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WO2006100177A1 - Alleles mutes du gene zwg (g6pdh) issus de bacteries coryneformes destines a augmenter la production de lysine - Google Patents

Alleles mutes du gene zwg (g6pdh) issus de bacteries coryneformes destines a augmenter la production de lysine Download PDF

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Publication number
WO2006100177A1
WO2006100177A1 PCT/EP2006/060519 EP2006060519W WO2006100177A1 WO 2006100177 A1 WO2006100177 A1 WO 2006100177A1 EP 2006060519 W EP2006060519 W EP 2006060519W WO 2006100177 A1 WO2006100177 A1 WO 2006100177A1
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seq
amino acid
acid sequence
isolated polynucleotide
nucleotide sequence
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Brigitte Bathe
Natalie Schischka
Georg Thierbach
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Evonik Operations GmbH
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Degussa GmbH
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Priority to EP06725146A priority Critical patent/EP1861493B8/fr
Priority to PL06725146T priority patent/PL1861493T3/pl
Priority to PCT/EP2006/060851 priority patent/WO2006100211A1/fr
Priority to ES06725146T priority patent/ES2394918T3/es
Priority to DK06725146.2T priority patent/DK1861493T3/da
Publication of WO2006100177A1 publication Critical patent/WO2006100177A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan

Definitions

  • the invention relates to mutants and alleles of the zwf gene coryneformer bacteria encoding variants of the Zwf subunit of glucose-6-phosphate dehydrogenase (EC: 1.1.1.49) and methods for the preparation of amino acids, in particular L-lysine and L-tryptophan using bacteria that contain these alleles.
  • Amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and especially in animal nutrition.
  • Process improvements may include fermentation measures such as stirring and oxygen supply, or nutrient media composition, such as sugar concentration during fermentation, or product form processing by, for example, ion exchange chromatography or the intrinsic performance characteristics of the microorganism itself.
  • Microorganisms are used methods of mutagenesis, selection and mutant selection. In this way, one obtains strains that are resistant to antimetabolites or auxotrophic for regulatory metabolites and produce the amino acids.
  • One known antimetabolite is the lysine analog S- (2-aminoethyl) -L-cysteine (AEC).
  • the microbial biosynthesis of L-amino acids in coryneform bacteria is a complex system and multi-layered with various other metabolic pathways in the cell. Therefore, it can not be predicted which mutation alters the catalytic activity of glucose-6-phosphate dehydrogenase such that the production of L-amino acids is improved. It is therefore it is desirable to have further variants of glucose-6-phosphate dehydrogenase available.
  • nucleotide sequence of the coding for the glucose-6-phosphate dehydrogenase or for the Zwf subunit of the glucose-6-phosphate dehydrogenase from Corynebacterium glutamicum zwf gene ("wild-type gene") according to the information of NCBI database in SEQ ID NO: 1 and the resulting amino acid sequence of the encoded glucose-6-phosphate dehydrogenase are shown in SEQ ID NO: 2 and 4.
  • SEQ ID NO: 3 are upstream and downstream nucleotide sequences additionally indicated.
  • the inventors have set themselves the task of providing new measures for the improved production of amino acids, in particular L-lysine and L-tryptophan.
  • the invention relates to generated or isolated mutants of coryneform bacteria which preferentially excrete amino acids and which contain a gene or allele which codes 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 of the amino acid sequence, each proteinogenic amino acid except glycine is contained. Replacement of glycine with L-serine is preferred.
  • the polypeptide contained in the mutants of the present invention may also be referred to as Zwf polypeptide or Zw-subunit of glucose-6-phosphate dehydrogenase.
  • amino acid-secreting strains which are based on the following types:
  • Corynebacterium efficiens such as the strain DSM44549,
  • Corynebacterium glutamicum such as strain ATCC13032,
  • thermoaminogenes such as strain FERM BP-1539, and
  • Corynebacterium ammoniagenes such as strain ATCC6871,
  • Corynebacterium glutamicum Some representatives of the species Corynebacterium glutamicum are also known in the art under other species. These include, for example:
  • Corynebacterium glutamicum BPS13 FermBP-1777
  • Corynebacterium glutamicum FermBP-3055 described in US 5,235,940.
  • ATCC American Type Culture Collection
  • DSM German Collection of Microorganisms and Cell Cultures
  • FERM European Collection of Microorganisms and Cell Cultures
  • the 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.
  • Proteinogenic amino acids are the amino acids found in natural proteins, ie proteins of microorganisms, plants, animals and humans. 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 L-amino acids also include L-homoserine.
  • the mutants according to the invention preferably excrete said proteinogenic amino acids, in particular L-lysine and L-tryptophan.
  • amino acids also includes their salts such as, for example, the lysine monohydrochloride or lysine sulfate in the case of the amino acid L-lysine.
  • the invention further provides mutants of coryneform bacteria containing a zwf allele which encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity comprising the nucleic acid sequence of SEQ ID NO: 2 excluding at position 321 any proteinogenic amino acid Glycine is included. Replacement of glycine with L-serine is preferred.
  • the amino acid sequence of the polypeptide further contains at position 8 an amino acid substitution L-serine for another proteinogenic amino acid, preferably L-threonine.
  • the invention furthermore relates to mutants of coryneform bacteria which contain a zwf allele which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which is active at position 321 of the
  • Amino acid sequence of SEQ ID NO: 2 containing any proteinogenic amino acid except glycine, preferably L-serine, the gene comprising a nucleotide sequence which is identical to the nucleotide sequence of a polynucleotide by a polymerase chain reaction (PCR) is obtainable using a primer pair whose nucleotide sequences each comprise at least 15 consecutive nucleotides, which consists of 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 can be selected. Examples of such suitable primer pairs are shown in SEQ ID NO: 17 and SEQ ID NO: 18 and in SEQ ID NO: 19 and SEQ ID NO: 20.
  • Chromosomal DNA of coryneform bacteria which have been treated in particular with a mutagen, is preferred as the starting material ("template" DNA). Particularly preferred is the chromosomal DNA of the genus Corynebacterium, and very particularly preferably of the species Corynebacterium glutamicum.
  • the invention further provides mutants of coryneform bacteria containing a zwf allele encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity comprising a nucleic acid sequence having a length corresponding to 514 L-amino acids, each proteinogenic at position 321 Amino acid except glycine, preferably L-serine, is included.
  • the amino acid sequence of the polypeptide further comprises an amino acid substitution of L-serine for another proteinogenic amino acid, preferably L-threonine, at position 8.
  • the invention furthermore relates to mutants of coryneform bacteria which contain a zwf allele which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which at position 312 to 330 of the amino acid sequence corresponds to the amino acid sequence corresponding to position 312 to 330 of SEQ ID NO : 6 or 8 contains.
  • the amino acid sequence of the encoded polypeptide contains an amino acid sequence corresponding to position 307 to 335 of SEQ ID NO: 6 or 8 or position 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 positions 247 to 395 of SEQ ID NO: 6 or 8 or position 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 positions 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 positions 2 to 514 of SEQ ID NO: 6 or 8.
  • the length of the encoded polypeptide comprises 514 Amino acids.
  • the invention furthermore relates to mutants of coryneform bacteria which contain a zwf allele which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which contains, at position 321 or at the corresponding position of the amino acid sequence, any amino acid except glycine, the
  • replacement with L-serine is preferred and its amino acid sequence is also identical to 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% Amino acid sequence of SEQ ID NO: 6.
  • An example of an amino acid sequence having at 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 the amino acid substitution at position 321, the amino acid substitution L-serine for L-threonine at position 8.
  • the invention furthermore relates to mutants of coryneform bacteria which contain a zwf allele which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which is active at position 321 or at the corresponding position of the Amino acid sequence contains any amino acid except glycine, preference being given to replacement against L-serine and its nucleotide sequence 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 98th % or at least 99% is identical to the nucleotide sequence of SEQ ID NO: 5.
  • An example of 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 shown.
  • Nucleotide sequence of this zwf allele has the nucleotide exchange thymine to adenine at position 22 (see SEQ ID NO: 7) in addition to the nucleotide exchange guanine to adenine at position 961 (see SEQ ID NO: 5).
  • SEQ ID NO: 9 Another example of 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: 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).
  • the zwf allele coding for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity contained in the mutants of the present invention may have, in addition to the amino acid sequence shown in SEQ ID NO: 6 and SEQ ID NO: 8 or SEQ ID NO: 10 contain one (1) or more conservative amino acid substitutions.
  • the polypeptide contains at most two (2), at most three (3), at most four (4) or at most five (5) conservative amino acid substitutions.
  • the aromatic amino acids are called conservative exchanges when phenylalanine, tryptophan and tyrosine are interchanged.
  • the hydrophobic amino acids are called conservative exchanges when leucine, isoleucine and valine are exchanged.
  • the polar amino acids are called conservative exchanges when glutamine and asparagine are interchanged.
  • the basic amino acids are called conservative exchanges when arginine, lysine and histidine are interchanged.
  • the acidic amino acids are called conservative exchanges when aspartic acid and glutamic acid are exchanged.
  • the hydroxyl-containing amino acids are called conservative substitutions when serine and threonine are interchanged.
  • An example of a conservative amino acid exchange is the exchange serine for threonine at position 8 of SEQ ID NO: 6, which leads to the amino acid sequence according to SEQ ID NO: 8 or SEQ ID NO: 10.
  • those mutants of coryneform bacteria containing a zwf allele coding for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which has at least one amino acid sequence selected from the group consisting of Val-Ile-Phe-Gly-Ali-AfA are preferred.
  • 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 the corresponding or comparable position of the amino acid sequence, any amino acid except glycine, preferably L-serine.
  • the amino acid sequence further contains an amino acid substitution L-serine for another proteinogenic amino acid, preferably L-threonine, at position 8 of SEQ ID NO: 2.
  • the amino acid sequence motif Val-Ile-Phe-Gly-Val-Thr-Gly-Asp-Leu is included, for example, in SEQ ID NO: 6, 8 or 10 from position 32 to 40.
  • 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.
  • the invention finally provides mutants of coryneform bacteria which contain a zwf allele which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO : 10 includes. It is known that by endogenous enzymes, so-called aminopeptidases, the terminal methionine is removed during protein synthesis.
  • a position corresponding to position 321 of the amino acid sequence or "a position comparable to position 321 of the amino acid sequence” is meant the fact that by inserting or deleting an amino acid coding codon in the N-terminal region (relative to the position 321 of SEQ ID NO: 6, 8 or 10) of the encoded polypeptide
  • the position and length are formally increased by one unit or reduced by one unit in the case of a deletion.
  • the L-serine moves from position 321 to position 320.
  • the length would then be: 513 amino acids.
  • insertion or deletion of a codon coding for an amino acid in the C-terminal region (relative to position 321) of the encoded polypeptide formally increases the length in the case of insertion, or decreases by one in the case of deletion.
  • Such comparable positions can be easily identified by comparison of the amino acid sequences in the form of "alignments", for example with the aid of the Clustal program.
  • the subject of the invention are also zwf alleles which, for polypeptide variants of SEQ ID NO: 6 or 8 or encode 10, containing one or more insertion (s) or deletion (s).
  • the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3 or a maximum of 2 insertions or deletions of amino acids.
  • mutants of the invention classical in vivo mutagenesis methods can be used with cell populations of coryneform bacteria using mutagenic substances such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), 5-bromouracil, or ultraviolet light become.
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • 5-bromouracil 5-bromouracil
  • ultraviolet light ultraviolet light become.
  • MNNG Use of MNNG include concentrations of 50 to 500 mg / l or even higher concentrations up to a maximum of 1 g / l, an incubation time of 1 to 30 minutes at a pH of 5.5 to 7.5. Under these conditions, the number of viable cells is reduced by a proportion of about 50% to 90% or about 50% to 99% or about 50% to 99.9% or more.
  • mutants are identified that in comparison to the parent strain or non-mutagenized parent strain increased amino acids in the nutrient medium or in the
  • Excrete cell interior include, for example, those mutants whose amino acid secretion is increased by at least 0.5%.
  • DNA is made available from the mutants or isolated and purified with the aid of the polymerase
  • the DNA is isolated from those mutants which excrete amino acids in an increased manner.
  • any primer pairs from the nucleotide sequence located upstream and downstream of the mutation according to the invention and the complementary thereto are provided.
  • Nucleotide sequence can be selected.
  • a primer of a primer pair here 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 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 positions 2100 and 1271 SEQ ID NO: 3 or SEQ ID NO: 11.
  • the primer pair is preferably selected 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 selected. If amplification of a portion of the coding region, as shown, for example, in SEQ ID NOS: 14 and 15, is desired, the primer pair is preferably selected from the nucleotide sequence between position 309 and 1267 of SEQ ID NO: 3 or SEQ ID NO: 11 and of complementary nucleotide sequence between position 1848 and 1271 of SEQ ID NO: 3 or SEQ ID NO: 11 selected.
  • primer pairs examples include the primer pair zwf-Kl and zwf-K2 reproduced under SEQ ID NO: 17 and SEQ ID NO: 18, or the primer pair zwf-L1 and zwf-L2 reproduced under SEQ ID NO: 19 and SEQ ID NO: 20.
  • the primer can also be equipped with recognition sites for restriction enzymes, with a biotin group or other accessories as described in the prior art.
  • the total length of the primer is generally at most 30, 40, 50 or 60 nucleotides.
  • thermostable DNA polymerases are used ("template DNA") by amplification by means of PCR
  • template DNA examples of such DNA polymerases are the Taq polymerase from Thermus aquaticus, which is sold, inter alia, by the company Qiagen (Hilden, Germany) Vent polymerase from Thermococcus litoralis, which is sold, inter alia, by the company New England Biolabs (Frankfurt, Germany) or the Pfuc polymerase from Pyrococcus furiosus, which is marketed inter alia by the company Stratagene (La Jolla, USA) Polymerases with "proof-reading" activity.
  • the conditions in the reaction mixture are set according to the manufacturer.
  • the polymerases are generally used by the manufacturer together with the usual ones
  • Buffer which usually has concentrations of 10 - 100 rtiM Tris / HCl and 6 - 55 rtiM KCl at pH 7.5 - 9.3. Magnesium chloride is added at a concentration of 0.5-10 mM if it is not included in the manufacturer's supplied buffer. Deoxynucleoside triphosphates are also added to the reaction mixture in a concentration of 0.1-16.6 rtiM.
  • the primers are presented in the reaction mixture with a final concentration of 0.1-3 ⁇ M and the template DNA in the optimal case with 10 2 to 10 5 copies. You can also use 10 6 to 10 7 copies.
  • the corresponding polymerase is added to the reaction mixture in an amount of 2-5 units. A typical reaction mixture has a volume of 20-100 ⁇ l.
  • bovine serum albumin Tween-20, gelatin, glycerol, formamide or DMSO may be added to the reaction (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.
  • Extension a synthesis step to extend the presented primer ("Extension") at the respective optimum activity for the polymerase, usually depending on the polymerase in the range of 73 0 C to 67 0 C preferably 72 0 C to 68 0 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.
  • the nucleotide sequence is then sequenced, for example, by the chain termination method of Sanger et al. (Proceedings of the National Academys of Sciences, USA, 74, 5463-5467 (1977)) with those of Zimmermann et al. (Nucleic Acids Research 18, 1067pp (1990)) and encoded by this nucleotide sequence Polypeptide analyzed in particular with respect to the amino acid sequence.
  • the nucleotide sequence is entered into a program for the translation of DNA sequence into an amino acid sequence.
  • Suitable programs include, for example, the patent program available from patent offices, such as the United States Patent Office (USPTO), or the Translate Tool available on the ExPASy Proteomics Server on the World Wide Web (Gasteiger et al., Nucleic Acids Research 31, 3784-3788 (2003)).
  • USPTO United States Patent Office
  • Translate Tool available on the ExPASy Proteomics Server on the World Wide Web
  • Alleles encode polypeptides having glucose-6-phosphate dehydrogenase enzyme activity which at position 321 of the amino acid sequence, or the corresponding or comparable position, contain any proteinogenic amino acid except glycine. Preference is given to replacement with L-serine.
  • the amino acid sequence further contains an amino acid substitution L-serine for another proteinogenic amino acid, preferably L-threonine, at position 8 or at the corresponding or comparable position.
  • the invention accordingly provides a mutant of a coryneform bacterium, characterized in that it is obtainable by the following steps:
  • mutants generated in this manner typically contain one (1) copy of the described zwf allele.
  • 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 the coding for the amino acid glycine GGC codon.
  • SEQ ID NO: 5 at position 961 contains the nucleobase adenine.
  • Adenine transition arises at position 961 to 963 the coding for the amino acid L-serine AGC codon.
  • 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 accordingly contains at positions 22 to 24 the coding for the amino acid serine TCC codon. SEQ ID NO: 7 contains at position 22 the nucleobase adenine. By this thymine adenine transversion is formed at position 22 to 24 coding for the amino acid L-serine AGC codon.
  • nucleotide sequences shown in SEQ ID NO: 5 and 7 may contain further base exchanges, which have resulted from the mutagenesis treatment, but do not manifest themselves in an altered amino acid sequence. Such mutations are also referred to in the art as silent or neutral mutations. These silent mutations may also be included in the coryneform bacterium used for mutagenesis treatment. Examples of such silent mutations are the cytosine-thymine transition at 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.
  • coryneform bacteria used for the mutagenesis preferably already have the ability to excrete the desired amino acid into the surrounding nutrient medium or fermentation broth or to enrich it in the cell interior.
  • Coryneform bacteria producing L-lysine typically have a "feed back” resistant or desensitized aspartate kinase.
  • "Feed back” resistant aspartate kinases are aspartate kinases, which are lower than wild type
  • lysC FBR alleles The genes or alleles coding for these desensitized aspartate kinases are also referred to as lysC FBR alleles.
  • Table 1 describes numerous lysC FBR alleles encoding aspartate kinase variants which have amino acid changes compared to the wild type protein.
  • 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.
  • L-lysine-secreting coryneform bacteria typically have one or more of the amino acid changes listed in Table 1.
  • lysC FBR alleles 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 against Valine), lysC S301F (replacement of serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 22 by phenylalanine), lysC T308I (replacement of threonine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 22 by isoleucine), lysC S301Y ( Exchange of serine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for tyrosine), lysC G345D (
  • Aspartate kinase protein according to SEQ ID NO: 22 against glycine lysC T311I (exchange of threonine at position 311 of the encoded aspartate kinase protein according to SEQ ID NO: 22 for isoleucine), lysC S381F (replacement of serine at position 381 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against phenylalanine) and lysC S317A (replacement of serine at position 317 of the encoded aspartate kinase protein according to SEQ ID NO: 22 against alanine).
  • lysC FBR Allel lysC T311I exchange of threonine at position 311 of the coded
  • 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 (replacement 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.
  • mutant DM1816 contains a zwf allele coding for a polypeptide containing L-serine at position 321 of the amino acid sequence.
  • the nucleotide sequence of the coding region of the zwf allele of the mutant DM1816 is shown as SEQ ID NO: 9 and the amino acid sequence of the encoded polypeptide as SEQ ID NO: 10 and 12, respectively.
  • the mutant DM1816 also contains nucleotide exchanges in the nucleotide sequence between position 1 and 307 of SEQ ID NO: 3. These nucleotide exchanges are shown in SEQ ID NO: 11.
  • SEQ ID NO: 11 contains at position 208 guanine instead of 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 further contains at position 329 adenine instead of thymine, at position 445 thymine instead of 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 Guanin.
  • L-lysine-secreting coryneform bacteria may be used which have an attenuated homoserine dehydrogenase or homoserine kinase or have other properties as known in the art.
  • L-tryptophan-producing coryneform bacteria typically have a "feed back” resistant or desensitized anthranilate synthase. "Feed back” resistant anthranilate synthase is understood
  • Anthranilate synthases which have a lower susceptibility to inhibition (5 to 10%, 10% to 15% or 10% to 20%) by tryptophan or 5-fluorotryptophan compared to the wild type (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 described, for example, in US Pat. No. 6,180,373 and EP0338474.
  • the mutants obtained show an increased excretion or production of the desired amino acid in a fermentation process compared to the starting strain or parent strain used.
  • the invention also provides an isolated polynucleotide which encodes a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which at position 321 or at a corresponding or comparable position of the amino acid sequence contains any proteinogenic amino acid except glycine; Serine is preferred.
  • the polynucleotide according to the invention can be isolated from a mutant according to the invention.
  • in vitro methods can be used for the mutagenesis of the zwf gene.
  • isolated polynucleotides which contain a coryneform bacterium gene, preferably the wild type gene of Corynebacterium glutamicum described in the prior art, are subjected to mutagenic treatment.
  • the isolated polynucleotides can be, for example, isolated total DNA or chromosomal DNA or also amplificates of the zwf gene, which were prepared by means of the polymerase chain reaction (PCR). Such amplicons are also referred to as PCR products.
  • PCR polymerase chain reaction
  • the skilled artisan will find, inter alia, in the handbook of Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag , Heidelberg, Germany, 1994).
  • the invention further provides an isolated polynucleotide encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity comprising the amino acid sequence of SEQ ID NO: 2, wherein at position 321 of the amino acid sequence any proteinogenic amino acid except glycine is contained. Preference is given to replacement with L-serine.
  • the amino acid sequence of the polypeptide further contains an amino acid substitution L-serine for another amino acid, preferably L-threonine, at position 8.
  • the invention further provides an isolated polynucleotide encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity comprising an amino acid sequence of 514 amino acids in length and wherein at position 321 any proteinogenic L-amino acid except glycine, preferably L-amino acid. Serine, is included.
  • the invention furthermore relates to an isolated polynucleotide which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which contains, 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 or 8.
  • the amino acid sequence of the encoded polypeptide comprises an amino acid sequence corresponding to positions 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 position 262 to 380 of SEQ ID NO: 6 or 8 or positions 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 positions 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 positions 2 to 514 of SEQ ID NO: 6 or 8.
  • the length of the encoded polypeptide comprises 514 amino acids.
  • the invention furthermore relates to an isolated polynucleotide which codes for a polypeptide with glucose-6-phosphate dehydrogenase enzyme activity which contains, at position 321 of the amino acid sequence or a corresponding or comparable position, any proteinogenic amino acid except glycine, preferably L-serine, and a nucleotide sequence associated with the nucleotide sequence of a polynucleotide which is obtainable by a polymerase chain reaction (PCR) using the primer pair whose nucleotide sequences each comprise at least 15 consecutive nucleotides selected from the nucleotide sequence between position 1 and 307 of SEQ ID NO: 3 or SEQ ID NO: 11 and from complementary nucleotide sequence between position 2100 and 1850 of SEQ ID NO: 3 or SEQ ID NO: 11 are selected.
  • PCR polymerase chain reaction
  • Suitable primer pairs are shown in SEQ ID NO: 17 and SEQ ID NO: 18 and in SEQ ID NO: 19 and SEQ ID NO: 20.
  • Chromosomal DNA of coryneform bacteria which have been treated in particular with a mutagen, is preferred as the starting material ("template" DNA).
  • template DNA is particularly preferred.
  • chromosomal DNA of the genus Corynebacterium is particularly preferred, and very particularly preferably of the species Corynebacterium glutamicum.
  • the invention further relates to an isolated polynucleotide which hybridizes with the nucleotide sequence complementary to SEQ ID NO: 5, 7 or 9 under stringent conditions and for a polypeptide with
  • Glucose-6-phosphate dehydrogenase enzyme activity encoding at position 321 of the amino acid sequence or a corresponding or comparable position any proteinogenic amino acid except glycine, preferably L-serine, and optionally at a position corresponding to position 8 any proteinogenic amino acid except L-serine, preferably L-threonine.
  • Hybridization takes place under stringent conditions, that is, only hybrids become in which the probe, ie a polynucleotide comprising the nucleotide sequence complementary to SEQ ID NO: 5, 7 or 9, and the target sequence, ie the polynucleotides treated or identified with the probe, are at least 90% identical.
  • the stringency of the hybridization including the washing steps is influenced or determined by varying the buffer composition, the temperature and the salt concentration.
  • the hybridization reaction is generally performed at relatively low stringency compared to the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).
  • probes can also hybridize with polynucleotides which have less than 90% identity to the nucleotide sequence of the probe used. Such hybrids are less stable and are removed by washing under stringent conditions.
  • This can, for example, by lowering the salt concentration to 2x SSC and 0.5x optionally thereafter SSC (The DIG System User's Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) can be achieved, with a temperature of about 5O 0 C - 68 0 C, about 52 0 C - 68 0 C, about 54 0 C - 68 0 C, about 56 0 C - 68 0 C, about 58 0 C - 68 0 C, about 6O 0 C - 68 0 C, about 62 0 C - 68 0 C, about 64 0 C - 68 0 C, about 66 0 C - 68 0 C is set.
  • Temperature ranges of about 64 0 C - 68 0 C or about 66 0 C - 68 0 C are preferred. It may be possible to lower the salt concentration to a concentration corresponding to 0.2x SSC or 0, Ix SSC.
  • the SSC buffer contains sodium dodecyl sulfate (SDS) at a concentration of 0.1%. By gradually increasing the hybridization temperature in steps of about 1 - 2 0 C of 5O 0 C to 68 0 C.
  • Polynucleotide fragments which are 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 sequence of the probe used and encode for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which comprises 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 available on the market in the form of so-called kits (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).
  • kits eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558.
  • the nucleotide sequences thus obtained code for polypeptides with glucose-6-phosphate
  • Dehydrogenase enzyme activity which is 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 or SEQ ID NO : 8 and containing the amino acid exchange according to the invention.
  • the invention furthermore relates to an isolated polynucleotide which codes for 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, the replacement of L-serine Is preferred and which comprises an amino acid 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 amino acid sequence of SEQ ID NO: 6.
  • An example of a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity comprising an amino acid sequence belonging to 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 furthermore relates to an isolated polynucleotide which codes for 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, the replacement of L-serine 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.
  • 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).
  • 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: 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).
  • those isolated polynucleotides encoding a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity containing at position 321 of the amino acid sequence or a corresponding or comparable position each amino acid except glycine, preferably L-serine, and 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.
  • Ali stands for the amino acids AIa or VaI
  • Alfa for the amino acids Lys or Thr
  • Bra for the amino acids He or Leu.
  • the invention furthermore relates to an isolated polynucleotide which codes for a polypeptide having glucose-6-phosphate dehydrogenase enzyme activity which comprises the amino acid sequence of SEQ ID NO: 6 or 8 or 10.
  • the encoded polypeptide contains one (1) or more conservative amino acid substitutions.
  • the polypeptide contains at most two (2), at most three (3), at most four (4) or at most five (5) conservative amino acid substitutions.
  • the invention furthermore relates to an isolated polynucleotide which is suitable for a polypeptide having glucose-6.
  • Phosphate dehydrogenase enzyme activity which comprises the amino acid sequence of SEQ ID NO: 6 or 8 or 10 including an extension at the N- or C-terminus by at least one (1) amino acid. This extension is not more than 50, 40, 30, 20, 10, 5, 3 or 2 amino acids or amino acid residues.
  • the subject of the invention is also zwf alleles which code for polypeptide variants of SEQ ID NO: 6, 8 or 10 which contain one or more insertions or deletions contain.
  • 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.
  • sequence motifs 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 preferably not ruptured by such insertions / deletions.
  • the invention furthermore relates to an isolated polynucleotide which comprises the nucleotide sequence according to SEQ ID NO: 5, 7, 9 or 11.
  • the invention furthermore relates to an isolated polynucleotide which has the nucleotide sequence between position 1 and 307 of SEQ ID NO: 11, preferably the nucleotide sequence between position 198 and 304 of SEQ ID NO: 11 and very particularly preferably the nucleotide sequence between position 208 and 299 of FIG SEQ ID NO: 11.
  • the subject of the invention is an isolated polynucleotide containing the zwf allele of the mutant DM1816.
  • the invention further provides an isolated polynucleotide comprising a portion of the coding region of a zwf allele of the invention, wherein the isolated polynucleotide in each case comprises the portion of the coding region containing the amino acid substitution at position 321 of the amino acid sequence of the encoded polypeptide.
  • 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 that 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 positions 202 to 440 of SEQ ID NO: 2, or that for at least one
  • Amino acid sequence corresponding to position 172 to 470 of SEQ ID NO: 2 or which codes for 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 positions 2 to 513 of SEQ ID NO: 2, or that encodes at least one amino acid sequence corresponding to positions 2 to 514 of SEQ ID NO: 2 or a corresponding one
  • Reading frame containing at position corresponding to 321 of SEQ ID NO: 2 any proteinogenic aminic acid except glycine, preferably L-serine, and optionally containing at the position corresponding to Figure 8 any proteinogenic amino acid other than L-serine, preferably L-threonine is.
  • a reading frame 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
  • SEQ ID NO: 13 The amino acid sequence encoded by this reading frame is shown as SEQ ID NO: 14 shown. Position 15 in SEQ ID NO: 14 corresponds to position 321 of SEQ ID NO: 6, 8, 10 or 12.
  • Nucleic acid molecules which are preferred for at least one amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 6 or 8 or 10, or at least corresponding to positions 292 to 350 of SEQ ID NO: 6 or 8 or 10, or at least corresponding to positions 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 or 10, or at least corresponding to positions 247 to 395 of SEQ ID NO: 6 or 8 or 10, or at least correspondingly 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 or 10, or at least corresponding to positions 172 to 470 of SEQ ID NO: 6 or 8 or 10 , or at least corresponding to position 82 to 500 of SEQ ID NO: 6 or 8 or 10, or at least corresponding to position 2 to 512 of SEQ ID NO: 6 or 8 , thereby
  • a reading frame according to the invention comprising a polynucleotide encoding at least the amino acid sequence corresponding to positions 307 to 335 of SEQ ID NO: 6 is listed below: gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt tgg cag
  • the reading frame is also shown as SEQ ID NO: 15.
  • SEQ ID NO: 16 shows the coded by this reading frame Amino acid sequence. Position 15 in SEQ ID NO: 16 corresponds to position 321 of SEQ ID NO: 6, 8, 10 or 12.
  • 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
  • the reading frames according to the invention may moreover contain one or more mutations which result in one or more mutations multiple conservative amino acid substitutions.
  • the mutations preferably lead to a maximum of 4%, to a maximum of 2% or to a maximum of 1% conservative amino acid substitutions.
  • the reading frames of the invention may contain one or more silent mutations. Preferably included the reading frames according to the invention at most 4% and more preferably at most 2% to at most 1% silent mutations.
  • the isolated polynucleotides of the invention can be used to produce recombinant strains of microorganisms that are compared to the
  • Parent or parent strain in an improved way amino acids in the surrounding medium release or accumulate in the cell interior.
  • a common method for incorporating mutations into genes of coryneform bacteria is that of allele exchange, also known as "gene replacement.”
  • a DNA fragment containing the mutation of interest is transformed into the desired strain of a coryneform bacterium transferred and the mutation by at least two recombination events or "cross over" events in the chromosome of the desired strain incorporated or exchanged existing in the relevant strain 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 Häfer 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)) used this method to incorporate various mutations from the isolated alleles into the C. glutamicum chromosome, thus allowing Nakagawa et al.
  • a polynucleotide according to the invention may be used which comprises the entire coding region, as shown for example in SEQ ID NO: 5, 7 or 9, or which comprises a part of the coding region, such as the nucleotide sequence corresponding to at least the amino acid sequence Position 307 to 335 of SEQ ID NO: 6, 8 or 10 encoded and represented 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.
  • Nucleic acid molecules encoding at least one amino acid sequence corresponding to position from 277 to 365 of SEQ ID NO: 6, 8 or 10. Very particular preference is given to those parts of the coding region whose length is ⁇ 357 nucleobases, such as, for example
  • Nucleic acid molecules encoding at least one amino acid sequence corresponding to positions 262 to 380 of SEQ ID NO: 6, 8 or 10.
  • the DNA fragment containing the mutation of interest in this method is typically present in a vector, in particular a plasmid, which is preferably not or only to a limited extent replicated by the strain to be mutated.
  • a bacterium of the genus Escherichia preferably of the species Escherichia coli, is generally used.
  • plasmid vectors examples include those of Shufer et al. (Gene 145, 69-73 (1994)) described pK * mob and pK * mobsacB vectors, such as pKl ⁇ mobsacB, and in WO 02/070685 and WO 03/014362 described vectors. These are replicative in Escherichia coli but not in coryneform bacteria.
  • Particularly suitable vectors are those which contain a conditionally negatively dominant gene such as the sacB gene (levansucrase gene) of, for example, Bacillus or the galK gene (galactose kinase gene) of, for example, Escherichia coli.
  • 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.
  • Nakamura et al. US Pat. No. 6,303,383 describe a temperature-sensitive plasmid for coryneform bacteria which can only replicate at temperatures below 31 ?? C.
  • the vector is then by conjugation, for example by the method of Schwarzer (Journal of Bacteriology 172, 1663-1666 (1990)) or transformation, for example, by the method of Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) or the Method of Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)) into the coryneform bacterium.
  • the transfer of the DNA can also be achieved by particle bombardment.
  • Another object of the invention is accordingly a process for the preparation of a coryneform bacterium, wherein
  • step c) the coryneform bacterium obtained according to step a) and b) increases.
  • Another inventive method for producing a microorganism is that one
  • a polynucleotide according to the invention which is a polypeptide with glucose-6-phosphate dehydrogenase Enzyme activity coded, transferred into a microorganism,
  • step c) increases the microorganism obtained in step a) and b).
  • a recombinant microorganism which contains at least one (1) copy or multiple copies of a polynucleotide of the invention encoding a glucose-6-phosphate dehydrogenase at position 321 or a comparable position of the amino acid sequence of the encoded polypeptide, contains any proteinogenic amino acid except glycine, with preference given to replacement with L-serine.
  • the polypeptide at position 8 or a comparable position contains any proteinogenic amino acid except L-serine, preferably the amino acid L-threonine.
  • hosts or host cells preferably microorganisms, particularly preferably coryneform bacteria and bacteria of the genus Escherichia, which contain the polynucleotides according to the invention.
  • the invention also relates to microorganisms prepared using the isolated polynucleotides. Such microorganisms or bacteria are also referred to as recombinant microorganisms or recombinant bacteria.
  • vectors containing the polynucleotides of the invention are the subject of the invention.
  • hosts containing these vectors are also the subject of the invention.
  • the isolated polynucleotides of the invention may also be used to obtain overexpression of the polypeptides encoded by them.
  • Overexpression is generally understood to mean an increase in the intracellular concentration or activity of a ribonucleic acid, a protein or an enzyme.
  • zwf alleles or polynucleotides are overexpressed for
  • Encode glucose-6-phosphate dehydrogenases containing at position 321 of the amino acid sequence of the encoded polypeptide any proteinogenic amino acid except glycine, with preference given to replacement with L-serine.
  • the encoded protein also contains an exchange of L-serine for another proteinogenic amino acid, preferably L-threonine at position 8 of the amino acid sequence.
  • N-terminal amino acids in particular the N-terminal methionine, can be cleaved off from the polypeptide formed by host-specific enzymes, so-called aminopeptidases.
  • the said increase in the concentration or activity of a gene product can be achieved, for example, by increasing the copy number of the corresponding polynucleotides by at least one copy.
  • a widely used method for increasing the copy number consists of incorporating the corresponding gene or allele in a vector, preferably a plasmid, which is replicated by a coryneform bacterium.
  • a vector preferably a plasmid, which is replicated by a coryneform bacterium.
  • Suitable plasmid vectors are, for example, pZl (Menkel et al.,
  • chromosomal gene amplification Another common method of overexpression is chromosomal gene amplification. In this method, at least one additional copy of the gene or allele of interest inserted into the chromosome of a coryneform bacterium.
  • a C. glutamicum non-replicative plasmid containing the gene of interest is transformed into a coryneform bacterium. After homologous recombination by means of a "cross over" event, the resulting strain contains at least two copies of the gene or allele in question.
  • one or more copies of the gene of interest are inserted into at least two recombination events in a desired location of the C. glutamicum chromosome.
  • a copy of a lysC allele which codes for an L-lysine-insensitive aspartate kinase was incorporated into the gluB gene of C. glutamicum.
  • 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 was achieved.
  • Another method for achieving overexpression is to link the corresponding gene or allele in a functional manner (operably linked) with a promoter or an expression cassette.
  • Suitable promoters for Corynebacterium glutamicum are described, for example, in the review article by Patek et al. (Journal of Biotechnology 104 (1-3), 311-323 (2003) described.
  • the well-known Mann et al. (Gene 69 (2), 301-315 (1988)) and Mann and Brosius (Gene 40 (2-3), 183-190 (1985)).
  • T3, T7, SP6, M13, lac, tac, and trc promoters become.
  • Such a promoter may, for example, be inserted upstream of the zwf allele, typically at intervals of about 1-500 or 1-307 nucleotides from the start codon of a recombinant coryneform bacterium which contains a different proteinogenic amino acid in place of the amino acid glycine naturally present at position 321.
  • Such a promoter may of course also be inserted upstream of the zwf allele of a mutant according to the invention.
  • the promoter and regulatory region or ribosome binding site located upstream of the structural gene can be mutated.
  • An example of a mutated promoter region of the zwf gene or zwf allele is the nucleotide sequence comprising position 208 to 299 of SEQ ID NO: 11.
  • the activity or concentration of the corresponding protein is generally increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000 % or 2000%, increased based on the activity or concentration of the protein in the parent microorganism or parent strain.
  • a starting microorganism or parent strain is meant a microorganism on which the measures of the invention are carried out.
  • 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 corresponding 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., CoId 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)).
  • the invention accordingly relates to processes for the overexpression of the glucose-6-phosphate dehydrogenases according to the invention.
  • a method according to the invention for overexpression consists inter alia of using the
  • Copy number of a polynucleotide according to the invention which codes for a glucose-6-phosphate dehydrogenase variant in which, at position 321 or the corresponding position of the encoded amino acid sequence, each proteinogenic amino acid except glycine and optionally at position 8 or the corresponding position, any proteinogenic amino acid other than L-serine is increased by at least one (1) or multiple copies.
  • a further method according to the invention consists in linking a promoter functionally to the polynucleotide.
  • the invention furthermore relates to microorganisms which have an increased concentration or activity of the glucose-6-phosphate dehydrogenase variants according to the invention in their cell interior.
  • L-amino acids in the mutants or recombinant strains according to the invention one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, the amino acid export and optionally overexpress regulatory proteins.
  • endogenous genes is generally preferred.
  • endogenous genes or “endogenous nucleotide sequences” is meant the genes or nucleotide sequences or alleles present in the population of a species.
  • a dapA gene coding for a dihydrodipicolinate synthase such as, for example, the wild-type dapA gene of Corynebacterium glutamicum described in EP 0 197 335,
  • 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,
  • a gene pyc coding for a pyruvate carboxylase such as, for example, the wild-type pyc gene of Corynebacterium glutamicum described in DE-A-198 31 609 and EP 1108790,
  • Fig ur 2A of WO 02/31158 is further an amino acid substitution A (alanine) against G (glycine) on Item 472 indicated.
  • 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.
  • Fig. 2B of WO 02/31158 is further an amino acid substitution D (aspartic acid) against E
  • a lysC FBR allele coding for a feedback-resistant aspartate kinase variant in particular correspondingly
  • 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,
  • a gene pgi coding for the glucose-6-phosphate isomerase such as, for example, the pgi gene of Corynebacterium glutamicum described in US Pat. Nos. 6,586,214 and 6,465,238,
  • a gene hom coding for the homoserine dehydrogenase such as, for example, the hom gene of Corynebacterium glutamicum described in EP-A-0131171,
  • 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),
  • the term "attenuation” in this context describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or by using a gene or allele, which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein) and optionally combines these measures.
  • the activity or concentration of the corresponding protein is generally 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein, respectively 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 occur as mutations for the generation of an attenuation
  • missense mutations results in an exchange of a given amino acid in one protein for another, in particular a non-conservative amino acid substitution.
  • the functionality or activity of the protein is impaired and reduced to a value of 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5%.
  • the nonsense mutation leads to a stop codon in the coding region of the gene and thus to a premature one
  • 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.
  • the isolated coryneform bacteria obtained by the measures of the invention show an increased excretion or production of the desired amino acid in a fermentation process compared to the starting strain or parent strain used.
  • mutants of the invention are the mutants of the invention, isolated or generated and recombinant bacteria, especially coryneform bacteria, containing a zwf allele encoding a glucose-6-phosphate dehydrogenase having the described amino acid substitution at position 321 of the amino acid sequence and optionally one
  • the performance of the isolated bacteria or fermentation process using the same with respect to one or more of the parameters selected from the group of product concentration (product per volume), product yield (product formed per carbon source consumed), and product formation (product formed per volume and time) or other process parameters and combinations thereof, is at least 0.5%, at least 1%, at least 1.5% or at least 2% based on the parent strain or parent strain or the fermentation process using the same improved.
  • the isolated coryneform bacteria according to the invention can be used continuously - as in the
  • PCT / EP2004 / 008882 described or batchwise in the batch process (sentence cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) are cultivated for the purpose of producing L-amino acids.
  • a general summary of known cultivation methods is in the textbook by Chmiel (bioprocess 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag,
  • the culture medium or fermentation medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are in the handbook Manual of Methods for General Bacteriology, of the American Society for Bacteriology (Washington, DC, USA, 1981). The terms culture medium and
  • Fermentation medium or medium are mutually exchangeable.
  • sugars and carbohydrates such as e.g. Glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from sugar beet or sugarcane production, starch, starch hydrolyzate and cellulose, oils and fats, such as soybean oil,
  • Sunflower oil peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerine, methanol and ethanol, and organic acids such as acetic acid. These substances can be used individually or as a mixture.
  • organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate,
  • Ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used.
  • the nitrogen sources can be used singly or as a mixture.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • the culture medium must further contain salts, for example, in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium and iron, such as magnesium sulfate or ferric sulfate, necessary for growth.
  • salts for example, in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium and iron, such as magnesium sulfate or ferric sulfate, necessary for growth.
  • essential growth substances such as amino acids such as homoserine and vitamins such as thiamine, biotin or pantothenic acid in addition to the above substances can be used.
  • suitable precursors of the respective amino acid can be added to the culture medium.
  • the said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.
  • basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used.
  • the pH is generally adjusted to a value of 6.0 to 9.0, preferably 6.5 to 8.
  • antifoams such as, for example, fatty acid polyglycol esters
  • suitable selective substances such as antibiotics
  • oxygen or oxygen-containing gas mixtures such as air, are introduced into the culture.
  • liquids enriched with hydrogen peroxide is also possible.
  • the fermentation at elevated pressure, for example at a pressure of 0.03 to 0.2 MPa, driven.
  • the temperature of the culture is normally from 2O 0 C to 45 0 C and preferably at 25 0 C to 40 0 C.
  • the cultivation is continued until a maximum of the desired amino acid has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes longer cultivation times are possible.
  • Suitable fermentation media are described inter alia in US 6,221,636, in US 5,840,551, in US 5,770,409, in US 5,605,818, in US 5,275,940 and in US 4,224,409.
  • Methods for the determination of L-amino acids are known from the prior art.
  • the analysis may be as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) described by anion exchange chromatography followed by ninhydrin derivatization, or it can be done by reversed phase HPLC, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
  • the invention accordingly provides a process for the preparation of an L-amino acid in which
  • the fermentation broth prepared in this way is then further processed to a solid or liquid product.
  • a fermentation broth is understood as meaning a fermentation medium in which a microorganism has been cultivated for a certain time and at a certain temperature.
  • the fermentation medium or the medium used during the fermentation contains / contain all substances or components which have a Propagation of the microorganism and ensures formation of the desired amino acid.
  • 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.
  • the organic by-products include substances which are optionally produced by the microorganisms used in the fermentation next to the respective desired L-amino acid and optionally excreted. These include L-amino acids, which are less than 30%, 20% or 10% compared to the desired amino acid. These include organic acids which carry one to three carboxyl groups such as acetic acid, lactic acid, citric acid, malic acid or fumaric acid. Finally, it also includes sugar such as trehalose.
  • Fermentation broths have an amino acid content of 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.
  • a group of L-lysine containing products comprises concentrated, aqueous, alkaline solutions of purified L-lysine (EP-B-0534865).
  • Another group as described, for example, in US Pat. Nos. 6,340,486 and 6,465,025, comprises 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 one described there
  • the product form contains most of the starting materials used and not consumed during the fermentative production and, if appropriate, the biomass of the microorganism used with a proportion of> 0% to 100%.
  • L-amino acid is collected from the fermentation broth, isolated or purified to produce the L-amino acid-containing product or the purified L-amino acid.
  • EP-B-0534865 describes a process for preparing aqueous, basic L-lysine-containing solutions from fermentation broths.
  • the biomass is separated from the fermentation broth and discarded.
  • a base such as sodium, potassium or ammonium hydroxide, a pH between 9 to 11 is set.
  • the mineral components are separated after concentration and cooling by crystallization from the broth and either used as fertilizer or discarded.
  • the biomass may be wholly or partially separated by separation methods such as e.g. centrifugation, filtration, decantation, or a combination thereof, from the fermentation broth or left completely in it.
  • separation methods such as e.g. centrifugation, filtration, decantation, or a combination thereof, from the fermentation broth or left completely in it.
  • the biomass or the biomass-containing fermentation broth is inactivated during a suitable process step, for example by thermal treatment (heating) or by acid addition.
  • the chemical constituents of the biomass include the cell envelope, for example the peptidoglycan and the arabinogalactan, the protein or polypeptide, for example the glucose-6-phosphate dehydrogenase polypeptide, lipids and phospholipids and nucleic acids (DNA and RNA), for example polynucleotides containing the inventive mutation.
  • the cell envelope for example the peptidoglycan and the arabinogalactan
  • the protein or polypeptide for example the glucose-6-phosphate dehydrogenase polypeptide, lipids and phospholipids and nucleic acids (DNA and RNA), for example polynucleotides containing the inventive mutation.
  • nucleic acids are typically in the form of fragments having a length of, inter alia, ⁇ 40-60 bp,> 60-80 bp,> 80-100 bp ,> 100-200 bp,> 200-300 bp,> 300-400 bp,> 400-500 bp,> 500-750 bp,> 750-1000 bp,> 1000-1250 bp,
  • the biomass is completely or almost completely removed so that 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.
  • 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. Accordingly, in a method according to the invention, the biomass is removed in proportions of ⁇ 0% to ⁇ 100%.
  • the fermentation broth obtained after the fermentation can be adjusted to an acidic pH before or after complete 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 (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).
  • the broth may also be stabilized by the addition of sodium bisulfite (NaHSO 3 , GB 1,439,728) or another salt, for example, ammonium, alkali or alkaline earth salt of the sulfurous acid.
  • organic or inorganic solids are partially or completely removed.
  • the organic by-products dissolved in the fermentation broth and the dissolved non-consumed constituents of the fermentation medium (starting materials) remain at least partially (> 0%), preferably at least 25%, more preferably at least 50% and most preferably at least 75% in the product. If appropriate, these also remain completely (100%) or almost completely ie> 95% or> 98% in the product. In this sense, the term means
  • Frermentation broth base means that a product contains at least part of the constituents of the fermentation broth.
  • the broth is removed by known methods, e.g. with the help of a rotary evaporator,
  • a desired product is isolated from the resulting granules by sieving or dust separation.
  • the fermentation broth directly d. H. without prior concentration by spray drying or spray granulation to dry.
  • free-flowing refers to powders which flow out freely from a series of glass outlet vessels with outlet openings of different sizes at least from the vessel with the opening 5 mm (mm) (Klein: Soaps, Oils, Fats, Wachse 94, 12 (1968)).
  • finely divided is meant a powder with a predominant proportion (> 50%) of a particle size of 20 to 200 ⁇ m in diameter.
  • coarse-grained is meant a product having a predominant proportion (> 50%) of a particle size of 200 to 2000 ⁇ m in diameter.
  • the particle size determination can be carried out using methods of laser diffraction spectrometry. The appropriate methods are in the textbook for
  • the free-flowing, finely divided powder can in turn be converted by suitable compacting or granulation process into a coarse-grained, free-flowing, storable and substantially dust-free product.
  • dust-free means that the product contains only small amounts ( ⁇ 5%) of particle sizes smaller than 100 ⁇ m in diameter.
  • storable means a product that can be stored in a dry and cool environment for at least one (1) year or more, preferably at least 1.5 years or longer, more preferably two (2) years or longer that a significant loss ( ⁇ 5%) of the respective amino acid occurs.
  • 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
  • step b) or c) an acid selected from the group sulfuric acid, phosphoric acid or hydrochloric acid is added.
  • step a) or b) water is removed from the L-amino acid-containing fermentation broth (concentration).
  • auxiliaries 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.
  • oils mineral oils vegetable oils or mixtures of vegetable oils can be used. Examples of such oils are soybean oil, olive oil, soybean oil / lecithin mixtures.
  • silicone oils, polyethylene glycols or hydroxyethycellulose are suitable.
  • the treatment of the surfaces with the oils mentioned achieves an increased abrasion resistance of the product and a reduction in the dust content.
  • the content of oil in the product is 0.02 to 2.0% by weight, preferably 0.02 to 1.0 wt .-%, and most preferably 0.2 to 1.0 wt .-% based on the total amount of the feed additive.
  • the proportion of dust d. H. Particles with a particle size ⁇ 100 microns is preferably> 0 to 1 wt .-%, more preferably at most 0.5 wt .-%.
  • the product can also be applied to one in the
  • Feed processing known and customary organic or inorganic carrier such as silicas, silicates, shot, brans, flours, starches sugar or other grown and / or mixed with conventional thickeners or binders and stabilized.
  • organic or inorganic carrier such as silicas, silicates, shot, brans, flours, starches sugar or other grown and / or mixed with conventional thickeners or binders and stabilized.
  • Application examples and processes for this purpose are described in the literature (Die Mühle + Mischfuttertechnik 132 (1995) 49, page 817).
  • the product can also be brought into a state by coating methods with film formers, such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920, in which it is stable to digestion by animal stomachs, especially the stomach of ruminants.
  • film formers such as, for example, metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C-4100920, in which it is stable to digestion by animal stomachs, especially the stomach of ruminants.
  • the corresponding amino acid can be added during the process in the form of a concentrate or, if appropriate, a substantially pure substance or its salt in liquid or solid form. These can be added individually or as mixtures to the obtained or concentrated fermentation broth, or also during the drying or granulation process.
  • the ratio of the ions is adjusted so that the ion ratio according to the following formula
  • the fermentation broth-based solid product thus prepared has a lysine content (as lysine base) of from 10% to 70% or 20% to 70%, preferably 30% by weight. % 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.
  • the fermentation broth-based solid product thus prepared has an amino acid content of at least 5% by weight, 10% by weight, 20% by weight, 30% by weight and maximum 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight or up to 95% by weight.
  • the water content of the solid product is up to 5 wt .-%, preferably up to 4 wt .-%, and particularly preferably less than 3 wt .-%.
  • the subject of the invention is therefore also a fermentation broth-based feed additive containing L-lysine, which has the following features
  • the subject of the invention is therefore also a fermentation-based feed additive containing L-tryptophan, which has the following characteristics
  • Designation DM1797 which contains the amino acid exchange lysC T311I in aspartate kinase, was deposited on October 28, 2004 with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) as DSM 16833.
  • the mutant Corynebacterium glutamicum DM1816 according to the invention which contains L-serine at position 321 of the amino acid sequence of the Zwf polypeptide, was deposited on 9 February 2005 with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) as DSM 17119.
  • the Corynebacterium glutamicum strain DM1797 was used as a starting strain for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis.
  • Strain DM1797 is an aminoethylcysteine-resistant mutant of Corynebacterium glutamicum ATCC13032 and under the name DSM16833 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).
  • Strain DM1797 was dissolved in 10 ml LB broth (Merck,
  • Example 1 The mutants obtained in Example 1 were cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined.
  • the clones were initially propagated on brain heart agar plates (Merck, Darmstadt, Germany) for 24 hours at 33 0 C. Starting from these agar plate cultures, one preculture was in each case inoculated (10 ml of medium in the 100 ml Erlenmeyer flask). As a medium for pre-culture was the Medium MM used. The preculture was incubated for 24 hours at 33 ° C. and 240 rpm on a shaker. From this preculture, a major culture was inoculated so that the initial OD (660 nm) of the major culture was 0.1 OD. The medium MM was also used for the main culture.
  • CSL Corn Steep Liquor
  • MOPS morpholinopropanesulfonic acid
  • saline solution was adjusted to pH 7 with ammonia water and autoclaved.
  • the sterile substrate and vitamin solutions as well as the dry autoclaved CaCO 3 were added. Culturing was done in volumes of 10 ml contained in 100 ml baffled Erlenmeyer flasks. The temperature was at 33 0 C, the number of revolutions was 250 rpm and the humidity 80%.
  • the optical density (OD) was determined at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Kunststoff).
  • the amount of lysine formed was determined using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection.
  • a mutant characterized by increased lysine formation was designated DM1816.
  • Clone DM1816 was isolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) isolated chromosomal DNA. With the help of the polymerase chain reaction, a DNA segment carrying the zwf gene was amplified. For this purpose, the following oligonucleotides were used as primers:
  • the primers shown were synthesized by the company MWG Biotech (Ebersberg, Germany). They allow the amplification of an approximately 1.95 kb DNA segment, which carries the zwf gene.
  • the primer zwf-L1 binds to the region corresponding to positions 59 to 78 of the strand complementary to SEQ ID NO: 3.
  • the primer zwf-L2 binds to the region enrovend position 2026 to 2007 the strand according to SEQ ID NO: 3.
  • the PCR reaction was carried out with the Phusion High Fidelity DNA Polymerase (New England Biolabs, Frankfurt, Germany).
  • the reaction mixture was prepared according to the manufacturer and contained at 50 ul total volume 10 ul of the supplied 5 x Phusion HF buffer, deoxynucleoside triphosphates in a concentration of 200 uM, primer in a concentration of 0.5 uM, about 50 ng template DNA and 2 units of phage polymerase. By adding H2O, the volume was adjusted to 50 ⁇ l.
  • the PCR approach was first subjected to preliminary denaturation at 98 ° C. for 30 seconds. This was followed 35x repetitively denaturation step at 98 0 C for 20 seconds, a step for binding the primer to the submitted DNA at 6O 0 C for 20 seconds and the
  • Extension step to extend the primers at 72 0 C for 60 seconds. After the final extension step for 5 minutes at 72 ° C., the PCR mixture was subjected to agarose gel electrophoresis (0.8% agarose). A DNA fragment of about 1.85 kb in length was identified, isolated from the gel and purified using the QIAquick Gel Extraction Kit from Qiagen, (Hilden, Germany).
  • the nucleotide sequence of the amplified DNA fragment or PCR product was obtained from Agowa (Berlin, Germany).
  • the obtained sequence of the coding region of the zwf allele is shown in SEQ ID NO: 9.
  • the amino acid sequence of the protein resulting from the program Patentin is shown in SEQ ID NO: 10.
  • the nucleotide sequence of the coding region of the zwf allele of mutant DM1816 at position 961 contains the nucleobase adenine (see SEQ ID NO: 5 or 9).
  • the wild-type gene (see SEQ ID NO: 1) contains the nucleobase guanine at this position. This guanine-adenine transition results in an amino acid exchange of glycine to serine at position 321 of the resulting amino acid sequence. This mutation is referred to below as zwfG321S.
  • the zwf allele of DM1816 still contains the nucleotide exchange thymine towards adenine at position 22 of the
  • Nucleotide sequence This thymine-adenine transversion results in an amino acid change from serine to threonine at position 8 of the resulting amino acid sequence.
  • zwf allele of DM1816 contains five more nucleotide exchanges that are not related to one
  • cytosine-thymine transition at position 138 a cytosine-thymine transition at position 279, a thymine-cytosine transition at position 738, a cytosine-thymine transition at position 777 and a Guanine adenine transition at position 906.
  • a part of the coding region, that is a so-called internal fragment or internal region, of the zwf allele was amplified, which carries the mutation zwfG321S.
  • the template was the one in Example 3 chromosomal DNA used.
  • the following oligonucleotides were selected as primers for the PCR:
  • nucleotides 11 to 30 of the primer zwf-intl-bam bind to the region corresponding to positions 546 to 565 of the strand complementary to SEQ ID NO: 3. Positions 546 and 565 of SEQ ID NO: 3 correspond to positions 239 and 258 in SEQ ID NO: 1. Nucleotides 11 to 30 of primer zwf-int2-bam bind to the region corresponding to positions 1527 to 1508 of the strand as shown in SEQ ID No. 3. Positions 1527 and 1508 of SEQ ID NO: 3 correspond to positions 1220 and 1201 of SEQ ID NO: 1.
  • the primers contain the sequences for restriction endonuclease BamHI sites which are underlined in the nucleotide sequence shown above.
  • the PCR reaction was performed with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Frankfurt, Germany). The reaction had the composition described above. The PCR was carried out with one exception as above: the 72 ° C extension step in the 35-fold repetition was performed for each 30 seconds only.
  • Restriction endonuclease BamHI treated and identified by electrophoresis in a 0.8% agarose gel. It was then isolated from the gel and purified with the QIAquick Gel Extraction Kit from Qiagen.
  • the thus purified DNA fragment contains the described zwfG32IS mutation and has BamHI compatible ends (zwfG32IS fragment or 'zwf in Figure 1). It was subsequently used in Schulfer et al. PK10mobsacB mobilized vector (Gene, 145, 69-73 (1994) to allow for allelic or mutational exchange by digesting pKl ⁇ mobsacB with the restriction enzyme BamHI and ending with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany). The prepared vector was mixed with the zwfG32IS fragment and the batch was treated with the Ready-To-Go T4 DNA ligase kit (Amersham-Pharmacia, Freiburg, Germany).
  • E. coli strain S17-1 (Simon et al., Bio / Technology 1: 784-791, 1993) was transformed with the ligation mixture (Hanahan, In. DNA cloning, A practical approach., Vol CoId Spring Habor, New York, 1989). Selection for plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al, Molecular Cloning. A Laboratory Manual 2 nd Ed Cold Spring Habor, New York, 1989..), The mg with 25/1 kanamycin had been supplemented.
  • Plasmid DNA was isolated from a transformant using the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction digestion in each case once with the enzyme BamHI and once with the enzyme SacI and subsequent agarose gel electrophoresis.
  • the plasmid was named pK18mobsacB_zwfG321S and is shown in FIG.
  • transconjugants ie clones with integrated pKl ⁇ mobsacB zwfG321S
  • the selection of transconjugants was carried out by plating out the conjugation mixture on LB agar supplemented with 25 mg / l kanamycin and 50 mg / l nalidixic acid. Kanamycin-resistant transconjugants were then streaked on kanamycin (25 mg / l) supplemented LB agar plates and incubated at 33 ° C. for 24 hours.
  • the clones were cultured unsupported in LB liquid medium for 30 hours, then streaked on LB agar supplemented with 10% sucrose for 24 hours 33 0 C incubated.
  • the plasmid pKl ⁇ mobsacB zwfG321S contains, like the starting plasmid pKl ⁇ mobsacB, in addition to the kanamycin resistance gene, a copy of the sacB gene coding for the levan sucrase from Bacillus subtilis.
  • the sucrose inducible expression of the sacB gene results in the formation of levan sucrase, which catalyzes the synthesis of the C. glutamicum toxic product Levan.
  • sucrose-supplemented LB agar therefore, only those clones grow in which the integrated pKl ⁇ mobsacB has excised zwfG321S as a result of a second recombination event.
  • the second recombination event Depending on the location of the second
  • the performance test was carried out as described in Example 2.
  • the DMl797_zwfG321S strain showed significantly increased lysine secretion compared to DM1797, similar to DM1816 (See Table 1).

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Abstract

L'invention concerne des mutants et des allèles du gène zwf de bactéries coryneformes, codant des variants de la sous-catégorie zwf de la glucose-6-phosphate-déshydrogénase (EC: 5 1.1.1.49), ainsi que des procédés de fabrication d'acides aminés, notamment de L-lysine, et de L-tryptophane, au moyen de bactéries contenant ces allèles.
PCT/EP2006/060519 2005-03-24 2006-03-07 Alleles mutes du gene zwg (g6pdh) issus de bacteries coryneformes destines a augmenter la production de lysine Ceased WO2006100177A1 (fr)

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EP06725146A EP1861493B8 (fr) 2005-03-24 2006-03-17 Alleles mutes du gene zwf (g6pdh) tire de corynebacteries pour la production accrue de lysine
PL06725146T PL1861493T3 (pl) 2005-03-24 2006-03-17 Zmutowane allele genu ZWG (G6PDH) z bakterii maczugowatych do zwiększonego wytwarzania lizyny
PCT/EP2006/060851 WO2006100211A1 (fr) 2005-03-24 2006-03-17 Alleles mutes du gene zwf (g6pdh) tire de corynebacteries pour la production accrue de lysine
ES06725146T ES2394918T3 (es) 2005-03-24 2006-03-17 Alelos mutados del gen zwf (G6PDH) de bacterias corineformes para aumentar la producción de lisina
DK06725146.2T DK1861493T3 (da) 2005-03-24 2006-03-17 Muterede allerer af zwf-genet (g6pdh) fra coryneforme bakterier til øget produktion af lysin

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DE102005013676.1 2005-03-24
DE102005013676A DE102005013676A1 (de) 2005-03-24 2005-03-24 Allele des zwf-Gens aus coryneformen Bakterien

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PCT/EP2006/060851 Ceased WO2006100211A1 (fr) 2005-03-24 2006-03-17 Alleles mutes du gene zwf (g6pdh) tire de corynebacteries pour la production accrue de lysine

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WO2007141111A3 (fr) * 2006-06-02 2008-03-06 Evonik Degussa Gmbh Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine
WO2008092956A1 (fr) * 2007-02-02 2008-08-07 Evonik Degussa Gmbh Production de l-lysine et d'additifs alimentaires contenant de la l-lysine
DE102008001874A1 (de) 2008-05-20 2009-11-26 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren
US7785840B2 (en) 2006-07-13 2010-08-31 Evonik Degussa Gmbh Method of production of L-amino acids
WO2022143763A1 (fr) * 2020-12-30 2022-07-07 宁夏伊品生物科技股份有限公司 Souche ayant une productivité d'acide l-glutamique améliorée, son procédé de construction et son application

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DE102009030342A1 (de) 2009-06-25 2010-12-30 Evonik Degussa Gmbh Verfahren zur fermentativen Herstellung von organisch chemischen Verbindungen
DE102010003419B4 (de) 2010-03-30 2019-09-12 Evonik Degussa Gmbh Verfahren zur fermentativen Herstellung von L-Ornithin
DE102011006716A1 (de) 2011-04-04 2012-10-04 Evonik Degussa Gmbh Mikroorganismus und Verfahren zur fermentativen Herstellung einer organisch-chemischen Verbindung
EP2762571A1 (fr) 2013-01-30 2014-08-06 Evonik Industries AG Microorganisme et procédé de fabrication par fermentation d'acides aminés
EP2940144A1 (fr) 2014-04-30 2015-11-04 Evonik Degussa GmbH Procédé de production de L-lysine en utilisant une bactérie alcaliphile

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1302537A1 (fr) * 2000-06-21 2003-04-16 Kyowa Hakko Kogyo Co., Ltd. Nouvelle glucose-6-phosphate deshydrogenase
WO2003042389A1 (fr) * 2001-11-13 2003-05-22 Basf Aktiengesellschaft Gene codant pour la proteine appelees glucose-6-phosphate deshydrogenase
US20030175911A1 (en) * 2000-03-20 2003-09-18 Stephen Hans Process for the preparation of L-amino acids with amplification of the zwf gene

Family Cites Families (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU507634A1 (ru) 1973-09-24 1976-03-25 Институт Микробиологии Имени А.Кирхенштейна Ан Латвийской Сср Способ получени -лизина
JPS54105293A (en) 1978-02-01 1979-08-18 Ajinomoto Co Inc Preparation of l-proline by fermentation
JPS559783A (en) 1978-07-10 1980-01-23 Ajinomoto Co Inc Preparation of l-lysine by fermentation
US4601983A (en) 1983-06-15 1986-07-22 Ajinomoto Co., Inc. Coryneform bacteria carrying recombinant plasmids and their use in the fermentative production of L-threonine and L-isoleucine
JPH0655149B2 (ja) 1985-03-12 1994-07-27 協和醗酵工業株式会社 Ｌ―リジンの製造法
US5250434A (en) 1987-03-27 1993-10-05 Ajinomoto Co., Inc. Microorganisms for production of glutamic acid
JP2656300B2 (ja) 1988-04-18 1997-09-24 協和醗酵工業株式会社 Ｌ−トリプトファンの製造法
GB2223754B (en) 1988-09-12 1992-07-22 Degussa Dna encoding phosphoenolpyruvate carboxylase
JP2990735B2 (ja) 1990-04-20 1999-12-13 味の素株式会社 Ｌ―リジンの発酵的製造法
JP3023615B2 (ja) 1990-08-30 2000-03-21 協和醗酵工業株式会社 発酵法によるｌ―トリプトファンの製造法
DE4100920A1 (de) 1991-01-15 1992-07-16 Degussa Wirkstoffzubereitung zur oralen verabreichung an wiederkaeuer
FR2679569B1 (fr) 1991-07-26 1995-05-19 Eurolysine Procede de separation de la lysine sous la forme de solutions aqueuses et utilisation de ces solutions pour l'alimentation animale.
DE4130868C2 (de) 1991-09-17 1994-10-13 Degussa Tierfuttermittelsupplement auf der Basis einer Aminosäure und Verfahren zu dessen Herstellung
DE4130867A1 (de) 1991-09-17 1993-03-18 Degussa Verfahren zur fermentativen herstellung von aminosaeuren
JPH05195736A (ja) 1992-01-20 1993-08-03 Mazda Motor Corp エンジンのバルブ駆動装置
DE4232468A1 (de) 1992-09-28 1994-03-31 Consortium Elektrochem Ind Mikroorganismen für die Produktion von Tryptophan und Verfahren zu ihrer Herstellung
JP3369231B2 (ja) 1992-12-03 2003-01-20 協和醗酵工業株式会社 芳香族アミノ酸の製造法
AU706285B2 (en) 1995-05-16 1999-06-10 Ajinomoto Co., Inc. Feed additive
DE19547361A1 (de) 1995-12-19 1997-06-26 Degussa Verfahren zur Herstellung von L-Aminosäuren durch Fermentation
DE19548222A1 (de) 1995-12-22 1997-06-26 Forschungszentrum Juelich Gmbh Verfahren zur mikrobiellen Herstellung von Aminosäuren durch gesteigerte Aktivität von Exportcarriern
JPH09224661A (ja) 1996-02-23 1997-09-02 Mitsubishi Chem Corp グルコース−６−リン酸デヒドロゲナーゼおよびそれをコードするｄｎａ
JP4075087B2 (ja) 1996-12-05 2008-04-16 味の素株式会社 Ｌ−リジンの製造法
DE19831609B4 (de) 1997-10-04 2009-11-12 Evonik Degussa Gmbh Verfahren zur Herstellung von Aminosäuren der Aspartat- und/oder Glutamatfamilie und im Verfahren einsetzbare Mittel
JP2000262288A (ja) 1999-03-16 2000-09-26 Ajinomoto Co Inc コリネ型細菌の温度感受性プラスミド
CN1236690C (zh) 1999-06-23 2006-01-18 德古萨股份公司 含有赖氨酸的含水动物饲料添加剂及其生产方法
KR20070087090A (ko) 1999-06-25 2007-08-27 바스프 악티엔게젤샤프트 탄소 대사 및 에너지 생산과 관련된 단백질을 코딩하는코리네박테리움 글루타미쿰 유전자
WO2001000843A2 (fr) 1999-06-25 2001-01-04 Basf Aktiengesellschaft Genes de corynebacterum glutamicum codant pour des proteines de voie metabolique
ATE340857T1 (de) 1999-07-23 2006-10-15 Archer Daniels Midland Co Verfahren zur herstellung von l-aminosäuren durch erhöhung des zellulären nadph
US6586214B1 (en) 1999-09-15 2003-07-01 Degussa Ag Method for increasing the metabolic flux through the pentose phosphate cycle in coryneform bacteria by regulation of the phosphoglucose isomerase (pgi gene)
DE19959328A1 (de) 1999-12-09 2001-06-13 Degussa Neue für das zwa1-Gen codierende Nukleotidsequenzen
JP4623825B2 (ja) 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド
CA2374261C (fr) 2000-03-20 2010-04-20 Degussa Ag Procede pour la preparation fermentative d'acides amines l avec amplification du gene zwf
PT1325135E (pt) 2000-10-13 2005-03-31 Archer Daniels Midland Co Gene da carboxilase do piruvato de corynebacterium resistente a retroacao
DE10109996A1 (de) 2001-03-01 2002-09-05 Basf Ag Verfahren zur Veränderung des Genoms von gram-positiven Bakterien mit einem neuen konditional negativ dominanten Markergen
US7160711B2 (en) 2001-08-06 2007-01-09 Degussa Ag Coryneform bacteria which produce chemical compounds I
DK1414986T3 (da) 2001-08-06 2009-01-19 Evonik Degussa Gmbh Coryforme bakterier, der frembringer kemiske forbindelser II
DE10137815A1 (de) 2001-08-06 2003-02-27 Basf Ag Verfahren zur Herstellung eines marker-freien mutierten Zielorganismus sowie dafür geeignete Plasmidvektoren
CA2455878A1 (fr) 2001-08-06 2003-05-15 Degussa Ag Bacteries coryneformes generatrices de composes chimiques
JP2003219807A (ja) 2002-01-25 2003-08-05 Ajinomoto Co Inc Ｌ−リジンを主成分とする造粒乾燥物
US20040115304A1 (en) 2002-12-16 2004-06-17 Frank Dubner Feesdstuffs additives containing L-lysine with improved abrasion resistance, and process for their production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175911A1 (en) * 2000-03-20 2003-09-18 Stephen Hans Process for the preparation of L-amino acids with amplification of the zwf gene
EP1302537A1 (fr) * 2000-06-21 2003-04-16 Kyowa Hakko Kogyo Co., Ltd. Nouvelle glucose-6-phosphate deshydrogenase
WO2003042389A1 (fr) * 2001-11-13 2003-05-22 Basf Aktiengesellschaft Gene codant pour la proteine appelees glucose-6-phosphate deshydrogenase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MORITZ B ET AL: "KINETIC PROPERTIES OF THE GLUCOSE-6-PHOSPHATE AND 6-PHOSPHOGLUCONATE DEHYDROGENASES FROM CORYNEBACTERIUM GLUTAMICUM AND THEIR APPLICATION FOR PREDICTING PENTOSE PHOSPHATE PATHWAY FLUXIN VIVO", EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN, DE, vol. 267, no. 12, June 2000 (2000-06-01), pages 3442 - 3452, XP000957993, ISSN: 0014-2956 *
OHNISHI J ET AL: "A novel gnd mutation leading to increased l-lysine production in Corynebacterium glutamicum", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 242, no. 2, 15 January 2005 (2005-01-15), pages 265 - 274, XP004696833, ISSN: 0378-1097 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007141111A3 (fr) * 2006-06-02 2008-03-06 Evonik Degussa Gmbh Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine
US7785840B2 (en) 2006-07-13 2010-08-31 Evonik Degussa Gmbh Method of production of L-amino acids
US8202706B2 (en) 2006-07-13 2012-06-19 Evonik Degussa Gmbh Method of production of L-amino acids
WO2008092956A1 (fr) * 2007-02-02 2008-08-07 Evonik Degussa Gmbh Production de l-lysine et d'additifs alimentaires contenant de la l-lysine
DE102008001874A1 (de) 2008-05-20 2009-11-26 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren
WO2022143763A1 (fr) * 2020-12-30 2022-07-07 宁夏伊品生物科技股份有限公司 Souche ayant une productivité d'acide l-glutamique améliorée, son procédé de construction et son application

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