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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
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

Definitions

• the invention relates to a process for the preparation of L-amino acids, in particular L-lysine, using coryneform bacteria in which the dctA gene, which codes for the C4- dicarboxylate transport protein is attenuated.
• L-Amino acids in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
• amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes . Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
• fermentation measures such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
• Methods of utagenesis, selection and mutant selection are used to improve the output properties of these microorganisms.
• Strains which are resistant to antimetabolites such as e.g. the lysine analogue S-(2- aminoethyl) -cysteine, or are auxotrophic for metabolites of regulatory importance and produce L- amino acids are obtained in this manner.
• Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium glutamicum strains which produce L-amino acids, by amplifying individual amino acid biosynthesis genes and investigating the effect on the L-amino acid production.
• the inventors had the object of providing new principles for improved processes for the preparation of L-amino acids, in particular L-lysine, with coryneform bacteria.
• the invention relates to a process for the preparation of L-amino acids using coryneform bacteria in which at least the nucleotide sequence which codes for the C4- dicarboxylate transport protein is attenuated, in particular eliminated or expressed at a low level.
• the C4- dicarboxylate transport protein is an integral membrane protein which transports dicarboxylates, such as, for example, succinate, fumarate and malate.
• This invention also provides a process for the preparation of L-amino acids, in which the following steps are carried out:
• coryneform bacteria employed preferably already produce L-amino acids, in particular L-lysine, before attenuation of the dctA gene, which codes for the C4-dicarboxylate transport protein.
• coryneform bacteria produce L-amino acids, in particular L-lysine, in an improved manner after attenuation of the dctA gene, which codes for the C4- dicarboxylate transport protein.
• nucleotide sequence of the dctA gene which codes for the C4-dicarboxylate transport protein of Corynebacterium glutamicum can be found in the patent application WO01/00805 under Identification Code AX066843 as SEQ ID No. 425.
• L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan and L-arginine. L-Lysine is particularly preferred.
• the term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or 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 combining these measures .
• the activity or concentration of the corresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.
• the microorganisms provided by the present invention can prepare amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol . They can be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, there may be mentioned in particular the species Corynebacterium glutamicum, which is known among experts for its ability to produce L-amino acids .
• Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains
• the gene expression can be reduced by suitable culturing 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 .
• the expert can find information on this e.g. in the patent application WO
• Possible mutations are transitions, transversions, insertions and deletions .
• "missense mutations” or "nonsense mutations” are referred to.
• Insertions or deletions of at least one base pair in a gene lead to "frame shift mutations", as a consequence of which incorrect amino acids are incorporated or translation is interrupted prematurely.
• Deletions of several codons typically lead to a complete loss of the enzyme activity. Instructions on generation of such mutations are prior art and can be found in known textbooks of genetics and molecular biology, such as e.g.
• a central part of the coding region of the gene of interest is cloned in a plasmid vector which can replicate in a host (typically E. coli) , but not in C. glutamicum.
• Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKl ⁇ ob or pKl9mob (Schafer et al., Gene 145, 69- 73 (1994)), pKl ⁇ mobsacB or pKl9mobsacB (Jager et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-T0P0
• a mutation such as e.g. a deletion, insertion or base exchange, is established in vitro in the gene of interest.
• the allele prepared is in turn cloned in a vector which is not replicative for C. glutamicum and this is then transferred into the desired host of C. glutamicum by transformation or conjugation.
• a deletion, insertion or a base exchange can be incorporated in this manner into the gene which codes for the C4-dicarboxylate transport protein.
• L-amino acids may enhance, in particular over-express, one or more enzymes of the particular biosynthesis pathway, of glycolysis, of anaplerosis, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export and optionally regulatory proteins, in addition to the attenuation of the gene which codes for the C4- dicarboxylate transport protein.
• enhancement or “enhance” in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or a gene which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures .
• the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.
• amino acids in particular L-lysine
• the gene which codes for the C4- dicarboxylate transport protein at the same time for one or more of the genes chosen from the group consisting of
• the invention also provides the microorganisms prepared according to the invention, and these can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of L-amino acids .
• batch culture batch culture
• feed process fed batch
• repetitive feed process repeated fed batch process
• the culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General
• Sugars and carbohydrates such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture.
• oils and fats such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat
• fatty acids such as e.g. palmitic acid, stearic acid and linoleic acid
• alcohols such as e.g. glycerol and ethanol
• organic acids such as e.g. acetic acid
• Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea
• inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen.
• the sources of nitrogen can be used individually or as a mixture.
• Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus.
• the culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins, can be employed in addition to the above-mentioned substances.
• Suitable precursors can moreover be added to the culture medium.
• the starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
• Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture.
• Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
• Suitable substances having a selective action such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids .
• oxygen or oxygen-containing gas mixtures such as e.g. air, are introduced into the culture.
• the temperature of the culture is usually 20 a C to 45 S C, and preferably 25 e C to 40 a C. Culturing is continued until a maximum of the desired product has formed. This target is usually reached within 10 hours to 160 hours.
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• chromosomal DNA is isolated by the method of Eikmanns et al. (Microbiology 140: 1817 - 1828 (1994)).
• the primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications,. 1990, Academic Press) with the Taq-polymerase from Boehringer Mannheim (Germany, Product Description Taq DNA polymerase, Product No. 1 146 165) . With the aid of the polymerase chain reaction, the primers allow amplification of an internal fragment of he dctA gene 297 bp in size. The product amplified in this way is tested electrophoretically in a 0.8% agarose gel.
• the amplified DNA fragment is ligated with the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, CA, USA; Catalogue Number K4500-01) in the vector pCR2.1-T0P0 (Mead at al. (1991) Bio/Technology 9:657-663).
• the E. coli strain TOP10 is then electroporated with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985) .
• Selection for plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), which had been supplemented with 50 mg/1 kanamycin.
• Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI and subsequent agarose gel electrophoresis (0.8%).
• the plasmid is called pCR2. IdctAint and is shown in Figure 1.
• the vector pCR2. IdctAint mentioned in example 1 is electroporated by the electroporation method of Tauch et al.(FEMS Microbiological Letters, 123:343-347 (1994)) in Corynebacterium glutamicum DSM 5715.
• the strain DSM 5715 is an AEC-resistant lysine producer, and the strain is described in EP-B-0435132.
• the vector pCR2. IdctAint cannot replicate independently in DSM5715 and is retained in the cell only if it has integrated into the chromosome of DSM 5715. Selection of clones with pCR2.
• IdctAint integrated into the chromosome is carried out by plating out the electroporation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which had been supplemented with 15 mg/1 kanamycin.
• the C. glutamicum strain DSM5715 : :pCR2.IdctAint obtained in example 2 is cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant is determined.
• the strain is first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/1) for 24 hours at 33 a C.
• a preculture is seeded (10 ml medium in a 100 ml conical flask) .
• the complete medium Cglll is used as the medium for the preculture.
• Kanamycin 25 mg/1 is added to this.
• the preculture is incubated for 16 hours at 33 a C at 240 rpm on a shaking machine.
• a main culture is seeded from this preculture such that the initial OD (660 am) of the main culture is 0.1.
• Medium MM is used for the main culture.
• MOPS morpholinopropanesulfonic acid
• the CSL, MOPS and the salt solution are brought to pH 7 with aqueous ammonia and autoclaved.
• the sterile substrate and vitamin solutions are then added, and the CaC0 3 autoclaved in the dry state is added.
• Culturing is carried out in a 10 ml volume in 100 ml conical flasks with baffles. Kanamycin (25 mg/1) is added. Culturing is carried out at 33 2 C and 80% atmospheric humidity. After 72 hours, the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Kunststoff) . The amount of lysine formed is determined with an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection.
• Figure 1 Map of the plasmid pCR2. IdctAint .
• the base pair numbers stated are approximate values obtained in the context of reproducibility of measurements .
• PStl Cleavage site of the restriction enzyme
• Pstl dctAint Internal fragment of the dctA gene
• ColEl Replication origin of the plasmid ColEl

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