in that it is obtainable by the following steps:

• a) treatment of a coryneform bacterium having the ability to eliminate amino acids with a mutagenic agent,
• b) isolation and propagation of the mutant produced in a),
• c) preferably determining the ability of the mutant in a medium to excrete at least 0.5% more amino acid or to enrich it in the cell interior than the coryneform bacterium used in a),
• d) providing nucleic acid from the mutant obtained in b),
• e) Preparation of a nucleic acid molecule / amplificate / nucleic acid fragment using the polymerase chain reaction, the nucleic acid from d), and a primer pair consisting of a first primer comprising at least 15 consecutive nucleotides selected from the nucleotide sequence between position 1 and 1267 of SEQ ID NO: 3 or SEQ ID NO: 11 and a second primer comprising at least 15 contiguous nucleotides selected from the complementary nucleotide sequence between position 2100 and 1271 of SEQ ID NO: 3 or 11.
• f) determination of the nucleotide sequence of the nucleic acid molecule obtained in e), and determination of the coded amino acid sequence,
• g) if appropriate, comparison of the amino acid sequence determined in f) with SEQ ID NO: 6, 8 or 10, and
• h) Identification of a mutant containing a polynucleotide encoding a polypeptide which at position 321 or comparable position excludes any proteinogenic amino acid except glycine, preferably L-serine, and which optionally at position 8 or comparable position excludes any proteinogenic amino acid L-serine, preferably L-threonine.

The mutants generated in this way typically contain one (1) Copy of the described zwf allele.

The coding regions of zwf alleles of mutants according to the invention are reproduced by way of example in SEQ ID NO: 5, 7 and 9. The coding region of the wild-type gene is represented as SEQ ID NO: 1. SEQ ID NO: 1 contains at position 961 the nucleobase guanine, at position 962 the nucleobase guanine and at position 963 the nucleobase cytosine. SEQ ID NO: 1 contains at position 961 to 963 that for the amino acid glycine ko forming GGC codon. SEQ ID NO: 5 at position 961 contains the nucleobase adenine. This guanine adenine transition produces at position 961 to 963 the AGC codon coding for the amino acid L-serine.

SEQ ID NO: 1 contains at position 22 the nucleobase thymine, at position 23 the nucleobase Cytosine and at position 24 the nucleobase cytosine. SEQ ID NO: 1 contains accordingly at position 22 to 24 the for the amino acid Serine-encoding TCC codon. SEQ ID NO: 7 contains the nucleobase at position 22 Adenine. Through this thymine adenine transversion arises at position 22 to 24 that for the amino acid L-serine encoding AGC codon.

Furthermore can the nucleotide sequences shown in SEQ ID NO: 5 and 7 further Base changes included by the mutagenesis treatment but do not express themselves in an altered amino acid sequence. such Mutations are also known in the professional world as silent or neutral Called mutations. These silent mutations can also in the for Mutagenesis treatment already employed coryneform bacteria be included. examples for such silent mutations are the cytosine-thymine transition Position 138, the cytosine-thymine transition at position 279, the Thymine-cytosine transition at position 738, the cytosine-thymine transition at position 777 and the guanine adenine Transition at position 906 as shown in SEQ ID NO: 9.

The for the Mutagenesis used coryneform bacteria have preferred already the ability the desired amino acid into the surrounding nutrient medium or excrete fermentation broth or to accumulate in the cell interior.

Typically, feed-back resistant aspartate kinases are aspartate kinases that are less susceptible to inhibition by mixtures of lysine and threonine or mixtures of AECs compared to wild-type (Aminoethylcysteine) and threonine or lysine alone or AEC alone. The genes or alleles coding for these desensitized aspartate kinases are also referred to as lysC ^FBR alleles. The prior art (Table 1) describes numerous lysC ^FBR alleles encoding aspartate kinase variants which have amino acid changes compared to the wild type protein. The coding region of the wild-type lysC gene of Corynebacterium glutamicum corresponding to accession number AX756575 of the NCBI database is shown in SEQ ID NO: 21 and the protein encoded by this gene is shown in SEQ ID NO: 22.

Table 1 lysC ^FBA alleles encoding feedback-resistant aspartate kinases

L-lysine excretory coryneform bacteria typically have one or more of the amino acid substitutions listed in Table 1.

The following lysC ^FBR alleles are preferred: lysC A279T (exchange of alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for threonine), lysC A279V (exchange of alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for valine ), lysC S301F (exchange of serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for phenylalanine), lysC T308I (exchange of threonine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for isoleucine), lysC S301Y (substitution of serine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against tyrosine), lysC G345D (exchange of glycine at position 345 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for aspartic acid), lysC R320G (replacement of arginine at position 320 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against glycine), lysC T311I (replacement of threonine at position 311 of the encoded aspartate k inase protein according to SEQ ID NO: 22 against isoleucine), lysC S381F (exchange of serine at position 381 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for phenylalanine) and lysC 5317A (replacement of serine at position 317 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against alanine).

Particular preference is given to the lysC ^FBR allele lysC T311I (replacement of threonine at position 311 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against isoleucine) and a lysC ^FBR allele containing at least one substitution selected from the group A279T (exchange of alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against threonine) and S317A (Exchange of serine at position 317 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against alanine).

The lysC ^FBR allele lysC T311I is contained in the strain DS1797 deposited with the DSMZ. DM1797 is a mutant of Corynebacterium glutamicum ATCC13032.

To the manner described above, starting from stem DM1797, a mutant designated DM1816, which is a zwf allele contains that for a polypeptide encoded at position 321 of the amino acid sequence Contain L-serine is. The nucleotide sequence of the coding region of the mutant zwf allele DM1816 is SEQ ID NO: 9 and the amino acid sequence of the encoded polypeptide shown as SEQ ID NO: 10 and 12, respectively. The mutant DM1816 contains about that In addition, nucleotide exchanges in the nucleotide sequence between position 1 to 307 of SEQ ID NO: 3. These nucleotide exchanges are shown in SEQ ID NO: 11 shown. SEQ ID NO: 11 contains guanine at position 208 instead Adenine, at position 235 adenine instead of guanine, at position 245 Cytosine instead of thymine, at position 257 guanine instead of adenine and at position 299 guanine instead of adenine. SEQ ID NO: 11 still contains at position 329 adenine instead of thymine, at position 445 thymine instead Cytosine, at position 586 thymine instead of cytosine, at position 1045 Cytosine instead of thymine, at position 1084 thymine instead of cytosine, at position 1213 adenine instead of guanine and at position 1268 adenine instead of guanine.

Furthermore can L-lysine secreting coryneform bacteria are used which a weakened Homoserine dehydrogenase or homoserine kinase or others Possess properties as known from the prior art are.

Cryptophan-producing coryneform bacteria typically have a "feed back" -resistant or desensitized anthranilate synthase. "Feed-back" -resistant anthranilate synthase is understood as meaning anthranilate synthases which have a lower susceptibility to inhibition (5 to 10%, 10% to 15%) compared to wild-type % or 10% to 20%) by tryptophan or 5-fluorotryptophan (Matsui et al., Journal of Bacteriology 169 (11): 5330-5332 (1987)) or similar analogs. The genes or alleles coding for these desensitized anthranilate synthases are also referred to as trpE ^FBR alleles. Examples of such mutants or alleles are for example in the US 6180373 and EP0338474 described.

The obtained mutants show a compared to the starting strain used or parent strain increased Elimination or production of the desired amino acid in a fermentation process.

object The invention is also an isolated polynucleotide useful for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity coded, which at position 321 or at a corresponding or comparable position of the amino acid sequence any proteinogenic amino acid contains glycine, substitution for L-serine being preferred.

The polynucleotide according to the invention may be from a mutant according to the invention be isolated.

Farther can for the Mutagenesis of the zwf gene can be used in vitro methods. at the use of in vitro methods become isolated polynucleotides, which a zwf gene a coryneform bacterium, preferably that of the prior art described wild-type gene of Corynebacterium glutamicum, subjected to a mutagenic treatment.

at the isolated polynucleotides may be isolated, for example Total DNA or chromosomal DNA or amplificates of the zwf gene, which by means of the polymerase chain reaction (PCR) were prepared. Such amplicons are also known as Labeled PCR products. Instructions for amplifying DNA sequences with the help of the polymerase chain reaction the skilled person will find in the Gait Handbook: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). It is also possible the zwf gene to be mutagenized first into a vector, for example in a bacteriophage or in a plasmid.

Suitable methods for in-vitro mutagenesis include treatment with hydroxyla According to Miller (Miller, JH: A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992), the use of mutagenic oligonucleotides (TA Brown: Genetic Engineering for Einsteiger, Spektrum Akademischer Verlag, Heidelberg, 1993 and RM Horton: PCR-Mediated Recombination and Mutagenesis, Molecular Biotechnology 3, 93-99 (1995)) and the use of a polymerase chain reaction using a DNA polymerase which has a high error rate. Such a DNA polymerase is, for example, Mutazyme DNA Polymerase (GeneMorph PCR Mutagenesis Kit, No. 600550) from Stratagene (LaJolla, CA, USA).

Further Instructions and overviews for the generation of mutations in vivo or in vitro, the state the technique and well-known textbooks genetics and molecular biology e.g. the textbook by Knippers ( "Molecular Genetics ", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ( "Gene and Clones ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann's ( "General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which encodes the amino acid sequence of SEQ ID NO: 2, wherein at position 321 of the amino acid sequence every proteinogenic amino acid except glycine is included. Preferably, the exchange is against L-serine. Optionally contains the amino acid sequence of the Polypeptids beyond an amino acid exchange L-serine against another amino acid, preferably L-threonine, at position 8.

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which encodes an amino acid sequence with a length of 514 amino acids and at position 321 except for any proteinogenic L-amino acid Glycine, preferably L-serine is included.

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which is from position 312 to 330 of the amino acid sequence the amino acid sequence according to position 312 to 330 of SEQ ID NO: 6 or 8. Preferably contains the amino acid sequence of the encoded polypeptide has an amino acid sequence corresponding to position 307 to 335 of SEQ ID NO: 6 or 8 or positions 292 to 350 of SEQ ID NO: 6 or 8 or positions 277 to 365 of SEQ ID NO: 6 or 8 or positions 262 to 380 of SEQ ID NO: 6 or 8 or position 247 to 395 of SEQ ID NO: 6 or 8 or positions 232 to 410 of SEQ ID NO: 6 or 8 or positions 202 to 440 of SEQ ID NO: 6 or 8 or positions 172 to 470 of SEQ ID NO: 6 or 8 or position 82 to 500 of SEQ ID NO: 6 or 8 or position 2 to 512 of SEQ ID NO: 6 or 8 or positions 2 to 513 of SEQ ID NO: 6 or 8 or Position 2 to 514 of SEQ ID NO: 6 or 8. Most particularly preferred includes the length of the encoded polypeptide 514 amino acids.

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which is at position 321 of the amino acid sequence, respectively a corresponding or comparable position any proteinogenic amino acid excluding glycine, preferably L-serine, contains and comprising a nucleotide sequence linked to the nucleotide sequence is identical to a polynucleotide produced by a polymerase chain reaction (PCR) is obtainable using the primer pair whose nucleotide sequences each comprise at least 15 consecutive nucleotides, from the nucleotide sequence between position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11 and from the complementary nucleotide sequence between Position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11 are selected. examples for Such suitable primer pairs are shown in SEQ ID NO: 17 and SEQ ID NO: 18 and shown in SEQ ID NO: 19 and SEQ ID NO: 20. As starting material ("Template" DNA) becomes chromosomal DNA coryneformer bacteria, in particular with a Mutagen have been treated. Particularly preferred is the chromosomal DNA of the genus Corynebacterium, and most preferably the of the species Corynebacterium glutamicum.

object The invention further relates to an isolated polynucleotide, which with a to SEQ ID NO: 5, 7 or 9 complementary nucleotide sequence below hybridized to stringent conditions and for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which is at position 321 of the amino acid sequence, respectively a corresponding or comparable position any proteinogenic Except amino acid Glycine, preferably L-serine, and optionally at one of the position 8 corresponding position excluded any proteinogenic amino acid L-serine, preferably L-threonine.

instructions for the hybridization of nucleic acids or polynucleotides, the skilled person finds inter alia in the manual "The DIG System Users Guide for Filter Hybridization "by 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, it will be only hybrids are formed in which the probe i. a polynucleotide comprising the nucleotide sequence complementary to SEQ ID NO: 5, 7 or 9 and the target sequence, d. H. those treated with the probe or identified polynucleotides are at least 90% identical. It It is known that the stringency of hybridization including Wash steps by varying the buffer composition, the temperature and the salt concentration is influenced or determined. The hybridization reaction is generally compared at relatively low stringency carried out to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).

For the hybridization reaction For example, a buffer corresponding to 5 × SSC buffer at a temperature from about 50 ° C - 68 ° C used become. It can Probes also hybridize with polynucleotides that are less than 90% identity to the nucleotide sequence of the probe used. Such hybrids are less stable and are washed by washing under stringent conditions away. This can be done, for example, by lowering the salt concentration on 2 × SSC and optionally subsequently 0.5 × SSC (The DIG System User's Guide for Filters Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) 50 ° C - 68 ° C, about 52 ° C - 68 ° C, about 54 ° C - 68 ° C, about 56 ° C - 68 ° C, about 58 60 ° C - 68 ° C, approx. 62 ° C - 68 ° C, approx. 64 ° C - 68 ° C, approx. 66 ° C - 68 ° C becomes. Temperature ranges from about 64 ° C - 68 ° C or about 66 ° C - 68 ° C prefers. It may be possible to adjust the salt concentration to a concentration corresponding to 0.2X SSC or 0.1X SSC reduce. Optionally contains the SSC buffer sodium dodecyl sulfate (SDS) in one concentration of 0.1%. By gradual increase The hybridization temperature in steps of about 1 - 2 ° C from 50 ° C to 68 ° C can polynucleotide fragments be isolated, which is at least 90% or at least 91%, preferably at least 92% or at least 93% or at least 94% or at least 95% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% identity to the sequence or complementary Have sequence of the probe used and for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity encode which contains the amino acid exchange according to the invention. The Nucleotide sequence of the polynucleotide thus obtained is determined by known methods. Further instructions for hybridization are commercially available in the form of so-called kits (e.g., DIG Easy Hyb of the company Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558). The nucleotide sequences thus obtained code for polypeptides with glucose-6-phosphate dehydrogenase Enzyme activity, at least 90% preferred at least 92% or at least 94% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% are identical to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 and the amino acid exchange according to the invention.

object The invention further provides an isolated polynucleotide encoding a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity coded, which at position 321 or a corresponding or comparable position of the amino acid sequence except any amino acid Contains glycine, wherein the exchange is given preference against L-serine and the an amino acid sequence that includes as well at least 90%, preferably at least 92% or at least 94% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 6. An example for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, which is a amino acid sequence which is at least 99% identical to that of SEQ ID NO: 6 is shown in SEQ ID NO: 8 and SEQ ID NO: 10.

The invention further relates to an isolated polynucleotide encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which contains at position 321 or a corresponding or comparable position of the amino acid sequence, each amino acid except glycine, wherein the replacement against L-serine of Is preferred and which comprises a nucleotide sequence which is also at least 90%, preferably at least 92% or at least 94% or at least 96%, and most preferably at least 97% or at least 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 5. An example of a polynucleotide encoding a polypeptide of the invention having glucose-6-phosphate dehydrogenase enzyme activity and having a nucleotide sequence at least 99% identical to that of SEQ ID NO: 5 is shown in SEQ ID NO: 7 , The nucleotide sequence of this zwf allele has, in addition to the nucleotide exchange guanine to adenine at position 961 (see SEQ ID NO: 5), the nucleotide exchange thymine to adenine at position 22 (see SEQ ID NO: 7). Another example of a nucleotide sequence of a zwf allele having at least 99% identity with the nucleus SEQ ID NO: 5 is shown in SEQ ID NO: 9. The nucleotide sequence of this zwf allele has in addition to the nucleotide exchange guanine to adenine at position 961 (see SEQ ID NO: 5) and the nucleotide exchange thymine to adenine at position 22 (see SEQ ID NO: 7) the nucleotide exchanges cytosine to thymine in position 138, cytosine to thymine at position 279, thymine to cytosine at position 738, cytosine to thymine at position 777 and guanine to adenine at position 906 (see SEQ ID NO: 9).

Furthermore those isolated polynucleotides are preferred which are for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which is at position 321 of the amino acid sequence, respectively a corresponding or comparable position except any amino acid Glycine, preferably L-serine contains and the at least one sequence motif or an amino acid sequence selected from the group Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys, and Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg contain.

The Designation "Ali" stands for the amino acids Ala or Val, the term "Afa" for the amino acids Lys or Thr and the term "Bra" for the amino acids Ile or Leu.

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which encodes the amino acid sequence of SEQ ID NO: 6 or 8 or 10, respectively. Possibly contains the encoded polypeptide contains one (1) or more conservative amino acid substitutions. Preferably contains the polypeptide at most two (2), at most three (3), at most four (4) or at most five (5) conservative amino acid substitutions.

object The invention further provides an isolated polynucleotide for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which encodes the amino acid sequence of SEQ ID NO: 6 or 8 or 10 inclusive renewal at the N- or C-terminus comprises at least one (1) amino acid. This extension is not more than 50, 40, 30, 20, 10, 5, 3 or 2 amino acids respectively Amino acid residues.

object Finally, the invention is also zwf alleles responsible for Polypeptide variants of SEQ ID NO: 6, 8 or 10 encode a or multiple insertions or deletions. Preferably, these contain a maximum of 5, a maximum of 4, a maximum of 3 or a maximum of 2 insertions or Deletions of amino acids. The sequence motif Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu and / or Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys and / or Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg is replaced by such inserts / deletions are preferably not torn.

object The invention further provides an isolated polynucleotide comprising Nucleotide sequence according to SEQ ID NO: 5, 7, 9 or 11.

object The invention further provides an isolated polynucleotide comprising Nucleotide sequence between position 1 and 307 of SEQ ID NO: 11, preferred the nucleotide sequence between position 198 and 304 of SEQ ID NO: 11 and most preferably the nucleotide sequence between position 208 and 299 of SEQ ID NO: 11.

object The invention is finally an isolated polynucleotide containing the zwf allele of the mutant DM1816.

object The invention is also an isolated polynucleotide that forms part of the coding region of a Zwelf allelic according to the invention wherein the isolated polynucleotide in each case the part the coding region comprising the amino acid substitution at the position 321 of the amino acid sequence of the encoded polypeptide.

In particular, a nucleic acid molecule or DNA fragment comprising at least one amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 2, or for at least one amino acid sequence corresponding to positions 292 to 350 of SEQ ID NO: 2, or for at least one amino acid sequence corresponding to position 277 to 365 of SEQ ID NO: 2, or that for at least one amino acid sequence corresponding to position 262 to 380 of SEQ ID NO: 2, or for at least one amino acid sequence corresponding to position 247 to 395 of SEQ ID NO: 2 , or that for at least one amino acid sequence corresponding to position 232 to 410 of SEQ ID NO: 2, or that encodes at least one amino acid sequence corresponding to position 202 to 440 of SEQ ID NO: 2, or that for at least one amino or encodes at least one amino acid sequence corresponding to position 82 to 500 of SEQ ID NO: 2, or for at least one amino acid sequence corresponding to position 2 to 512 of SEQ ID NO: 2 or encodes at least one amino acid sequence corresponding to position 2 to 513 of SEQ ID NO: 2, or which encodes at least one amino acid sequence corresponding to position 2 to 514 of SEQ ID NO: 2 or includes a corresponding reading frame, corresponding to the position 321 of SEQ ID NO: 2 is any proteinogenic amino acid except glycine, preferably L-serine, and wherein optionally at the position corresponding to 8 each proteinogenic amino acid except L-serine, preferably L-threonine, is included.

An example of a reading frame according to the invention comprising a polynucleotide encoding at least the amino acid sequence from position 307 to 335 corresponding to SEQ ID NO: 2, wherein at the position corresponding to 321 of the amino acid sequence each proteinogenic amino acid (Xaa) is excluded glycine is listed below:

It is also shown as SEQ ID NO: 13. The of this reading frame encoded amino acid sequence is shown as SEQ ID NO: 14. Position 15 in SEQ ID NO: 14 corresponds to position 321 of SEQ ID NO: 6, 8, 10 or 12.

Prefers be nucleic acid molecules, the for at least an amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 6 or 8, respectively 10, or at least corresponding to positions 292 to 350 of SEQ ID NO: 6 or 8 or 10, or at least according to position 277 to 365 of SEQ ID NO: 6 or 8 or 10, or at least corresponding to positions 262 to 380 of SEQ ID NO: 6 or 8, respectively 10, or at least corresponding to positions 247 to 395 of SEQ ID NO: 6 or 8 or 10, or at least according to position 232 to 410 of SEQ ID NO: 6 or 8 or 10, or at least corresponding to positions 202 to 440 of SEQ ID NO: 6 or 8, respectively 10, or at least corresponding to position 172 to 470 of SEQ ID NO: 6 or 8 or 10, or at least according to position 82 to 500 of SEQ ID NO: 6 or 8 or 10, or at least according to position 2 to 512 of SEQ ID NO: 6 or 8 respectively 10, or at least corresponding to position 2 to 513 of SEQ ID NO: 6 or 8 or 10, or at least according to position 2-514 of SEQ ID NO: 6 or 8 and 10, respectively.

An example of a reading frame according to the invention comprising a polynucleotide which codes for at least the amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 6 is listed below:

The Reading frame is also shown as SEQ ID NO: 15. SEQ ID NO: 16 shows the amino acid sequence encoded by this reading frame. The position 15 in SEQ ID NO: 16 corresponds to the position 321 of SEQ ID NO: 6, 8, 10 or 12.

Very particular preference is given to nucleic acid molecules which have at least one nucleotide sequence corresponding to positions 919 to 1005 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 874 to 1050 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 829 to 1095 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 784 to 1140 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 739 to 1185 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to position 694 to 1230 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to position 604 to 1320 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence entspre at positions 514 to 1410 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 244 to 1500 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to positions 4 to 1536 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to position 4 to 1539 of SEQ ID NO: 5, 7 or 9, or at least one nucleotide sequence corresponding to position 4 to 1542 of SEQ ID NO: 5, 7 or 9.

The reading frame according to the invention, as exemplified in SEQ ID NO: 13 and 15 as a nucleotide sequence and in SEQ ID NO: 14 and SEQ ID NO: 16 in the form of the encoded amino acid sequence can be shown Furthermore contain one or more mutations leading to one or more conservative amino acid substitution to lead. Preferably lead the mutations to a maximum of 4%, to a maximum of 2% or to a maximum of 1% conservative Amino acid substitutions. Furthermore you can the reading frame of the invention contain one or more silent mutations. Preferably contain the reading frame according to the invention maximum 4% and most preferably at most 2% to at most 1% silent mutations.

The according to the invention, isolated Polynucleotides can used to make recombinant strains of microorganisms, which in comparison to the parent or parent in improved Way amino acids into the surrounding medium or accumulate in the cell interior.

A common method for incorporation of mutations in genes coryneformer Bacteria is that of allele exchange, also known as "gene replacement" This method is a DNA fragment containing the interest Contains mutation, in the desired Strain of a coryneform bacterium transferred and the mutation through at least two recombination events or "cross over "events into the chromosome of the desired one Tribe incorporated or existing in the relevant strain Replaced sequence of a gene against the mutated sequence.

Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991) used this method to place a lysA allele that was deletion and a lysA allele that carried an insertion into the chromosome of C. glutamicum Schäfer et al., Gene 145, 69-73 (1994)) used this method to incorporate a deletion into the hom-thrB operon of C. glutamicum., by Nakagawa et al. EP 1108790 ) and Ohnishi et al. (Applied Microbiology and Biotechnology 58 (2), 217-223 (2002)), this method was used to incorporate various mutations from the isolated alleles into the chromosome of C. glutamicum. In this way, Nakagawa et al. a mutation designated as Val59Ala into the homoserine dehydrogenase gene (hom), a mutation designated as Thr311I1 into the aspartate kinase gene (lysC or ask), a mutation designated Pro458Ser into the pyruvate carboxylase gene (pyc) and a Ala213Thr mutation into the glucose-6-phosphate dehydrogenase gene (zwf) of C. glutamicum strains incorporate.

For a method according to the invention may be a polynucleotide of the invention which encompasses the entire coding region, such as For example, shown in SEQ ID NO: 5, 7 or 9, or which has a Part of the coding region, such as the nucleotide sequence, the for at least the amino acid sequence coded according to position 307 to 335 of SEQ ID NO: 6, 8 or 10 and shown as SEQ ID NO: 13 and 15. The part of the coding region corresponding to SEQ ID NO: 13 and 15 has a length of ≥ 87 nucleobases. To be favoured those parts of the coding region whose length is ≥ 267 nucleobases, such as For example, nucleic acid molecules, the for at least an amino acid sequence corresponding to position from 277 to 365 of SEQ ID NO: 6, 8 or 10 encode. Very particular preference is given to those parts of the coding region, their length ≥ 357 nucleobases is, such as nucleic acid molecules that are responsible for at least an amino acid sequence according to position from 262 to 380 of SEQ ID NO: 6, 8 or 10 encode.

The DNA fragment containing the mutation of interest lies with this Method typically in a vector, in particular a plasmid, preferably, from the strain to be mutated is not or only partially replicated. As auxiliary or intermediate host, where the vector is replicable will generally become a bacterium the genus Escherichia prefers the species Escherichia coli used.

Examples of such plasmid vectors are those of Schäfer et al. (Gene 145, 69-73 (1994)) described pK · mob and pK · mobsacB vectors, such as pKl8mobsacB, and the vectors described in WO 02/070685 and WO 03/014362. These are replicative in Escherichia coli but not in coryneform bacteria. Especially suitable are vectors which have a conditionally negatively dominant gene such as the sacB gene (levansucrase gene) of, for example, Bacillus or the galK gene (Galaktoseki Nase gene) of, for example, Escherichia coli. (A conditionally negatively dominant gene refers to a gene which, under certain conditions, is, for example, toxic to the host, but under other conditions has no negative effect on the host carrying the gene.) These allow selection for recombination events in which the vector is eliminated from the chromosome. Furthermore, Nakamura et al. (US Pat. No. 6,303,383) describe a temperature-sensitive plasmid for coryneform bacteria that can only replicate at temperatures below 31 ° C.

Of the Vector will follow by conjugation, for example, by the method of Schäfer (Journal of Bacteriology 172, 1663-1666 (1990)) or transformation, for example, according to the method of Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) or the method by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)) into the coryneform bacterium. If necessary, the transfer of the DNA can also be achieved by particle bombardment.

To homologous recombination by means of a first, cross-over event causing integration and a suitable second excision-causing "cross-over" event in the target gene or in the target sequence one achieves the incorporation of the mutation and receives a recombinant bacterium.

to Identification and characterization of the resulting strains can be carried out under other methods of Southern blotting hybridization, the polymerase chain reaction, Sequence determination, the method of "Fluorescence Resonance Energy Transfer "(FRET) (Lay et al., Clinical Chemistry 43, 2262-2267 (1997)) or methods of enzymology.

• a) ein erfindungsgemäßes Polynukleotid in ein coryneformes Bakterium überführt,
• b) das im Chromosom des coryneformen Bakteriums vorhandene Glucose-6-Phosphat-Dehydrogenase Gen, das für eine Aminosäuresequenz mit Glycin an Position 321 oder an einer vergleichbaren Position der Aminosäuresequenz kodiert, gegen das Polynukleotid aus a) austauscht, das für eine Aminosäuresequenz kodiert, die an der Position 321 oder an einer vergleichbaren Position der Aminosäuresequenz eine andere L-Aminosäure, vorzugsweise L-Serin, und gegebenenfalls an Position 8 oder an einer vergleichbaren Position jede proteinogene Aminosäure ausgenommen L-Serin, bevorzugt die Aminosäure L-Threonin, besitzt, und
• c) das gemäß Schritt a) und b) erhaltene coryneforme Bakterium vermehrt.

Another object of the invention is accordingly a process for the preparation of a coryneform bacterium, wherein

• a) converting a polynucleotide according to the invention into a coryneform bacterium,
• b) exchanging the glucose 6-phosphate dehydrogenase gene present in the chromosome of the coryneform bacterium which code for an amino acid sequence with glycine at position 321 or at a comparable position of the amino acid sequence, against the polynucleotide from a) which codes for an amino acid sequence, which at position 321 or at a comparable position of the amino acid sequence has another L-amino acid, preferably L-serine, and optionally at position 8 or at a comparable position any proteinogenic amino acid except L-serine, preferably the amino acid L-threonine, and
• c) the coryneform bacterium obtained according to step a) and b) increases.

On get that way a recombinant coryneform bacterium, which instead of the Wild-type zwf gene contains one (1) zwf allele according to the invention.

• a) ein erfindungsgemäßes Polynukleotid, das für ein Polypeptid mit Glucose-6-Phosphat-Dehydrogenase Enzymaktivität kodiert, in einen Mikrorganismus überführt,
• b) das Polynukleotid in dem Mikroorganismus repliziert, und
• c) den gemäß Schritt a) und b) erhaltenen Mikroorganismus vermehrt.

Another inventive method for producing a microorganism is that one

• a) converting a polynucleotide according to the invention, which codes for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity, into a microorganism,
• b) replicating the polynucleotide in the microorganism, and
• c) increases the microorganism obtained in step a) and b).

On get that way a recombinant microorganism containing at least one (1) Copy or multiple copies of a polynucleotide of the invention contains that for encodes a glucose-6-phosphate dehydrogenase at position 321 or a comparable position of the amino acid sequence of the encoded polypeptide, every proteinogenic amino acid contains glycine, substitution for L-serine being preferred. Possibly contains the polypeptide at position 8 or a comparable position every proteinogenic amino acid except L-serine, preferably the amino acid L-threonine.

Further objects The invention accordingly hosts or host cells, preferably microorganisms, more preferably coryneform bacteria and bacteria of the genus Escherichia containing the polynucleotides of the invention contain. The invention likewise relates to microorganisms, made using the isolated polynucleotides. Such microorganisms or bacteria are also called recombinant Called microorganisms or recombinant bacteria. In the same Wise are vectors containing the polynucleotides of the invention, Subject of the invention. After all, hosts are these Contain vectors, also the subject of the invention.

The isolated polynucleotides of the invention may also be used to achieve overexpression be used on the polypeptides coded by them.

Under overexpression Generally one understands an increase the intracellular Concentration or activity a ribonucleic acid, a protein or an enzyme. In the case of the present invention zwf alleles or polynucleotides are overexpressed, the for Glucose-6-phosphate dehydrogenases encoding at position 321 the amino acid sequence of the encoded polypeptide except any proteinogenic amino acid Glycine, with the replacement of L-serine is preferred. Optionally contains the encoded protein as well replacement of L-serine with another proteinogenic amino acid is preferred L-threonine at position 8 of the amino acid sequence. It is known, that by our own enzymes - so-called Aminopeptidases - N-terminal Amino acids, in particular the N-terminal methionine, of the polypeptide formed can be split off. The mentioned increase the concentration or activity of a gene product For example, achieve by the copy number of the corresponding polynucleotides increased by at least one copy.

A widespread method of increase The number of copies is that you have the appropriate gene or Allele in a vector, preferably a plasmid, built by one coryneform bacterium is replicated. Suitable plasmid vectors For example, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or that of Tauch et al. (Journal of Biotechnology 99, 79-91 (2002)) described pSELF vectors. A review on the topic Plasmids in Corynebacterium glutamicum can be found in Tauch et al. (Journal of Biotechnology 104, 27-40 (2003)).

A Another common method for achieving overexpression is the method the chromosomal gene amplification. In this method, at least an additional Copy of the gene or allele of interest into the chromosome of a coryneform bacterium inserted.

In an embodiment, as described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for The hom-thrB operon is described as being a non-replicative in C. glutamicum Plasmid, which contains the gene of interest, into a coryneform bacterium. To Homologous recombination by means of a "cross over" event contains the resulting Strain at least two copies of the gene in question, respectively Allele.

In another embodiment, which is described in WO 03/040373 and US 2003-0219881-A1, is one or more copies of the gene of interest by means of at least two recombination events in a desired Location of the chromosome of C. glutamicum inserted. That way, for example a copy of a lysC allele, that for encoded an L-lysine insensitive aspartate kinase into the gluB gene of C. glutamicum incorporated.

In a further embodiment, which is described in WO 03/014330 and US-2004-0043458-A1, at least one further copy, preferably in tandem arrangement to the already existing gene or allele, of the at least two recombination events at the natural site incorporated gene of interest. In this way, for example, a tandem duplication of a lysC ^FBR allele at the natural lysC locus was achieved.

A another way to achieve overexpression is to the corresponding gene or allele in a functional way (operably linked) with a promoter or an expression cassette to link. Suitable promoters for Corynebacterium glutamicum are for example in the review article by Patek et al. (Journal of Biotechnology 104 (1-3), 311-323 (2003)) can the adequate Known by Amann et al. (Gene 69 (2), 301-315 (1988)) and Amann and Brosius (Gene 40 (2-3), 183-190 (1985)) promoters T3, T7, SP6, M13, lac, tac and trc be used. Such a promoter may, for example upstream of the zwf allele, typically at intervals of about 1-500 or 1-307 Nucleotides from start codon, a recombinant coryneform bacterium added which instead of the amino acid glycine naturally present at position 321 another proteinogenic amino acid contains. Naturally, such a promoter may also be upstream of the promoter zwf alleles of a mutant according to the invention added become. It is still possible an isolated according to the invention Polynucleotide used for a variant of the invention the glucose-6-phosphate dehydrogenase encoded with a promoter to link and the expression unit obtained into an extrachromosomally replicating Plasmid or into the chromosome of a coryneform bacterium.

In addition, the promoter and regulatory region or ribosome binding site that may be present upstream of the structural gene. An example of a mutant promoter region of the zwf gene or zwf allele is the nucleotide sequence comprising position 208 to 299 of SEQ ID NO: 11. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. Alternatively, overexpression of the gene or allele concerned can be achieved by altering the composition of the medium and culture.

By the measures overexpression becomes the activity or concentration of the corresponding protein in general at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, maximum to 1000% or 2000%, based on activity or concentration of the protein in the initial microorganism or parent strain elevated. By a parent microorganism or parent strain is meant a microorganism on which the measures of the invention are carried out.

A Method for determining the enzymatic activity of glucose-6-phosphate dehydrogenase in Moritz et al. (European Journal of Biochemistry 267, 3442-3452 (2000) described.

The concentration of the protein can be determined by 1 and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software in the gel. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712-23 (2001)). The protein concentration can also be determined by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual, 2 ^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and subsequent optical evaluation with corresponding software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5: 32-39, Lottspeich, Angewandte Chemie 321: 2630-2647 (1999)).

object The invention accordingly relates to methods for overexpression the glucose-6-phosphate dehydrogenases according to the invention. One inventive Method of overexpression consists inter alia in that the copy number of a polynucleotide according to the invention, that for encodes a glucose-6-phosphate dehydrogenase variant in which at position 321 or the corresponding position of the encoded amino acid sequence every proteinogenic amino acid excluding glycine and where appropriate at position 8 or except in the appropriate position any proteinogenic amino acid L-serine is included at least one (1) or multiple copies elevated. Another inventive The method consists of using a promoter functionally linked to the polynucleotide.

object The invention furthermore relates to microorganisms which have an increased concentration or activity the glucose-6-phosphate dehydrogenase according to the invention Have variants in their cell interior.

In addition, can it for the improved production of L-amino acids advantageous be in the mutants of the invention or recombinant strains one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, of amino acid export and optionally overexpress regulatory proteins. The usage endogenous genes are generally preferred.

Under "endogenous Genes "or" endogenous Nucleotide sequences " the genes present in the population of a species Nucleotide sequences or alleles.

• • ein für ein Dihydrodipicolinat-Synthase kodierendes Gen dapA, wie beispielsweise das in der EP 0 197 335 beschriebene dapA-Gen des Wildtyps von Corynebacterium glutamicum,
• • ein für eine Glucose-6-Phosphat Dehydrogenase kodierendes Gen zwf, wie beispielsweise das in der JP-A-09224661 und EP-A-1108790 beschriebene zwf-Gen des Wildtyps von Corynebacterium glutamicum,
• • die in der US-2003-0175911-A1 beschriebenen zwf-Allele von Corynebacterium glutamicum, die für ein Protein kodieren, bei dem beispielsweise das L-Alanin an Position 243 der Aminosäuresequenz durch L-Threonin ersetzt ist oder bei dem die L-Asparaginsäure an Position 245 durch L-Serin ersetzt ist,
• • ein für eine Pyruvat-Carboxylase kodierendes Gen pyc, wie beispielsweise das in der DE-A-198 31 609 und EP 1108790 beschriebene pyc-Gen des Wildtyps von Corynebacterium glutamicum, • das für das in der EP 1 108 790 beschriebene pyc-Allel von Corynebacterium glutamicum, das für ein Protein kodiert, bei dem L-Prolin an Position 458 der Aminosäuresequenz durch L-Serin ersetzt ist,
• • die in der WO 02/31158 beschriebene pyc-Allele von Corynebacterium glutamicum, die für Proteine kodieren, welche gemäß Anspruch 1 einen oder mehrere der Aminosäureaustausche ausgewählt aus der Gruppe L-Glutaminsäure an Position 153 ersetzt durch L-Asparaginsäure, L-Alanin an Position 182 ersetzt durch L-Serin, L-Alanin an Position 206 ersetzt durch L-Serin, L-Histidin an Position 227 ersetzt durch L-Arginin, L-Alanin an Position 452 ersetzt durch Glycin und L-Asparaginsäure an Position 1120 ersetzt durch L-Glutaminsäure tragen (2A von WO 02/31158 gibt zwei verschiedene Startpositionen für die Pyruvat-Carboxylase an, die sich um eine Länge entsprechend 17 Aminosäuren unterscheiden. Dementsprechend entspricht die Position 153 nach Anspruch 1 von WO 02/31158 der Position 170 von 2A von WO 02/31158, die Position 182 nach Anspruch 1 der Position 199 von 2A, die Position 206 nach Anspruch 1 der Position 223 von 2A, die Position 227 nach Anspruch 1 der Position 244 von 2A, die Position 452 nach Anspruch 1 der Position 469 von 2A, die Position 1120 nach Anspruch 1 der Position 1137 von 2B. In 2A von WO 02/31158 ist weiterhin ein Aminosäureaustausch A (Alanin) gegen G (Glycin) an Position 472 angegeben. Die Position 472 des Proteins mit der N terminalen Sequenz MTA entspricht der Position 455 des Proteins mit der N-terminalen Sequenz MST gemäß 2A. In 2B von WO 02/31158 ist weiterhin ein Aminosäureaustausch D (Asparaginsäure) gegen E (Glutaminsäure) an Position 1133 des Proteins mit dem N-Terminus MTA angegeben.),
• • ein für eine Aspartatkinase kodierendes lysC Gen wie beispielsweise das als SEQ ID NO:281 in der EP-A-1108790 (Siehe auch Zugangsnummer AX120085 und 120365) und das als SEQ ID NO:25 in der WO 01/00843 (Siehe Zugangsnummer AX063743) beschriebene von lysC-Gen des Wildtyps von Corynebacterium glutamicum,
• • ein für eine feed-back resistente Aspartatkinase Variante kodierendes lysC^FBR Allel, insbesondere entsprechend Tabelle 1,
• • ein für ein Lysin-Export-Protein kodierendes Gen lysE, wie beispielsweise das in der DE-A-195 48 222 beschriebene lysE-Gen von des Wildtyps Corynebacterium glutamicum,
• • das für das Zwa1-Protein kodierende Gen zwa1 des Wildtyps von Corynebacterium glutamicum ( US 6,632,644 )

überexprimiert werden.Thus, for the production of L-lysine, one or more of the genes selected from the group

• A dapA gene coding for a dihydrodipicolinate synthase, such as that in the EP 0 197 335 described wild-type dapA gene of Corynebacterium glutamicum,
• A gene encoding a glucose-6-phosphate dehydrogenase, such as, for example, the wild-type zwf gene of Corynebacterium glutamicum described in JP-A-09224661 and EP-A-1108790,
• • the zwf alleles of Corynebacterium glutamicum described in US-2003-0175911-A1, which code for a protein in which, for example, the L-alanine at position 243 of the amino acid sequence is replaced by L-threonine or in which the L-aspartic acid replaced at position 245 by L-serine,
• A gene pyc coding for a pyruvate carboxylase, such as, for example, that described in DE-A-198 31 609 and US Pat EP 1108790 described pyc gene of the wild type of Corynebacterium glutamicum, • for the in the EP 1 108 790 described pyc allele of Corynebacterium glutamicum, which codes for a protein in which L-proline at position 458 of the amino acid sequence is replaced by L-serine,
• • the pyc alleles of Corynebacterium glutamicum described in WO 02/31158, which co-proteins according to claim 1, one or more of the amino acid substitutions selected from the group L-glutamic acid at position 153 replaced by L-aspartic acid, L-alanine at position 182 replaced by L-serine, L-alanine at position 206 replaced by L-serine , L-histidine at position 227 replaced by L-arginine, L-alanine at position 452 replaced by glycine and L-aspartic acid at position 1120 replaced by L-glutamic acid ( 2A WO 02/31158 indicates two different starting positions for the pyruvate carboxylase, which differ by a length corresponding to 17 amino acids. Accordingly, position 153 of claim 1 of WO 02/31158 corresponds to position 170 of FIG 2A from WO 02/31158, the position 182 according to claim 1 of the position 199 of 2A the position 206 according to claim 1 of the position 223 of 2A , the position 227 of claim 1 of position 244 of 2A , item 452 according to claim 1 of item 469 of 2A 1120 of claim 1 of item 1137 of 2 B , In 2A WO 02/31158 further discloses an amino acid substitution A (alanine) for G (glycine) at position 472. The position 472 of the protein with the N terminal sequence MTA corresponds to the position 455 of the protein with the N-terminal sequence MST according to FIG 2A , In 2 B WO 02/31158 further discloses an amino acid substitution D (aspartic acid) for E (glutamic acid) at position 1133 of the protein with the N-terminus MTA.),
• A lysC gene coding for an aspartate kinase such as SEQ ID NO: 281 in EP-A-1108790 (see also accession numbers AX120085 and 120365) and SEQ ID NO: 25 in WO 01/00843 (see accession number AX063743 ) of the wild-type lysC gene of Corynebacterium glutamicum,
• A lysC ^FBR allele coding for a feedback-resistant aspartate kinase variant, in particular according to Table 1,
• A lysE gene coding for a lysine export protein, such as, for example, the lysE gene of the wild-type Corynebacterium glutamicum described in DE-A-195 48 222,
• The zwa1 wild-type gene of Corynebacterium glutamicum (Zwa1) coding for the Zwa1 protein ( US 6,632,644 )

be overexpressed.

• • ein für die Glucose-6-Phosphat-Isomerase kodierendes Gen pgi, wie beispielsweise das in der US 6,586,214 und US 6,465,238 beschriebene pgi-Gen von Corynebacterium glutamicum,
• • ein für die Homoserin-Dehydrogenase kodierendes Gen hom, wie beispielsweise das in der EP-A-0131171 beschriebene hom-Gen von Corynebacterium glutamicum,
• • ein für die Homoserin-Kinase kodierendes Gen thrB, wie beispielsweise das von Peoples et al. (Molecular Microbiology 2 (1988): 63–72)) beschriebene thrB-Gen von Corynebacterium glutamicum und
• • ein für die Phosphofruktokinase kodierendes Gen pfkB, wie beispielsweise das in der WO 01/00844 (Sequenz Nr. 57) beschriebene pfkB-Gen von Corynebacterium glutamicum,

abzuschwächen oder auszuschalten.Furthermore, it may be advantageous for the production of L-lysine, in addition to the use of the alleles of the zwf gene according to the invention simultaneously one or more of the endogenous genes selected from the group

• • a gene coding for the glucose-6-phosphate isomerase gene pgi, such as that in the US 6,586,214 and US 6,465,238 described pgi gene of Corynebacterium glutamicum,
• A gene hom coding for the homoserine dehydrogenase, such as, for example, the hom gene of Corynebacterium glutamicum described in EP-A-0131171,
• • a homoserine kinase encoding gene thrB, such as that of Peoples et al. (Molecular Microbiology 2 (1988): 63-72)) described thrB gene of Corynebacterium glutamicum and
• A gene pfkB coding for the phosphofructokinase, such as, for example, the pfkB gene of Corynebacterium glutamicum described in WO 01/00844 (Sequence No. 57),

to attenuate or switch off.

Of the Term "weakening" describes in this Related to the reduction or elimination of intracellular activity of a or more enzymes (proteins) in a microorganism, which are caused by the corresponding DNA are encoded by, for example, a weak promoter used or used a gene or allele, that for encodes a corresponding enzyme with a low activity or the corresponding gene or enzyme (protein) inactivated and where appropriate, these measures combined.

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

Transitions, transversions, insertions and deletions of at least one (1) base pair or nucleotide may be considered as mutations for the generation of an attenuation. Depending on the effect of mutation-induced amino acid exchange on enzyme activity, one speaks of missense mutations or nonsense mutations. The missense mutation results in the replacement of a given amino acid in one protein for another, in particular a non-conservative amino acid substitution. As a result, the functionality or activity of the protein is impaired and to a value of 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% reduced. The nonsense mutation leads to a stop codon in the coding region of the gene and thus to premature termination of translation. Insertions or deletions of at least one base pair in a gene result in frameshift mutations that lead to the incorporation of incorrect amino acids or premature termination of translation, resulting in a stop codon in the coding region as a result of the mutation this also leads to a premature termination of translation.

instructions for generating such mutations belong to the prior art and can known textbooks genetics and molecular biology e.g. the textbook by Knippers ("Molecular genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ( "Gene and Clones ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann's ( "General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986). Further measures are described in the prior art.

The isolated coryneforms obtained by the measures of the invention Bacteria show one compared to the starting strain used or parent strain increased Elimination or production of the desired amino acid in one Fermentation process.

Under isolated bacteria are the inventive, isolated or produced mutants and recombinant bacteria, especially coryneformer Bacteria, which contain a zwf allele, are responsible for a glucose-6-phosphate dehydrogenase encoding the described amino acid substitution at position 321 the amino acid sequence and optionally an amino acid exchange L-serine against another proteinogenic amino acid, preferably L-threonine, at position 8.

The Performance of the isolated bacteria or the fermentation process using the same with respect to one or more of the parameters selected from the group of product concentration (Product by volume), the product yield (product formed per spent carbon source) and product formation (formed carbon Product per volume and time) or other process parameters and combinations thereof, by at least 0.5%, at least 1%, at least 1.5% or at least 2% in relation to the parent strain or parent strain or improves the fermentation process using the same.

The according to the invention, isolated Coryneform bacteria can continuously - like For example, in PCT / EP2004 / 008882 described or discontinuous in the batch process (Sentence cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) for the purpose of Production of L-amino acids be cultivated. A general 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 to be used culture medium or fermentation medium must be in Appropriate to the claims of the respective strains suffice. Descriptions of culture media of various microorganisms are in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms Culture medium and fermentation medium or medium mutually exchangeable.

When Carbon source can Sugars and carbohydrates, e.g. Glucose, sucrose, lactose, fructose, Maltose, molasses, sucrose-containing solutions from sugar beet or cane production, Strength, 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 glycerol, methanol and ethanol and organic acids, such as acetic acid be used. These substances can used singly or as a mixture.

When Nitrogen source can 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 used singly or as a mixture.

When 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 Salts, for example in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium and iron contain, such as magnesium sulfate or iron sulfate, the for the Growth are necessary. After all can essential growth substances such as amino acids, for example, homoserine and vitamins, for example, thiamine, biotin or pantothenic acid in addition to the above substances are used.

the Culture medium can also suitable precursors of the respective amino acid are added.

The mentioned feedstocks can added to culture in the form of a unique approach or in suitable way during fed to the cultivation become.

to pH control of the culture are basic compounds such as sodium hydroxide, Potassium hydroxide, ammonia or ammonia water or acid Compounds such as phosphoric acid or sulfuric acid in used appropriately. The pH is generally set to one Value of 6.0 to 9.0, preferably 6.5 to 8 set. For control the foam development can Antifoams, such as fatty acid polyglycol used become. To maintain the stability of plasmids, the Medium suitable selective substances, such as antibiotics added become. To maintain aerobic conditions, oxygen is used or oxygen-containing gas mixtures, such as air in the culture registered. The use of liquids containing hydrogen peroxide Enriched is also possible. If necessary, will the fermentation at overpressure, for example, at a pressure of 0.03 to 0.2 MPa, driven. The Temperature of the culture is usually 20 ° C to 45 ° C, and preferably 25 ° C to 40 ° C. In batch process the cultivation is continued until a maximum the desired amino acid has formed. This goal will normally be within 10 hours reached up to 160 hours. In continuous processes are longer culture times possible.

Suitable fermentation media are inter alia in the US 6,221,636 , in the US 5,840,551 , in the US 5,770,409 , in the US 5,605,818 , in the US 5,275,940 and in the US 4,224,409 described.

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

• a) ein isoliertes coryneformes Bakterium in einem geeignetem Medium fermentiert, wobei das Bakterium ein für ein Polypeptid mit Glucose-6-Phosphat-Dehydrogenase Enzymaktivität kodierendes Gen enthält, wobei in den Aminosäuresequenzen des Polypeptids das Glycin an der Position 321 oder der entsprechenden Position durch eine andere proteinogene Aminosäure, bevorzugt L-Serin, ersetzt ist und wobei gegebenenfalls das L-Serin an Position 8 oder der entsprechenden Position gegen eine andere proteinogene Aminosäure, bevorzugt L-Threonin, ersetzt ist, und
• b) die L-Aminosäure in der Fermentationsbrühe oder in den Zellen des isolierten coryneformen Bakteriums anreichert.

The invention accordingly provides a process for the preparation of an L-amino acid in which

• a) fermenting an isolated coryneform bacterium in a suitable medium, the bacterium containing a gene coding for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity, wherein in the amino acid sequences of the polypeptide, the glycine at position 321 or the corresponding position by a other proteinogenic amino acid, preferably L-serine, is replaced and where appropriate the L-serine at position 8 or the corresponding position is replaced by another proteinogenic amino acid, preferably L-threonine, and
• b) enriching the L-amino acid in the fermentation broth or in the cells of the isolated coryneform bacterium.

The The fermentation broth produced in this way is subsequently added a solid or liquid Product further processed.

Under a fermentation broth one understands a fermentation medium in which a microorganism for a certain Time and at a certain temperature was cultivated. The fermentation medium or during the The medium used in the fermentation contains / contain all Substances or components that cause an increase of Ensures microorganism and formation of the desired amino acid.

At the conclusion of the fermentation, the resulting fermentation broth accordingly contains a) the biomass of the microorganism resulting from the multiplication of the cells of the microorganism, b) the desired amino acid formed during the fermentation, c) the organic by-products formed during the fermentation and d) the by the fermentation unused components of / the fermentation medium used / fermentation media or the starting materials such as Vitamins such as biotin, amino acids such as homoserine or salts such as magnesium sulfate.

To The organic by-products include substances that are from the optionally adjacent to the fermentation used microorganisms the respective desired L-amino acid produced and optionally excreted. These include L-amino acids, the compared to the desired amino acid less than 30%, 20% or 10%. This still applies organic acids, which carry one to three carboxyl groups such as acetic acid, lactic acid, citric acid, malic acid or Fumaric acid. Finally, belong to it also sugar such as trehalose.

typical for industrial Purpose of suitable fermentation broths have an amino acid content from 40 g / kg to 180 g / kg or 50 g / kg to 150 g / kg. The content of Biomass (as dried biomass) is generally 20 to 50 g / kg.

in the Trap of the amino acid L-lysine is essentially four different in the art Product forms known.

A group of L-lysine containing products comprises concentrated aqueous alkaline solutions of purified L-lysine (EP-B-0534865). Another group, such as in the US 6,340,486 and US 6,465,025 describes aqueous, acidic, biomass-containing concentrates of L-lysine-containing fermentation broths. The most common group of solid products comprises powdered or crystalline forms of purified or pure L-lysine, which is typically in the form of a salt such as L-lysine monohydrochloride. Another group of solid product forms is described for example in EP-B-0533039. The product form described there contains, in addition to L-lysine, the major part of the starting materials used during the fermentative production and not consumed and optionally the biomass of the microorganism used with a proportion of> 0% -100%.

Corresponding of the various product forms one knows various procedures, where the L-amino acid from the fermentation broth collected, isolated or purified to contain the L-amino acid Product or the purified L-amino acid.

For the preparation of solid, pure L-amino acids, essentially methods of ion exchange chromatography are optionally applied using activated carbon and methods of crystallization. In the case of lysine, the corresponding base or a corresponding salt is obtained in this way, for example the monohydrochloride (Lys-HCl) or the lysine sulfate (Lys ₂ -H ₂ SO ₄ ).

in the Case of lysine is in EP-B-0534865 a process for the preparation aqueous, basic L-lysine containing solutions from fermentation broths described. In the method described there, the biomass from the fermentation broth separated and discarded. By means of a base such as Sodium, potassium or ammonium hydroxide will have a pH between 9 to 11 set. The mineral components (inorganic salts) are after concentration and cooling by crystallization from the broth separated and either as a fertilizer used or discarded.

at Process for the preparation of lysine using the bacteria according to the invention preference is given to those processes in which products are obtained, contain the components of the fermentation broth. These will especially used as animal feed additives.

ever Upon request, the biomass may be wholly or partly by separation methods such as. centrifugation, filtration, decanting or a combination thereof is removed from the fermentation broth or completely to be left in her. Optionally, the biomass or the biomass contained fermentation broth during a suitable process step inactivated for example by thermal treatment (heating) or by acid addition.

In In one procedure, the biomass is completely or almost completely removed, so no (0%) or at most 30%, at most 20%, at most 10%, at most 5%, at most 1% or at most 0.1% Biomass remains in the product produced. In a further procedure the biomass is not removed or only in minor proportions so that all (100%) or more than 70%, 80%, 90%, 95%, 99% or 99.9% biomass remains in the product produced. In a method according to the invention Accordingly, the biomass is removed in proportions ≥ 0% to ≤ 100%.

Finally, the fermentation broth obtained after the fermentation before or after the full partial or partial removal of the biomass with an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid or organic acid such as propionic acid are made to an acidic pH ( GB 1,439,728 or EP 1 331 220 ). It is also possible to acidify the fermentation broth with the completely contained biomass ( US 6,340,486 or US 6,465,025 ). Finally, the broth can also be prepared by addition of sodium bisulfite (NaHSO ₃ , GB 1,439,728 ) or another salt, for example ammonium, alkali metal or alkaline earth metal salt of sulfurous acid.

at the separation of the biomass are optionally contained in the fermentation broth organic or inorganic solids partially or completely removed. The in the fermentation broth dissolved organic By-products and the dissolved ones Unused components of the fermentation medium (starting materials) stay at least partially (> 0%), preferably at least 25%, more preferably at least 50% and most preferably at least 75% in the product. Possibly they stay complete (100%) or almost complete that is> 95% or> 98% in the product. In In this sense, the term "fermentation broth base" means that a product contains at least part of the constituents of the fermentation broth.

Subsequently, will the broth with known methods such as e.g. with the aid of a rotary evaporator, thin-film evaporator, Falling film evaporator, by reverse osmosis or by nanofiltration Drained or concentrated or concentrated water. These concentrated fermentation broth can subsequently by freeze drying, spray drying, spray granulation or by other methods such as in the circulating Fluidized bed according to PCT / EP2004 / 006655 described, too free-flowing Products in particular to a finely divided powder or preferably coarse-grained Granules are worked up. Optionally, from the obtained Granulate by sieving or dust separation a desired Product isolated.

It is also possible the fermentation broth directly d. H. without prior concentration by spray drying or spray granulation to dry.

By "free-flowing" is meant powder, the from a series of glass outlet vessels with different sized outlet openings at least from the vessel with the opening 5 mm (millimeters) unrestricted leak (small: soaps, oils, fats, Wachse 94, 12 (1968)).

By "finely divided" is a powder with predominant Share (> 50%) of one Grain size of 20 up to 200 μm diameter meant.

With "coarse-grained" is a product with a prevalent Share (> 50%) of one Grain size from 200 to 2000 μm Meant diameter.

The Particle size determination can be performed with laser diffraction spectrometry methods. The corresponding methods are described in the textbook on particle size measurement in laboratory practice "by R.H. Müller and R. Schuhmann, Scientific Publishing Company Stuttgart (1996) or in the textbook "Introduction to Particle Technology "by M. Rhodes, Publisher Wiley & Sons (1998).

The free-flowing, finely divided powder can in turn by suitable Kompaktier- or Granulation process into a coarse-grained, good flowable, be stored storable and largely dust-free product.

Of the Term "dust-free" means that the product only small proportions (<5 %) on granule sizes smaller than 100 μm in diameter contains.

"Storable", in the sense of this Invention, means a product that is at least one (1) year or so longer, preferably at least 1.5 years or more, more preferably two (2) years or more in a dry and cool environment can be stored without a substantial loss (<5%) of the respective Amino acid occurs.

• a) Kultivierung und Fermentation eines L-Aminosäure ausscheidenden coryneformen Bakteriums, welches mindestens ein zwf-Allel enthält, das für ein Polypeptid mit Glucose-6-Phosphat-Dehydrogenase Aktivität kodiert, welches eine Aminosäuresequenz umfasst, bei der an Position 321 oder der vergleichbaren Position jede proteinogene Aminosäure ausgenommen Glycin, bevorzugt L-Serin enthalten ist, und wobei gegebenenfalls an Position 8 oder der vergleichbaren Position jede proteinogene Aminosäure ausgenommen L-Serin, bevorzugt L-Threonin, enthalten ist, in einem Fermentationsmedium,
• b) Entfernung der während der Fermentation gebildeten Biomasse in einer Menge von 0 bis 100 Gew.-%, und
• c) Trocknung der gemäß a) und/oder b) erhaltenen Fermentationsbrühe, um das Produkt in der gewünschten Pulver- oder Granulatform zu erhalten

wobei gegebenenfalls vor Schritt b) oder c) eine Säure ausgewählt aus der Gruppe Schwefelsäure, Phosphorsäure oder Salzsäure hinzugefügt wird.Another object of the invention is accordingly a process for the preparation of an L-amino acid, preferably L-lysine or L-tryptophan, containing product, preferably animal feed additive, from fermentation broths, characterized by the steps

• a) culturing and fermenting a L-amino acid secreting coryneform bacterium containing at least one zwf allele encoding a polypeptide having glucose-6-phosphate dehydrogenase activity comprising an amino acid sequence at position 321 or the comparable posi each proteinogenic amino acid except glycine, preferably L-serine is contained, and wherein optionally at position 8 or the comparable position each proteinogenic amino acid except L-serine, preferably L-threonine, is contained in a fermentation medium,
• b) removal of the biomass formed during the fermentation in an amount of 0 to 100 wt .-%, and
• c) drying the fermentation broth obtained according to a) and / or b) in order to obtain the product in the desired powder or granular form

wherein optionally before step b) or c) an acid selected from the group sulfuric acid, phosphoric acid or hydrochloric acid is added.

Preferably will contain water from the L-amino acid following step a) or b) fermentation broth removed (concentration).

Advantageous in granulation or compaction is the use of customary organic or inorganic auxiliaries, or carriers such as starch, gelatin, cellulose derivatives or similar substances, such as are commonly used in food or feed processing as binders, gelling or thickening agents, or of other substances such as silicas, silicates ( EP0743016A ) Stearates.

Farther it is beneficial the surface the granules obtained with oils to be provided as described in WO 04/054381. As oils can mineral oils, vegetable oils or Mixtures of vegetable oils be used. examples for such oils are soybean oil, Olive oil, Soybean oil / lecithin mixtures. In the same way, silicone oils, polyethylene glycols or Hydroxyethycellulose suitable. By treating the surfaces with the said oils you get an increased Abrasion resistance of the product and a reduction of the dust content. The content of oil in the product 0.02 to 2.0% by weight, preferably 0.02 to 1.0% by weight, and more particularly preferably 0.2 to 1.0 wt .-% based on the total amount of the feed additive.

Prefers are products in a proportion of ≥ 97 wt .-% of a particle size of 100 up to 1800 μm or a share of ≥ 95 Wt .-% of a grain size of 300 up to 1800 um diameter. The proportion of dust d. H. Particles with a particle size <100 microns is preferred at> 0 to 1% by weight particularly preferably at a maximum of 0.5 wt .-%.

alternative But the product can also be applied to feed processing known and usual organic or inorganic carrier such as silicas, silicates, Crumbs, brans, flours, starches Sugar or other grown and / or with conventional thickening or binding agents mixed and stabilized. Application examples and methods For this purpose, the literature (Die Mühle + Mischfuttertechnik 132 (1995) 49, page 817).

Finally, can the product also by coating processes ("coating") with film formers such as Metal carbonates, silicas, Silicates, alginates, stearates, starches, Gums and cellulose ethers as described in DE-C-4100920, be brought into a state in which it is stable against the Digestion by animal stomachs especially the stomach of ruminants.

to Setting a desired amino acid concentration in the product, depending on the requirement, the corresponding amino acid can be used during the Process in the form of a concentrate or possibly a largely pure substance or its salt in liquid or added to solid form become. these can individually or as mixtures to the obtained or concentrated Fermentation broth or even while be added to the drying or granulation process.

In the case of lysine, in the production of lysine-containing products, the ratio of the ions is adjusted so that the ion ratio according to the following formula 2 × [SO ₄ ^2- ] + [Cl ^- ] - [NH ₄ ⁺ ] - [Na ⁺ ] - [K ⁺ ] - 2 × [Mg ⁺ ] - 2 × [Ca ²⁺ ] / [L-Lys] 0.68 to 0.95, preferably 0.68 to 0.90, as described by Kushiki et al. in the US 20030152633 described.

In the case of lysine, the fermentation broth-based solid product thus prepared has a lysine content (as lysine base) of from 10% to 70% or 20% to 70% by weight 30 wt .-% to 70 wt .-% and most preferably from 40 wt .-% to 70 wt .-% based on the dry weight of the product. Maximum levels of lysine base of 71% by weight, 72% by weight, 73% by weight are also possible.

in the Trap of an electrically neutral amino acid such as L-tryptophan has the fermentation broth-based solid product thus prepared an amino acid content of at least 5% by weight, 10% by weight, 20% by weight, 30% by weight and maximum 50 wt%, 60 wt%, 70 wt%, 80 wt%, 90 wt%, or up to 95 Wt .-%.

Of the Water content of the solid product is up to 5 wt .-%, preferably up to 4% by weight, and more preferably less than 3% by weight.

A Mutant of Corynebacterium glutamicum designated DM1797, the amino acid exchange lysC T311I in aspartate kinase was published on October 28, 2004 at the German Collection for Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) deposited as DSM 16833.

The mutant according to the invention Corynebacterium glutamicum DM1816, the L-serine at position 321 of the amino acid sequence of the Zwf polypeptide, was on 9 February 2005 at the German Collection of Microorganisms and cell cultures (DSMZ, Braunschweig, Germany) as DSM 17119 deposited.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (64)

Isolated mutants of coryneform bacteria which contain a gene coding for a polypeptide having glucose-6-phosphate dehydrogenase activity, characterized in that the polypeptide comprises an amino acid sequence in which at position 321 or a corresponding or comparable position each proteinogenic amino acid, excluding glycine. Mutant coryneform bacteria according to claim 1, characterized in that it is in the coryneform bacteria selected for a bacterium from the group Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium thermoaminogenes and Corynebacterium aminogenes is. Mutant coryneform bacteria according to claim 2, characterized in that it is Corynebacterium glutamicum is. Mutant coryneform bacteria according to claim 1, 2, or 3, characterized in that it is L-amino acid leaving Bacteria act. Mutant coryneform bacteria according to claim 4, characterized in that it is L-lysine or L-tryptophan leaving bacteria. Mutant coryneform bacteria according to claim 1 to 5, characterized in that the encoded polypeptide to Item 321 or a comparable item contains L-serine. Mutant coryneform bacteria according to claim 1 to 5, characterized in that the encoded polypeptide the amino acid sequence of SEQ ID NO: 2, wherein at position 321 any proteinogenic amino acid except glycine is included. Mutant coryneform bacteria according to claim 7, characterized in that the amino acid L-serine at position 8 against another proteinogenic amino acid is exchanged. Mutant coryneform bacteria according to claim 1 to 8, characterized in that the gene comprises a nucleotide sequence identical to the nucleotide sequence of a polynucleotide consisting of a polymerase chain reaction (PCR) using DNA obtained from a coryneform bacterium and using a primer pair of a first primer comprising at least 15 contiguous nucleotides selected from the nucleotide sequence between position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11, and a second primer comprising at least 15 contiguous nucleotides selected from the complementary nucleotide sequence between position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11. Mutant coryneform bacteria according to claim 1 to 6 or 9, characterized in that the encoded polypeptide an amino acid sequence with a length corresponding to 514 L-amino acids includes. Mutant coryneform bacteria according to claim 1 to 6 or 9 to 10, characterized in that the coded Polypeptide from position 312 to 330 of the amino acid sequence, the amino acid sequence corresponding to positions 312 to 330 of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 contains. Mutant coryneform bacteria according to claim 1 to 6 or 9 to 11, characterized in that the coded Polypeptide an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. Mutant coryneform bacteria according to claim 1 to 6 or 9 to 11, characterized in that the gene a Nucleotide sequence which is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7. Mutant coryneform bacteria according to claim 6, characterized in that the encoded protein has an amino acid sequence selected from the group a) Amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, b) Amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 including one or more conservative Amino acid substitution (s), and c) amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 including one or more insertions or deletions of amino acids, includes. Mutant coryneform bacteria according to claim 7 or 8, characterized in that the amino acid sequence of SEQ ID NO: 2 at position 321 contains L-serine. Mutant coryneform bacteria according to claim 15, characterized in that the gene is the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. Mutant coryneformer bacteria, characterized that these are available through the following steps: a) treatment a coryneform bacterium that has the ability to excrete amino acids with a mutagenic agent, b) isolation and propagation of in a) generated mutant, c) providing nucleic acid the mutant obtained in b), d) Preparation of a nucleic acid molecule under Use of the polymerase chain reaction, the nucleic acid c), and a primer pair consisting of a first primer comprising at least 15 consecutive nucleotides selected from the nucleotide sequence between position 1 and 1267 of SEQ ID NO: 3 or SEQ ID NO: 11 and a second primer comprising at least 15 consecutive nucleotides selected from the complementary nucleotide sequence between positions 2100 and 1271 of SEQ ID NO: 3 or 11, e) Determination of the nucleotide sequence of the nucleic acid molecule obtained in d), and Determination of the coded amino acid sequence, f) if necessary, comparison of the amino acid sequence determined in f) with SEQ ID NO: 6, 8 or 10, and g) identification of a mutant, which contains a polynucleotide, that for a polypeptide encoding at position 321 or comparable Position any proteinogenic amino acid except glycine, preferred L-serine, contains. Mutant coryneform bacteria according to claim 17, characterized in that following step b) a Mutant selected that will be the ability has in a medium at least 0.5% more L-amino acid excrete or inside the cell to accumulate as the coryneform bacterium used in step a) of claim 17. An isolated polynucleotide encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which at position 321 of the amino acid sequence excludes any proteinogenic amino acid Contains glycine. Isolated polynucleotide according to claim 19, characterized in that that the proteinogenic amino acid is L-serine. Isolated polynucleotide according to claim 19, characterized in that the encoded polypeptide has the amino acid sequence of SEQ ID NO: 2 at position 321 of the amino acid sequence, each proteinogenic amino acid except glycine is included. Isolated polynucleotide according to claim 21, characterized in that the encoded polypeptide at position 8 excludes any proteinogenic amino acid Contains L-serine. An isolated polynucleotide according to claim 19 or 20, characterized in that the encoded polypeptide has an amino acid sequence with a length of 514 amino acids includes. Isolated polynucleotide according to claim 20, characterized that the encoded polypeptide from position 324 to 334 of the amino acid sequence the amino acid sequence corresponding to position 324 to 334 of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 contains. An isolated polynucleotide according to claim 19 or 20, characterized characterized in that it is linked to the nucleotide sequence of a polynucleotide identical, which by a polymerase chain reaction (PCR) under Use of DNA obtained from a coryneform bacterium and using a primer pair consisting of a first primer, comprising at least 15 consecutive nucleotides selected from Nucleotide sequence between position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11, and a second primer comprising at least 15 successive nucleotides selected from the complementary nucleotide sequence between position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11 available is. An isolated polynucleotide according to claim 19 or 20, characterized in that it is identified by SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 complementary Nucleotide sequence hybridized under stringent conditions. An isolated polynucleotide according to claim 19 or 20, characterized in that the encoded polypeptide has an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. An isolated polynucleotide according to claim 19 or 20, characterized characterized in that the polynucleotide comprises a nucleotide sequence, which is at least 90% identical to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. An isolated polynucleotide according to claim 19 or 20, characterized in that the encoded protein has an amino acid sequence selected from the group a) Amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, b) Amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 including one or more conservative Amino acid substitution (s), and c) amino acid sequence according to SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 including one or more insertions or deletions of amino acids includes. Isolated polynucleotide according to one or more of claims 21, 22 or 29, characterized in that the encoded polypeptide the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. Isolated polynucleotide according to claim 30, characterized in that in that the nucleotide sequence is SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 includes. Isolated polynucleotide according to Claim 31, characterized the nucleotide sequence comprises SEQ ID NO: 11. An isolated polynucleotide comprising a nucleic acid molecule encoding at least one reading frame having an amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 2, wherein the position corresponding to position 321 of SEQ ID NO: 2 is any proteinogenic amino acid except glycine. Isolated polynucleotide according to Claim 33, characterized that it is at least for a reading frame with an amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 encoded. Isolated polynucleotide according to Claim 33, characterized that the amino acid sequence is a or more conservative amino acid substitutions contains wherein the conservative amino acid substitutions do not affect item 321. Isolated polynucleotide according to Claim 33, characterized that it contains one or more silent mutations. Isolated polynucleotide according to Claim 34, characterized that there is at least the nucleotide sequence corresponding to position 919 to 1005 of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. An isolated polynucleotide containing the nucleotide sequence between position 208 and 299 of SEQ ID NO: 11. Process for producing a recombinant coryneform Bacterium, characterized in that one a) an isolated one Polynucleotide according to claim 19 to 32 or according to claim Transferred 33 to 37 into a coryneform bacterium, b) that in the chromosome of the coryneform bacterium existing glucose-6-phosphate dehydrogenase Gene that for an amino acid sequence with glycine at position 321 and optionally L-serine at position 8 or a comparable position of the amino acid sequence against the polynucleotide from a) exchanges that for an amino acid sequence which encodes another L-amino acid at the Position 321 and optionally another amino acid on the Has position 8 or a comparable position, and c) that according to step a) and b) increased coryneform bacterium. Method according to claim 39, characterized in that the isolated polynucleotide according to claim 19 to 32 used. Method according to claim 39, characterized in that the isolated polynucleotide according to claim 33 to 37 used. Method according to claim 39, characterized in that it is in position at the other L-amino acid 321 is L-serine and that the other amino acid is in position 8 is L-threonine. Process for producing a recombinant coryneform Bacterium, characterized in that one a) the isolated one Polynucleotide according to claim 38 converted into a coryneform bacterium, b) those in the chromosome of the coryneform bacterium nucleotide sequence present according to position 208 to 299 of SEQ ID NO: 3 against the isolated polynucleotide a) exchanges, and c) the product obtained according to step a) and b) Recombinant coryneform bacterium multiplies. Process for the preparation of a recombinant microorganism, characterized in that one a) an isolated polynucleotide according to claim 19 to 33, transferred to a microorganism, b) the isolated one Polynucleotide replicated in the microorganism, and c) the according to step a) and b) increased microorganism. A recombinant microorganism that isolated the Polynucleotide according to claim 19 to 33 or 34 to 37 contains. Recombinant microorganism according to claim 45, characterized in that it is a coryneform bacterium or a bacterium of the genus Escherichia. Recombinant microorganism according to claim 46, characterized in that it is in the Coryneform bacterium to the genus Corynebacterium acts. Recombinant microorganism according to claim 47, characterized in that it is in the bacterium of the genus Corynebacterium is the species Corynebacterium glutamicum. A vector containing the isolated polynucleotide according to claim 19 to 33 or 34 to 37 or 38. A recombinant microorganism containing the vector according to claim 49 contains. Recombinant microorganism according to claim 50, characterized in that it is a coryneform bacterium or a bacterium of the genus Escherichia. Recombinant microorganism according to claim 51, characterized in that it is in the coryneform bacterium is the genus Corynebacterium. Recombinant microorganism according to claim 52, characterized in that it is in the bacterium of the genus Corynebacterium is the species Corynebacterium glutamicum. Method of overexpression a glucose-6-phosphate dehydrogenase in a microorganism, characterized in that the copy number a polynucleotide according to claim 19 to 33 in a microorganism by at least one (1) copy elevated. Method of overexpression a glucose-6-phosphate dehydrogenase in a microorganism, characterized in that one Promoter or an expression cassette functional with a polynucleotide connected, that for encodes a polypeptide having glucose-6-phosphate dehydrogenase activity, at position 321 or at comparable position of the amino acid sequence every proteinogenic amino acid excluding glycine, and where appropriate Position 8 or comparable position excludes any proteinogenic amino acid L-serine is included. A process for producing an L-amino acid thereby marked that one a) an isolated coryneform bacterium fermented in a suitable medium, the bacterium being at least a copy one for a polypeptide encoding glucose-6-phosphate dehydrogenase enzyme activity Contains gene, wherein in the amino acid sequences of the polypeptide glycine at position 321 or comparable Position and optionally the L-serine at position 8 or the like Position replaced by another proteinogenic amino acid, and b) the L-amino acid in the fermentation broth or in the cells of the bacteria. Method according to claim 56, characterized in that it is in the isolated coryneform bacterium to a mutant according to claim 1 to 18 is. Method according to claim 56, characterized in that it is in the isolated coryneform bacterium is a recombinant coryneform bacterium that is an isolated bacterium Polynucleotide according to claim 19 to 37 contains or using the same. Method according to claim 56, characterized in that that one is the L-amino acid isolated or collect 60. The method according to claim 59, characterized that one is the L-amino acid cleans. Method according to claim 56, characterized in that that one is the L-amino acid together with constituents from the fermentation broth and / or the biomass (> 0 to 100%) isolated or collected. Method according to claim 56, characterized in that that he a) from that obtained in step b) of claim 55 fermentation broth removes the formed biomass in an amount of 0 to 100%, and b) from the broth obtained in step a) a substantially dry and shaped product by a method selected from the group granulation, Compaction, spray drying and extrusion. Method according to claim 62, characterized in that that one of the fermentation broth before or after step a) an acid selected from the group sulfuric acid, hydrochloric acid and phosphoric acid adds. Method according to claim 62, characterized in that from the broth obtained before or after step a) away. Procedure according to step 62, characterized in that the in or during step b) molded product obtained sprayed with an oil.