WO2007141111A2 - Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine - Google Patents

Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine Download PDF

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WO2007141111A2
WO2007141111A2 PCT/EP2007/054566 EP2007054566W WO2007141111A2 WO 2007141111 A2 WO2007141111 A2 WO 2007141111A2 EP 2007054566 W EP2007054566 W EP 2007054566W WO 2007141111 A2 WO2007141111 A2 WO 2007141111A2
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amino acid
seq
lysine
acid sequence
polypeptide
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PCT/EP2007/054566
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German (de)
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WO2007141111A3 (fr
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Brigitte Bathe
Stephan Hans
Caroline Kreutzer
Hermann Lotter
Georg Thierbach
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Evonik Degussa Gmbh
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Priority to EP07729018A priority Critical patent/EP2026662A2/fr
Publication of WO2007141111A2 publication Critical patent/WO2007141111A2/fr
Publication of WO2007141111A3 publication Critical patent/WO2007141111A3/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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)

Definitions

  • the invention relates to a process for the preparation of light and thermally stable, granulated animal feed additives based on fermentation broth with a high content of L-lysine and low-loss processes for the preparation of broths obtained by fermentation using selected coryneform bacteria.
  • Animal feeds are supplemented with individual amino acids according to the needs of the animals.
  • z. B. with L-lysine far predominantly the L-lysine monohydrochloride is used with an L-lysine content of about 80%. Since the L-lysine is produced by fermentation, it must be for
  • EP 0 533 039 relates to processes for the preparation of an amino acid animal feed supplement based on fermentation broth, which supplement can be obtained directly from the fermentation broth by spray drying. For this purpose, a part of the biomass is separated before spray drying in a variant. By a very clean fermentation, d. H. when obtaining a fermentation broth low in organic substances, the broth can be dried even without the biomass and without additional carrier excipient to a manageable granules.
  • L-lysine-containing solid concentrates Approximately 20 wt .-% L-lysine-containing solid concentrates are known from GB 1 439 121, in which also L-lysine-containing fermentation broths are described with a pH of 4.5 and addition of sodium bisulfite.
  • EP 0 615 693 discloses a process for the preparation of an animal feed additive based on fermentation broth, in which the fermentation broth, if appropriate after removal of some of the ingredients, is converted into a fine grain which contains at least 70% by weight has a maximum particle size of 100 microns, spray-dried, and that one builds up this fine grain in a second stage to a granulate containing at least 30 wt .-% of the fine grain.
  • a concentrate containing L-lysine is prepared from a fermentation broth which is acidified to a pH of about 6.4 with HCl prior to concentration and to which bisulfite is added for stabilization. After evaporation, the mixture is further acidified to pH 4.0 and the desired product is obtained by spray-drying.
  • EP-A 1 331 220 (US 2003/152633) relates to granulated feed additives which contain L-lysine as main component.
  • the object of the invention is to provide a method for producing a feed additive containing L-lysine with improved properties using selected coryneform bacteria.
  • the invention relates to a process for the preparation of a feed additive containing L-lysine, characterized in that the following steps are carried out
  • the mixture thus obtained is concentrated, dried and preferably granulated to give a product having an L-lysine content of 10 to 70% by weight (determined as lysine base based on the total amount), and
  • Nucleotide sequence preferably in the chromosome, which codes for a lysine insensitive aspartate kinase, and wherein the isolated coryneform bacterium contains one or more of the nucleotide sequences, preferably in the chromosome, selected from the group:
  • Phosphogluconate dehydrogenase which has an amino acid sequence which is ⁇ 95% identical to SEQ ID NO: 6 and at position 329 contains any proteinogenic amino acid except L-valine,
  • nucleotide sequence encoding a polypeptide having glucose-6-phosphate dehydrogenase activity which has an amino acid sequence which is ⁇ 95% identical to SEQ ID NO: 12 and contains at position 321 any proteinogenic amino acid except glycine and optionally at position 8 any proteinogenic amino acid except L-serine,
  • Position 107 of the amino acid sequence any proteinogenic amino acid except L-tyrosine, at position 219 of the amino acid sequence any proteinogenic amino acid except L-lysine at position 233 of the amino acid sequence any proteinogenic amino acid except L-proline and at position 261 of the amino acid sequence any proteinogenic amino acid except L-tyrosine contains, and
  • the OpcA subunit of glucose-6-phosphate dehydrogenase may also be referred to as OpcA polypeptide, Glc-6-phosphate dehydrogenase opcA polypeptide or OpcA polypeptide subunit.
  • the OpcA polypeptide is also referred to as "glucose 6-phosphate dehydrogenase assembly protein".
  • the isolated coryneform bacteria are preferably those of the genus Corynebacterium. Particularly preferred are 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 names. These include, for example:
  • Strains designated "ATCC” can be purchased from the American Type Culture Collection (Manassas, Va.) Strains designated “DSM” may be purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ,
  • strains called "FERM” can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan).
  • the said strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A 5,250,434.
  • Strains designated "NRRL” can be obtained from the Agricultural Research Service Patent Culture Collection (ARS, Peoria, Ill., US).
  • the chromosome of Corynebacterium glutamicum was completely sequenced some time ago (Kalinowski et al., Journal of Biotechnology 104, 5-25 (2003)). The chromosome of Corynebacterium efficiens has also been sequenced (Nishio et al., Genome Res. 13 (7), 1572-1579 (2003)).
  • Suitable databases are, for example, the database of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), that of the Swiss Institute of Bioinformatics (Swissprot , Geneve, Switzerland), the Protein Information Resource Database (PIR, Washington, DC, USA) and the DNA Data Bank of Japan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).
  • Corynebacterium can be found in the articles by Ikeda, by Pfefferle et al. and by Mueller and Huebner in the book “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering 79, (2003), Springer Verlag, Berlin, Germany, publisher: T. Scheper), in the
  • Proteinogenic amino acids are understood to be the L-form amino acids found in natural proteins, that is to say in the proteins of microorganisms, plants, animals and humans.
  • 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 and their salts.
  • protein and polypeptide are interchangeable.
  • L-lysine-producing coryneform bacteria coryneform bacteria which are able to precipitate L-lysine into the surrounding nutrient medium or into the surrounding fermentation broth and / or to enrich the L-lysine in its cells.
  • a lysine-insensitive aspartate kinase is understood as meaning a polypeptide or protein having aspartate kinase activity (EC No. 2.7.2.4), which has a lower sensitivity to inhibition by comparison with the wild-type
  • aspartate kinases are also referred to as "feed back" resistant or desensitized aspartate kinases, and the desensitized aspartate kinases or
  • Aspartate kinase variants encoding nucleotide sequences are also referred to as lysC FBR alleles. About Numerous lysC FBR alleles are available in public databases.
  • SEQ ID NO: 1 The coding region of the wild-type lysC gene of Corynebacterium glutamicum corresponding to accession number AX756575 of the NCBI database is shown in SEQ ID NO: 1 and the polypeptide encoded by this gene is shown in SEQ ID NO: 2. Also shown in SEQ ID NO: 3 are the nucleotide sequences located upstream of the 5 'end and downstream of the 3' end of the coding region. SEQ ID NO: 4 corresponds to SEQ ID NO: 2.
  • coryneform bacteria used for the measures of the invention preferably have a lysC allele which codes for an aspartate kinase variant which has the amino acid sequence of SEQ ID NO: 2, this one or more of the amino acid substitutions selected from the group:
  • lysC A279T replacement of L-alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-threonine, see US 5,688,671 and accession numbers E06825, E06826, E08178 and 174588 to 174597
  • lysC A279V replacement of L-alanine at position 279 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-valine, see JP 6-261766 and accession number E08179
  • lysC L297Q replacement of L-leucine at position 297 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-glutamine; see DE 102006026328,
  • lysC S301F replacement of L-serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-phenylalanine, see US 6,844,176 and accession number E08180
  • lysC S301Y replacement of L-serine at position 301 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-tyrosine, see Kalinowski et al (Molecular and General Genetics 224, 317-324 (1990)) and accession number X57226)
  • lysC T308I exchange of L-threonine at position 308 of the encoded aspartate kinase protein according to SEQ ID NO: 2 for L-isoleucine, see JP 6-261766 and accession number E08181)
  • lysC S317A replacement of L-serine at position 317 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-alanine, see US 5,688,671 and accession number 174589
  • lysC R320G replacement of L-arginine at position 320 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against glycine; see Jetten et al
  • lysC G345D replacement of glycine at position 345 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-aspartic acid, see Jetten et al (Applied Microbiology and Biotechnology 43, 76-82 (995)) and accession number L16848),
  • lysC T380I exchange of L-threonine at position 380 of the encoded aspartate kinase protein according to SEQ ID NO: 2 for L-isoleucine, see WO 01/49854 and accession number AX192358
  • lysC S381F replacement of L-serine at position 381 of the encoded aspartate kinase protein according to SEQ ID NO: 2 against L-phenylalanine, see EP 0435132
  • the aspartate kinase variant contained in the isolated coryneform bacteria used may contain one (1) or more conservative amino acid substitutions in addition to the indicated 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 substitutions when aspartic acid and
  • Glutamic acid are exchanged for each other.
  • the hydroxyl-containing amino acids are called conservative substitutions when serine and threonine are interchanged.
  • Aspartate kinase variants are essentially unaffected. "Essentially unaffected” means that the enzymatic activity of said aspartate kinase variants is conservative Amino acid exchange increased or decreased by a maximum of 10%, a maximum of 7.5%, a maximum of 5%, a maximum of 2.5% or a maximum of 1%.
  • a) be obtained from the publicly accessible core collections, or
  • d) are obtained and identified by classical mutagenesis methods, optionally followed by selection on AEC-containing agar plates and sequencing of the lysC gene.
  • a mutant of Corynebacterium glutamicum, which contains the amino acid exchanges lysC A279T and lysC S381F in the aspartate kinase, is for example under the
  • a mutant of Corynebacterium glutamicum, which contains the amino acid substitutions lysC S301Y in the aspartate kinase, is described in EP 0387527 with the designation DM58-1 and deposited in the form of the strain DM58-l / pDM6 as DSM4697 (EP 0358940) in the DSM.
  • a mutant of Corynebacterium glutamicum containing the amino acid exchange lysC T311I in aspartate kinase is described in PCT / EP2005 / 012417 and deposited as DSM 16833 in the DSM.
  • a mutant of Corynebacterium glutamicum, which contains the amino acid substitution lysC S317A in aspartate kinase, is deposited, for example, under the name FERM P-6464 (JP 58-170487) at the National Institute of Advanced Industrial Science and Technology.
  • a mutant of Corynebacterium glutamicum which contains the amino acid exchange lysC T380I in the aspartate kinase, for example, under the name ATCC21529 deposited with the ATCC.
  • a mutant of Corynebacterium glutamicum containing the amino acid substitution lysC S381F in aspartate kinase is known by the designation MH20-22B (Menkel et al., Applied and Environmental Microbiology 55, 684-688 (1989)). This strain is deposited with the DSMZ under the designation DSM 16835.
  • nucleotide sequences of lysC FBR alleles can be found in the published publications and the sequences available in public databases.
  • a DNA fragment containing the mutation of interest is transferred to the desired strain of a coryneform bacterium, and the mutation is incorporated into the chromosome of the desired strain through at least two recombination events or crossover events, respectively relevant strain Existing sequence of a gene exchanged for the mutated sequence.
  • the desired alleles can be obtained from the deposited strains by means of the polymerase chain reaction or directly from stored plasmid DNA.
  • DNA of the lysC FBR allele coding for the aspartate kinase variant with the amino acid exchange lysC T311I is available in the form of the strain FERM BP-6689 (US Pat. No. 6,893,848).
  • Nucleotide sequence can be detected by sequencing or also by the fluorescence resonance energy transfer (FRET) method by melting curve analysis (Lay et al., Clinical Chemistry, 43: 2262-2267 (1997)).
  • FRET fluorescence resonance energy transfer
  • mutagenic oligonucleotides (TA Brown: Genetic Engineering for Beginners, Spektrum Akademischer Verlag, Heidelberg, 1993) or the method of Papworth et al. (Strategies 9 (3), 3-4 (1996)) using the Quik Change Site-directed Mutagenesis Kit from Stratagene (La Jolla, California, USA).
  • the DNA of the desired lysC FBR alleles can be prepared by chemical synthesis, for example, by the phosphoramidite method (Beaucage et al., Tetrahedon Letters 22, 1859-1862 (1981).
  • the obtained alleles can in turn be incorporated into the chromosome of coryneform bacteria by the method of allele exchange described above.
  • mutants For the preparation of mutants, classical in vivo mutagenesis methods 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 can be used. Mutagenesis methods are described, for example, in the Manual of Methods for General Bacteriology (Gerhard et al. (Eds.), American Society for Microbiology, Washington, DC, USA, 1981) or Tosaka et al.
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • 5-bromouracil 5-bromouracil
  • Typical mutageneses using 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 taken from the mutagenized cell population. Subsequently, the lysC gene of the mutants is amplified by means of the polymerase chain reaction (PCR), the nucleotide sequence of the lysC gene of these mutants is determined and the amino acid sequence of the encoded aspartate kinase variant determined from the resulting nucleotide sequence.
  • PCR polymerase chain reaction
  • primers are advantageously selected which bind to the DNA portion located upstream of the lysC gene and to the DNA portion located downstream of the complementary strand of the lysC gene (see SEQ ID NO: 3). Possibly It is also possible to select primers which bind within the coding region of the lysC gene.
  • the mutagenized cell population is previously subjected to selection for minimal agar supplemented with AEC or mixtures of AEC and L-threonine.
  • the corresponding strains resistant to AEC are used for the further selection procedure.
  • the activity of aspartate kinase can be characterized. It is also possible to characterize the strains by fermentation in a suitable medium. Instructions on this can be found, for example, in US Pat. No. 6,893,848.
  • suitable robotic systems such as Zimmermann et al. (VDI Reports No. 1841, VDI-Verlag, Dusseldorf, Germany 2004, 439-443) or Zimmermann (Chemie Ingenieurtechnik 77 (4), 426-428 (2005)) described, numerous mutants can be examined in a short time.
  • isolated coryneform bacteria whose aspartate kinase variants contain one or more of the amino acid substitutions selected from the group lysC A279T, lysC L297Q, lysC S317A, lysC T380I and lysC S381F.
  • SEQ ID NO: 31 describes the coding region of a lysC allele encoding an aspartate kinase variant containing L-threonine (lysC A279T) at position 279 and L-alanine at position 317 (lysC S317A).
  • SEQ ID NO: 32 shows the amino acid sequence of the polypeptide.
  • SEQ ID NO: 33 describes the coding region of a lysC allele encoding an aspartate kinase variant containing at position 297 L-glutamine (lysC L297Q) and at position 317 L-alanine (lysC S317A).
  • SEQ ID NO: 34 shows the amino acid sequence of the polypeptide.
  • the invention also relates to an isolated polynucleotide which codes for an aspartate kinase variant which, at position 297 of SEQ ID NO: 2, any proteinogenic amino acid except L-leucine, preferably L-glutamine and optionally one or more of the amino acid substitutions selected from the group lysC A279T , lysC A279V, lysC S301F, lysC S301Y, lysC T308I, lysC T311I, lysC S317A, lysC R320G, lysC G345D, lysC T380I, lysC S381F, and lysC S317A.
  • the invention also provides vectors which contain the polynucleotide.
  • the invention furthermore relates to coryneform bacteria, preferably of the genus Corynebacterium, more preferably of the species
  • Corynebacterium glutamicum which contain the polynucleotide and in which it is optionally overexpressed.
  • the invention also relates to processes for the production of feed additives containing L-lysine or L-lysine by fermentation of said bacteria in a suitable nutrient medium, wherein L-lysine accumulates in the cells of the bacteria or in the nutrient medium.
  • the L-lysine formed is then collected and optionally isolated or further processed together with the major part (> 50%) of the biomass by dehydration to a solid product.
  • the isolated coryneform bacteria used for the method according to the invention moreover comprise one or more, preferably at least two, more preferably at least three and most preferably four of the nucleotide sequences selected from the group:
  • a nucleotide sequence which codes for a polypeptide having glucose-6-phosphate dehydrogenase activity which has an amino acid sequence which is ⁇ 95%, preferably ⁇ 97%, ⁇ 98% or ⁇ 99% and most preferably 100% identical to SEQ ID NO: 12 and at position 321 any proteinogenic amino acid except glycine, preferably L-serine and optionally at position 8 any proteinogenic amino acid except L-serine, preferably L-threonine contains;
  • a nucleotide sequence encoding a polypeptide having malate quinone oxidoreductase activity which is a
  • nucleotide sequence encoding a polypeptide having 6-phosphogluconate dehydrogenase activity having the amino acid sequence of SEQ ID NO: 6 containing at position 329 L-methionine instead of L-valine is> 90% or> 95% , preferably> 97% or> 98% identical to SEQ ID NO: 5.
  • SEQ ID NO: 9 shows an example of such a nucleotide sequence.
  • nucleotide sequence encoding a polypeptide having glucose-6-phosphate dehydrogenase activity having the amino acid sequence of SEQ ID NO: 12 is that at position 321 L-serine instead of glycine and optionally at position 8 L-threonine instead L-serine contains> 90% or> 95%, preferably> 97% or> 98% identical to SEQ ID NO: 11.
  • SEQ ID NO: 15 shows an example of such a nucleotide sequence.
  • nucleotide sequence encoding an OpcA subunit of glucose-6-phosphate dehydrogenase having the amino acid sequence of SEQ ID NO: 18 is one or more of the amino acid substitutions selected from the group at position 107 L-histidine L-tyrosine, at position 219 L-asparagine instead of L-lysine, at position 233 L-serine instead of L-proline and at position 261 contains L-histidine instead of L-tyrosine, to ⁇
  • SEQ ID NO: 21 an example of such a nucleotide sequence is shown.
  • the nucleotide sequence is for a polypeptide having malate quinone oxidoreductase activity which has the amino acid sequence of SEQ ID NO: 24, which contains at position 111 L-phenylalanine or L-alanine instead of L-serine and optionally at position 201 L-alanine instead of L-serine, to> 90% or> 95 %, preferably> 97% or> 98% identical to SEQ ID NO: 23.
  • SEQ ID NOS: 27 and 29 show examples of such nucleotide sequences.
  • Isolated coryneform bacteria containing a nucleotide sequence coding for a polypeptide with 6-phosphogluconate dehydrogenase, which is the indicated
  • SEQ ID NO: 5 shows the coding region of the wild-type 6-phosphogluconate dehydrogenase gene of Corynebacterium glutamicum.
  • the 6-phosphogluconate dehydrogenase gene is also commonly referred to as the gnd gene.
  • SEQ ID NO: 6 shows the amino acid sequence of the encoded polypeptide. In SEQ ID NO: 7, in addition to the coding region, the upstream and downstream nucleotide sequences are indicated.
  • SEQ ID NO: 8 is identical to SEQ ID NO: 6.
  • SEQ ID NO: 9 shows the nucleotide sequence of the coding region of the gnd allele present in the mutant DSM16834. In SEQ ID NO: 10, the amino acid sequence of the 6-phosphogluconate dehydrogenase variant is given.
  • Isolated coryneform bacteria containing a nucleotide sequence encoding a polypeptide having glucose-6-phosphate dehydrogenase activity containing the indicated amino acid substitution at position 321 and optionally at position 8 are described in PCT / EP2006 / 060851.
  • the mutant DSM17119 which contains a nucleotide sequence which codes for a glucose-6-phosphate dehydrogenase variant which contains the amino acid L-serine at position 321 instead of glycine and optionally the amino acid L-serine at position 8 instead of L-threonine.
  • Amino acid exchange at position 321 is also referred to as zwf G321S and that at position 8 as zwf S8T.
  • SEQ ID NO: 11 shows the coding region of the wild-type glucose-6-phosphate dehydrogenase gene of Corynebacterium glutamicum.
  • Phosphate dehydrogenase gene is also commonly referred to as the zwf gene.
  • SEQ ID NO: 12 shows the amino acid sequence of the encoded polypeptide. In SEQ ID NO: 13, in addition to the coding region, the upstream and downstream nucleotide sequences are indicated.
  • SEQ ID NO: 14 is identical to SEQ ID NO: 12.
  • SEQ ID NO: 15 shows the nucleotide sequence of the coding region of the zwf allele present in the mutant DSM17119.
  • SEQ ID NO: 16 indicates the amino acid sequence of the 6-phosphogluconate dehydrogenase variant.
  • Isolated coryneform bacteria containing a nucleotide sequence encoding an OpcA subunit of glucose-6-phosphate dehydrogenase having the indicated amino acid substitutions at one or more of positions 107, 219, 233 and 261 of the amino acid sequence are described in DE 102005023829 described.
  • This patent application describes the mutant DSM 17223 which codes for an OpcA polypeptide which is at position 107 L-histidine instead of L-tyrosine, at position 219 L-asparagine instead of L-lysine, at position 233 L-serine instead of L- Proline and at position 261 contains L-histidine instead of L-tyrosine.
  • These amino acid substitutions are also referred to as opcA Y107H, opcA K219N, opcA P233S and opcA Y261H.
  • SEQ ID NO: 17 shows the nucleotide sequence of the coding region of the wild-type OpcA polypeptide of Corynebacterium glutamicum.
  • the gene that for the OpcA polypeptide is also commonly referred to as the opcA gene.
  • SEQ ID NO: 18 shows the amino acid sequence of the encoded polypeptide. In SEQ ID NO: 19, in addition to the coding region, the upstream and downstream nucleotide sequences are indicated.
  • SEQ ID NO: 20 is identical to SEQ ID NO: 18.
  • SEQ ID NO: 21 shows the nucleotide sequence of the coding region of the opcA allele present in the mutant DSM17223.
  • SEQ ID NO: 22 indicates the amino acid sequence of the OpcA variant.
  • Isolated coryneform bacteria containing a nucleotide sequence encoding a malate quinone oxidoreductase polypeptide polypeptide having the indicated amino acid substitutions at position III and optionally at position 201 are described in PCT / EP2005 / 057216.
  • This patent application describes the mutant DSM 16937 which contains a nucleotide sequence which codes for a malate quinone oxidoreductase variant which contains the amino acid L-penylalanine at position III instead of L-serine. This amino acid exchange is also referred to as Mqo SlIF.
  • PCT / EP2005 / 057216 describes a mutant which contains a nucleotide sequence coding for a malate quinone oxidoreductase variant which, at position III instead of L-serine, contains the amino acid L-alanine and at position
  • 201 contains the amino acid L-serine instead of L-alanine. These amino acid substitutions are also referred to as Mqo SlIlA and mqo A201S.
  • SEQ ID NO: 24 shows the amino acid sequence of the encoded polypeptide.
  • SEQ ID NO: 25 in addition to the coding region, the upstream and downstream nucleotide sequences are specified.
  • SEQ ID NO: 26 is identical to SEQ ID NO: 24.
  • SEQ ID NO: 27 represents the nucleotide sequence of the coding region of the mqo allele present in the mutant DSM 16937.
  • SEQ ID NO: 28 reproduces the amino acid sequence of the malate quinone oxidoreductase variant.
  • SEQ ID NO: 29 describes the nucleotide sequence of the coding region of the mqo allele of a mutant containing the amino acid substitutions mqo SlIlA and mqo A201S.
  • SEQ ID NO: 30 the amino acid sequence of this malate quinone oxidoreductase variant is reproduced.
  • the isolated coryneform bacteria containing one or more of the indicated amino acid substitutions in the 6-phosphogluconate dehydrogenase, the glucose-6-phosphate dehydrogenase, the OpcA polypeptide and the malate quinone oxidoreductase, the classical mutagenesis methods given and in vitro mutagenesis and allelic exchange methods. If necessary, the methods are combined.
  • Polymerase chain reaction preferably using primers that bind to the upstream and downstream of the genes concerned polynucleotide sequences amplified and then sequencing the resulting PCR products.
  • the following isolated coryneform bacteria can be used for a method according to the invention:
  • the mutant DM1910 was prepared by two rounds of mutagenesis and screening from Corynebacterium glutamicum NRRL B-11474.
  • the strain DM1910 contains besides the Apartatkinase-related mutations lysC A279T and lysC S381F a gnd allele encoding a 6-phosphogluconate dehydrogenase containing the amino acid exchange gnd V329M and a zwf allele encoding a glucose-6-phosphate dehydrogenase encoding the amino acid substitution Contains G321S.
  • strain DM1917 was prepared by the allele exchange method using the exchange vector pKl ⁇ mobsacB opcAaa4ex described in US Patent Application 60/710314.
  • This strain contains an opcA allele encoding an OpcA polypeptide containing the amino acid exchanges opcA Y107H, opcA K219N, opcA P233S and opcA Y261H.
  • the mutant DM1913 was isolated from ATCC14067 by several rounds of mutagenesis, screening and analysis of selected genes. It contains a gnd allele encoding a 6-phosphogluconate dehydrogenase containing the amino acid substitution gnd V329M, a zwf allele encoding a glucose-6-phosphate dehydrogenase containing the amino acid substitution zwf G321S, an opcA- Allele encoding an OpcA polypeptide containing the amino acid exchanges opcA Y107H and opcA K219N, an mqo allele encoding a malate quinone oxidoreductase containing the amino acid substitutions mqo SlIlA and mqo A201S and a lysC allele, which encodes an aspartate kinase containing the amino acid substitution lysC S317A.
  • a strain was prepared by the allele exchange method, the aspartate kinase of which contains the amino acid exchange lysC T279A in addition to the amino acid exchange lysC S317A.
  • This strain was named DM1918.
  • coryneform bacteria in which the mentioned variants of glucose-6-phosphate dehydrogenase, the OpcA subunit of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are used individually or in combination with one another overexpressed.
  • Escherichia coli Bacillus subtilis, Bacillus megaterium, Mycobacterium tuberculosis, Mycobacterium bovis, Streptomyces coeliclor, Lactobacillus acidophilus, Lactobacillus johnsonii, Bifidobacterium longum, and Saccharomyces cerevisiae.
  • the endogenous genes or polynucleotides of coryneform bacteria are used.
  • Endogenous genes or polynucleotides are understood as meaning the open reading frame (ORF), genes or alleles or their polynucleotides present in the population of a species such as, for example, Corynebacterium glutamicum.
  • Uberexpression is generally understood to mean an increase in the intracellular concentration or activity of a ribonucleic acid, a protein or an enzyme.
  • the increase in concentration or activity can be achieved, for example, by increasing the copy number of the corresponding polynucleotides chromosomally or extrachromosomally by at least one copy.
  • a widely used method for increasing the copy number consists of incorporating the corresponding polynucleotide into a vector, preferably a plasmid, which is replicated by a coryneform bacterium.
  • Suitable plasmid vectors are, for example, pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554) or those described by Tauch et al. (Journal of Biotechnology 99, 79-91 (2002)) described pSELF vectors.
  • pZl Mach et al., Applied and Environmental Microbiology (1989) 64: 549-554
  • Tauch et al. Journal of Biotechnology 99, 79-91 (2002)
  • transposons insertion elements
  • phages phages
  • Such genetic systems are described for example in the patents US 4,822,738, US 5,804,414 and US 5,804,414.
  • the IS element ISaB1 described in WO 92/02627 or the transposon Tn 45 of the plasmid pXZ10142 can be used.
  • Another common method for achieving overexpression is the method of chromosomal gene amplification.
  • this method at least one additional copy of the polynucleotide of interest is inserted into the chromosome of a coryneform bacterium.
  • Such amplification methods are described, for example, in WO 03/014330 or WO 03/040373.
  • Another method for achieving an 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 Pätek et al. (Journal of Biotechnology 104 (1-3), 311-323 (2003))
  • the well-known Amann et al. (Gene 69 (2), 301-315 (1988)) and Amann and Brosius (Gene 40 (2-3), 183-190 (1985)), such as T3, T7, SP6, M13, lac, tac, and trc.
  • Such a promoter may be, for example, upstream of the subject gene, typically in the Distance of about 1 - 500 nucleotides are inserted from the start codon.
  • the activity or concentration of the corresponding polypeptide will generally be at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000 % or 2000% based on the activity or concentration of the polypeptide in the strain prior to overexpression.
  • one or more of the genes or alleles is / are selected from the group
  • Very particularly preferred is the common overexpression of two or more of the genes selected from the group lysC FBR allele, dapA gene, dapB gene, ddh gene and lysA gene.
  • attenuation or “attenuation” in this context describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme or protein and optionally combines these measures.
  • Gene expression can be reduced by appropriate culture or by genetic modification (mutation) of the signal structures of gene expression.
  • Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and Terminators.
  • An example of the targeted regulation of gene expression is the cloning of the gene to be attenuated under the control of an inducible by the addition of metered amounts of IPTG (isopropyl - /? - D-thiogalactopyranoside)
  • Promoter such as the trc promoter or the tac promoter.
  • vectors such as, for example, the Escherichia coli expression vector pXK99E (WO0226787, deposited under the Budapest Treaty on July 31, 2001 in DH5alpha / pXK99E as DSM14440 in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) or pVWEx2 (Wendisch, Ph. D. thesis, reports of Anlagens congress Anlagen, Jülich, Jül-3397, ISSN 0994-2952, Garlich, Germany (1997)), which enable an IPTG-dependent expression of the cloned gene in Corynebacterium glutamicum.
  • Another method for specifically reducing gene expression is the antisense technique, with short ones Oligodeoxynukleotide or vectors for the synthesis of longer antisense RNA are brought into the target cells.
  • the antisense RNA can there bind to complementary sections of specific mRNAs and reduce their stability or block the translatability. An example of this is found by the person skilled in Srivastava et al. (Applied Environmental Microbiology 66 (10): 4366-4371 (2000)).
  • Mutations are transitions, transversions, insertions and deletions of at least one (1) base pair or nucleotide. Depending on the effect of the mutation caused
  • missense mutations Amino acid exchanges on enzyme activity are referred to as missense mutations or nonsense mutations.
  • the missense mutation results in the replacement of a given amino acid in one protein for another, in particular a non-conservative amino acid substitution.
  • 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 of the gene and thus to a premature termination of translation.
  • Insertions or deletions of at least one base pair in a gene result in frameshift mutations that lead to the incorporation of incorrect amino acids or premature termination of translation, resulting in a stop codon in the coding region as a result of the mutation This also leads to a premature termination of the translation Deletions of at least one (1) or more codons also typically lead to a complete
  • the measures mentioned are preferably carried out in the 5 '-terminal part of the coding region which codes for the N-terminus of the polypeptide.
  • the total length of a polypeptide (measured as the number of chemically linked L-amino acids) is 100%, part of the amino acid sequence belonging to the N-terminus of the polypeptide, which is calculated starting from the starting amino acid L, belongs within the scope of the present invention -Formyl-methionine contains 80% of the subsequent L-amino acids.
  • the activity or concentration of the corresponding polypeptide is generally set at 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10%, 0 to 5% or 0 to 1% of the activity or concentration of the Wild-type protein, or activity, or
  • a “starting microorganism” is meant the microorganism on which the attenuation measures are carried out.
  • one or more of the genes is selected from the group
  • Oxidoreductase coding poxB gene (EP 1 096 013)
  • the fermentation of coryneform bacteria can be continuous - as in the PCT / EP
  • 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 set forth in the Manual of Methods for General Bacteriology, of the American Society for Bacteriology (Washington, DC, USA, 1981). contain. The terms culture medium and
  • Fermentation medium or medium are mutually exchangeable.
  • sugars and carbohydrates such as e.g. Glucose, sucrose, lactose, fructose, maltose,
  • sucrose-containing solutions from sugarcane or sugar cane production starch, starch hydrolyzate and cellulose, oils and fats such as soya oil, sunflower oil, peanut oil and coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid,
  • Alcohols such as glycerin, methanol and ethanol and organic acids such as acetic acid can be used. These substances can be used individually or as a mixture.
  • the nitrogen source there may be used 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 ammonia, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium nitrate, preferably ammonia or ammonium sulfate.
  • organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea
  • inorganic compounds such as ammonia, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium nitrate, preferably ammonia or ammonium sulfate.
  • the nitrogen sources can be used singly or as a mixture.
  • phosphorus source can phosphoric acid
  • the culture medium must further contain salts, for example in the form of sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, for example magnesium sulphate or iron sulphate, which are necessary for growth.
  • salts for example in the form of sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, for example magnesium sulphate or iron sulphate, which are necessary for growth.
  • essential growth substances such as amino acids such as homoserine and vitamins such as thiamine, biotin or pantothenic acid can be used in addition to the above-mentioned substances.
  • suitable precursors of the respective amino acid can be added to the culture medium.
  • the said starting materials can be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.
  • pH of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, preferably ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are used in a suitable manner.
  • the pH is generally adjusted to a value of 6.0 to 9.0, preferably 6.5 to 8.
  • Foaming can be used anti-foaming agents, such as Fettsaurepolyglykolester.
  • suitable selective substances such as antibiotics may optionally be added to the medium.
  • oxygen or oxygen-containing gas mixtures such as air, are introduced into the culture.
  • the use of liquids enriched with hydrogen peroxide is also possible.
  • the fermentation is at
  • Overpressure for example, at a pressure of 0.03 to 0.2 MPa, driven.
  • the temperature of the culture is normally from 20 0 C to 45 0 C and preferably at 25 0 C to 40 0 C.
  • the cultivation is continued until a maximum of L-lysine has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes longer cultivation times are possible.
  • the fermentation broth produced in this way is subsequently further processed according to the invention.
  • 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 media used during the fermentation contains / contain all substances or components which ensure an increase of the microorganism and a formation of the desired amino acid.
  • 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 in addition to L-lysine and optionally excreted. These include L-amino acids, which are less than 30%, 20% or 10% compared to the desired L-lysine. These include organic Acids bearing 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 L-lysine 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.
  • the biomass is partially or completely removed from the fermentation broth before the further process steps.
  • the resulting fermentation broth is then further processed according to the invention, by carrying out a process which comprises at least the following steps:
  • Steps b) and c) sets a sulfide / L-lysine ratio of 0.85 to 1.2, preferably 0.9 to 1.0, more preferably> 0.9 to ⁇ 0.95 in the broth, and
  • step c) can also be carried out before step b).
  • Sulfate-containing compounds in the sense of the abovementioned process steps are in particular ammonium sulfate and sulfuric acid.
  • a product having an L-lysine content of 10 to 70 wt .-% (calculated as lysine base, based on the total amount) and a molar sulfate / L-lysine ratio of 0.85 to 1.2 , preferably 0.9 to 1.0, more preferably> 0.9 to ⁇ 0.95.
  • V 2 ⁇ [SO 4 2 ] / [L-lysine].
  • the fermentation broth of one or more of the salts of sulphurous acid (sulphites) is selected from the group
  • Ammonium, alkali, and alkaline earth metal salt in an amount of 0.01 to 0.5 wt .-%, preferably 0.1 to 0.3 wt .-%, particularly preferably 0.1 to 0.2 wt .-% added to the fermentation broth. It is preferred Alkali hydrogen sulfite and particularly preferably used sodium hydrogen sulfite.
  • the sulfites in particular sodium hydrogen sulfite are preferably added as a solution before the concentration of the fermentation broth.
  • the amount used is preferably taken into account in the adjustment of the sulfide / L-lysine ratio.
  • the pH adjustment in the fermentation broth was found to be pH> 4 to 5.2, the sulphate / L-lysine ratio increased to 0.85 to 1.2 and the sulphite addition to 0.01 to 0.5 wt .-% in the fermentation broth loss of L-lysine during the
  • 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.
  • 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 manufactured product ,
  • 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 manufactured product.
  • Fermentation broths from which the biomass has been partially or totally removed can also be used for standardization in the production of the product.
  • this also applies to the pure compounds L-lysine base and lysine sulfate.
  • the fermentation broth obtained is acidified with sulfuric acid prior to concentration and, if appropriate, admixed with ammonium sulfate.
  • the broth may also be stabilized and lightened by the addition of preferably sodium bisulfite (sodium bisulfite) or another salt, for example, ammonium, alkali or alkaline earth salt of the sulfurous acid.
  • biomass is completely or partially separated, this is preferably carried out before the reduction of the pH according to the invention and the addition of ammonium sulfate and sulfite salt.
  • 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.
  • the term "fermentation broth base" means that a product contains at least a portion of the components of the fermentation broth.
  • the broth with known methods such as heating or heating, for example with the aid of a Rotary evaporator, Dunn Anlagenverdampfers or falling film evaporator, or withdrawn or thickened or concentrated by reverse osmosis or nanofiltration water.
  • This concentrated broth can then be worked up by freeze-drying, spray-drying, spray granulation or other methods, for example in the circulating fluidized bed according to PCT / EP 2004/006655, into free-flowing, fine-particle or coarse-grained products, in particular granules.
  • a product having the desired grain size is isolated from the resulting granules by sieving or dust separation.
  • the free-flowing, finely divided powder can in turn be converted by suitable compacting or granulating process into a coarse-grained, readily pourable, storable and substantially dust-free product.
  • the granules are z. B. can be prepared by the method according to EP-B 0 615 693 or EP-B 0809 940, US 5,840,358 or WO 2005/006875 or WO 2004/054381.
  • Free-flowing refers to powders that flow out of a series of glass outlet vessels with different sized outflow openings at least from the vessel with the opening 5 mm (millimeters) unhindered (Klein: Soaps, Ole, Fats, Wachse 94, 12 (1968)).
  • finely divided is meant a powder with a predominant proportion (> 50%) of a grain size of 20 to 200 ⁇ m in diameter.
  • coarse grained is meant a product having a predominant proportion (> 50%) of a grain size of 200 to 2000 ⁇ m in diameter.
  • dust-free means that the product contains only small amounts ( ⁇ 5%) of grain sizes below 100 microns in diameter.
  • the proportion of dust, ie particles having a particle size of ⁇ 100 ⁇ m, is preferably> 0 to 1% by weight, more preferably not more than 0.5% by weight.
  • the bulk density of the preferred products is generally 600 to 950 kg / m 3 , in particular 650 to 900 kg / m 3 .
  • 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 (EP-A 0 743 016) or stearates.
  • oils mineral oils, vegetable oils or mixtures of vegetable oils can be used become. Examples of such oils are soya oil, olive oil, soyaol / lecithin mixtures. In the same way, silicone oils, polyethylene glycols or hydroxyethycellulose are also suitable.
  • the content of oil in the product is 0.02 to 2.0 wt .-%, preferably 0.02 to 1.0 wt .-%, and most preferably 0.2 to 1.0 wt .-% based on the Total amount of feed additive.
  • the product can also be applied to a known and customary in animal feed processing organic or inorganic carrier material such as silicas, silicates, shot, bran, flour, starch, sugar or other and / or with conventional organic or inorganic carrier material such as silicas, silicates, shot, bran, flour, starch, sugar or other and / or with conventional organic or inorganic carrier material such as silicas, silicates, shot, bran, flour, starch, sugar or other and / or with conventional
  • Thickening or binding agents are mixed and stabilized. Application examples and processes for this purpose are described in the literature (Die Mühle + Mischfuttertechnik 132 (1995) 49, page 817).
  • Coating with film formers such as metal carbonates, silicas, silicates, alginates, stearates, starches, gums and cellulose ethers, as described in DE-C 41 00 920, be brought into a state in which it is stable to digestion by animal stomach in particular the stomach of Wiederkauern is.
  • the L-lysine may be added 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 solid products produced by the process according to the invention are preferably granules and have, inter alia, the following properties:
  • L-lysine ratio in the range of, for example, 0.75 to 0.87, as known in the art.
  • the color values or the "degree of whiteness" of the products are determined according to the tristimulus method (L * a * b * color measurement) defined in the German Industrial Standard 5033.
  • a 3-range color measuring instrument for dyeing and remission measurement such as, for example, the Micro Color II LMC colorimeter from Dr. Lange (Düsseldorf, Germany), in which the diffuse reflection of the sample is measured at an angle of 8 ° Transfer the device to the exactly defined standard color filters for splitting.
  • the color values are preferably in the ranges:
  • lysine base 10% by weight to 70% by weight, preferably 30 to 60% by weight or 30 to 65% by weight and very particularly preferably 40% by weight up to 60% by weight or 40% by weight to 65% by weight, based on the total amount of the product.
  • the ratio of sulfate to L-lysine in the product is 0.85 to 1.2, preferably 0.9 to 1.0, particularly preferably> 0.9 to ⁇ 0.95.
  • the water content is between 0.1 wt .-% and at most 5 wt .-%.
  • the water content is preferably at most 4% by weight, more preferably at most 3% by weight and most preferably at most 2.5% by weight. Water contents of a maximum of 2 wt .-% are also possible.
  • DM1913 was deposited on May 15, 2006 with the German Collection of Microorganisms and Cell Cultures under the Budapest Treaty (DSMZ, Braunschweig, Germany) as DSM 18256.
  • the lysC allele contained in strain DM1918 was duplicated by the method of tandem duplication at the lysC locus described in WO 03/014330. Another copy of the lysC allele was incorporated into the aecD gene of the resulting strain as described in WO 03/040373.
  • strain DM1918_3xlysC obtained in this way was then used in a fed batch process for the preparation of an L-lysine-containing fermentation broth.
  • the fermentation was carried out in accordance with the patent EP 0 533 039.
  • Corn steep liquor was used instead of corn gluten hydrolyzate.
  • Ammonium sulfate was added using a sterile ammonium sulfate solution (37% by weight) as needed.
  • the defoamer was also dosed separately according to requirements.
  • L-lysine By adding L-lysine, a content of L-lysine of 57.5% by weight in dry matter was adjusted.
  • the pH of the broth was 7.8 and the sulfate / L-lysine ratio V was 0.80.
  • the broth was then heated to about 60 0 C, concentrated under vacuum and then, as in the
  • Patent EP 0 809940 described, granulated.
  • the content of L-lysine in the obtained product (product 1) was 52.6 wt .-%.
  • the residual water content in the product was about 2% by weight.
  • the lysine loss was thus about 4.9 wt .-%.
  • the sulphate / L-lysine ratio V was increased to about 0.90 (based on the dry mass) by adding ammonium sulfate using a 37 percent solution. Subsequently, the pH was adjusted to about 5.1 by addition of concentrated sulfuric acid. As a result, the sulphate / L-lysine ratio V increased to about 0.92 (based on the dry mass). The content of L-lysine in the dry matter decreased due to the dilution effect to a value of 55.4 wt .-%.
  • the broth was then heated to about 60 0 C, concentrated under vacuum and then granulated as described in the patent EP 0809940.
  • the content of L-lysine in the obtained product (product 2) was 52.7% by weight.
  • the residual water content in the product was about 2% by weight.
  • the lysine loss was thus about 2.4 wt .-%.
  • Product 2 was optically brighter than product 1.

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Abstract

L'invention concerne un procédé de production d'additifs alimentaires pour animaux, utilisant un bouillon de fermentation à haute teneur en L-lysine et des procédés à pertes moindres pour produire des bouillons obtenus par fermentation en utilisant des bactéries corynéformes contenant des mutations sélectionnées, ainsi que ces additifs alimentaires pour animaux eux-mêmes.
PCT/EP2007/054566 2006-06-02 2007-05-11 Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine WO2007141111A2 (fr)

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EP2246415A2 (fr) * 2008-01-28 2010-11-03 CJ CheilJedang Corporation Promoteur amélioré et procédé de production de l-lysine au moyen de ce promoteur
WO2013167659A1 (fr) 2012-05-09 2013-11-14 Evonik Industries Ag Additif d'aliment pour animaux contenant un acide aminé l, sous forme de granulé, à base de bouillon de fermentation et son procédé de production
EP2865275A1 (fr) * 2013-10-24 2015-04-29 Evonik Industries AG Additifs de nourriture pour animaux contenant des acides animés L
EP2865274A1 (fr) 2013-10-24 2015-04-29 Evonik Industries AG Additifs de nourriture pour animaux contenant des acides animés L
EP2940144A1 (fr) * 2014-04-30 2015-11-04 Evonik Degussa GmbH Procédé de production de L-lysine en utilisant une bactérie alcaliphile
WO2016030441A1 (fr) * 2014-08-29 2016-03-03 Chr. Hansen A/S Acides aminés essentiels fournis par bacillus dans une charge liquide
EP3395827A1 (fr) 2017-04-27 2018-10-31 Universität Bielefeld Biosynthèse d'acides aminés et de caroténoïdes à l'aide de corynebacterium glutamicum recombiné
WO2020049313A1 (fr) 2018-09-06 2020-03-12 3F Bio Ltd Processus et produit de celui-ci
CN112662713A (zh) * 2020-12-28 2021-04-16 天津科技大学 一种高密度发酵生产l-赖氨酸的培养基及其方法

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EP2826384A1 (fr) 2013-07-16 2015-01-21 Evonik Industries AG Procédé destiné au séchage de biomasse
US11324234B2 (en) 2014-10-02 2022-05-10 Evonik Operations Gmbh Method for raising animals
DK180016B1 (da) * 2014-10-02 2020-01-22 Evonik Degussa Gmbh Feedstuff of high abrasion resistance and good stability in water, containing PUFAs
CA2958460C (fr) 2014-10-02 2022-09-13 Evonik Industries Ag Procede de production d'un aliment pour animaux contenant des agpi par extrusion d'une biomasse contenant des agpi
CA2958457C (fr) 2014-10-02 2022-10-25 Evonik Industries Ag Procede de production d'une biomasse contenant des agpi qui presente une haute stabilite cellulaire

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EP2246415A2 (fr) * 2008-01-28 2010-11-03 CJ CheilJedang Corporation Promoteur amélioré et procédé de production de l-lysine au moyen de ce promoteur
JP2011510625A (ja) * 2008-01-28 2011-04-07 シージェイ チェイルジェダン コーポレイション 改良されたプロモーターおよびこれを用いたｌ−リシンの生産方法
EP2246415A4 (fr) * 2008-01-28 2011-06-01 Cj Cheiljedang Corp Promoteur amélioré et procédé de production de l-lysine au moyen de ce promoteur
US8426577B2 (en) 2008-01-28 2013-04-23 Cj Cheiljedang Corporation Promoter and a production method for L-lysine using the same
WO2013167659A1 (fr) 2012-05-09 2013-11-14 Evonik Industries Ag Additif d'aliment pour animaux contenant un acide aminé l, sous forme de granulé, à base de bouillon de fermentation et son procédé de production
US20130302470A1 (en) * 2012-05-09 2013-11-14 Evonik Industries Ag L-amino-acid-containing feed additive in the form of fermentation-broth-based granules, and processes for its preparation
CN104270957A (zh) * 2012-05-09 2015-01-07 赢创工业集团股份有限公司 基于发酵液的粒状材料形式的包含l-氨基酸的动物饲料添加剂及其制备方法
CN104270957B (zh) * 2012-05-09 2016-08-24 赢创德固赛有限公司 基于发酵液的粒状材料形式的包含l-氨基酸的动物饲料添加剂及其制备方法
EP2865275A1 (fr) * 2013-10-24 2015-04-29 Evonik Industries AG Additifs de nourriture pour animaux contenant des acides animés L
EP2865274A1 (fr) 2013-10-24 2015-04-29 Evonik Industries AG Additifs de nourriture pour animaux contenant des acides animés L
WO2015058951A1 (fr) * 2013-10-24 2015-04-30 Evonik Industries Ag Additif d'aliment pour animaux contenant un l-aminoacide
WO2015058949A1 (fr) 2013-10-24 2015-04-30 Evonik Industries Ag Additif d'aliment pour animaux contenant un l-aminoacide
US11723384B2 (en) 2013-10-24 2023-08-15 Evonik Operations Gmbh L-amino acid-containing feedstuff additive
US20160255862A1 (en) * 2013-10-24 2016-09-08 Evonik Degussa Gmbh L-amino acid-containing feedstuff additive
US11076616B2 (en) 2013-10-24 2021-08-03 Evonik Operations Gmbh L-amino acid-containing feedstuff additive
CN105658080A (zh) * 2013-10-24 2016-06-08 赢创德固赛有限公司 包含l-氨基酸的饲料添加剂
WO2015165743A1 (fr) * 2014-04-30 2015-11-05 Evonik Degussa Gmbh Procédé de production de lysine l au moyen d'une bactérie alcalophile
EP2940144A1 (fr) * 2014-04-30 2015-11-04 Evonik Degussa GmbH Procédé de production de L-lysine en utilisant une bactérie alcaliphile
WO2016030441A1 (fr) * 2014-08-29 2016-03-03 Chr. Hansen A/S Acides aminés essentiels fournis par bacillus dans une charge liquide
EP3395827A1 (fr) 2017-04-27 2018-10-31 Universität Bielefeld Biosynthèse d'acides aminés et de caroténoïdes à l'aide de corynebacterium glutamicum recombiné
WO2018197608A1 (fr) 2017-04-27 2018-11-01 Universität Bielefeld Biosynthèse de caroténoïdes et d'acides aminés au moyen de corynébactérium glutamicum
US11312981B2 (en) 2017-04-27 2022-04-26 Universität Bielefeld Carotenoid and amino acid biosynthesis using recombinant corynebacterium glutamicum
WO2020049313A1 (fr) 2018-09-06 2020-03-12 3F Bio Ltd Processus et produit de celui-ci
CN112662713A (zh) * 2020-12-28 2021-04-16 天津科技大学 一种高密度发酵生产l-赖氨酸的培养基及其方法

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