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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
• • 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
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • 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
  • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
• • 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

Definitions

• the invention relates to a process for the preparation of L-amino acids.
• L-amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and in animal nutrition.
• the recombinant DNA technique is additionally used to enhance the intrinsic properties of L-amino acid producing strains of Corynebacterium. It is thus described that amplification of the expression of the biosynthesis genes ilvBN, ilvC, ilvD are advantageously used for L-valin formation (EP 1155139B1, EP 0356739B1). It is also known that the attenuation or elimination of the threonine dehydratase gene ilvA and / or genes of pantothenate synthesis for L-valine formation can be used (EP 1155139B1).
• L-isoleucine L-valine and L-lysine needed.
• alanine may still be formed as an undesirable by-product, and relatively low yields of the target amino acid, e.g. B. L-valine reached.
• L-amino acids which are preferably formed from pyruvate, which are associated with higher product yields.
• the yield in the production of L-valine, L-isoleucine and L-lysine should be increased.
• the object is achieved in that the alanine transaminase is weakened in its activity against the naturally occurring strain or completely eliminated, or that the Alaninpro- reduction is reduced. Furthermore, the object is achieved by identifying an alanine transaminase.
• the alanine transaminase according to the invention is to be understood in particular as the L-alanine transaminase.
• amino acids in particular L-valine, L-lysine and L-isoleucine.
• Fig.2 Plasmid, which is used for the deletion of the alanine transaminase gene according to Example 4
• Sequence Listing 1 A gene sequence coding for the alanine transaminase.
• Sequence Listing 2 The amino acid sequence of alanine transaminase.
• Sequence Listing No.1 codes from nucleotide 101 to 1414 for the alanine transaminase.
• Sequence Listing 2 shows the sequence of the alanine transaminase encoded by nucleotides 101 to 1414 of Sequence Listing 1.
• the elimination of the alanine transaminase gene can be effected by deletion or disruption.
• the invention also includes gene structures which contain the sequence according to sequence listing 1. These can be chromosomes, plasmids, vectors, phages, viruses. Furthermore, the gene sequence or nucleotide sequence itself is encompassed by the invention.
• sequence according to Sequence Listing 1 encodes the alanine transaminase and can therefore also be used for their preparation.
• methods known to the person skilled in the art for example overexpression, amplification of promoters or start codons, can be used.
• the organisms disclosed in this application can be used for the production of alanine transaminase.
• any gene encoding the alanine transaminase can be deleted or subject to disruption.
• Blocking the catalytic center of the alanine transaminase for example by adding substrates blocking this center or chemical see substitution, for example due to mutation.
• Coryneform bacteria particularly preferably Corynebacterium glutamicum, are preferably used according to the invention.
• the invention also provides a plasmid which is used for the deletion or inactivation of the gene coding for the alanine transaminase gene.
• the plasmid contains internal sequences of the alanine transaminase gene, or adjacent sequences to the 3 'and 5 "ends of the alanine transaminase gene.
• alanine transaminase gene or adjacent to the alanine transaminase gene, preferably the regions immediately adjacent to the 3 'and 5' ends of the gene, and ideally is the plasmid shown in FIG.
• the amino acid L-valine is used in human medicine, in the pharmaceutical industry, in the food industry and in animal nutrition.
• the invention also relates to a microorganism or a transformed cell or a recombinant cell, in which the production of alanine is reduced or completely eliminated or in which the alanine transaminase activity is reduced or completely eliminated.
• strains used preferably produce L-valine or L-isoleucine or L-lysine before the deletion of the alanine transaminase gene. Preferred embodiments can be found in the claims.
• modification in this context describes the reduction of the intracellular activity of one or more biosynthetic enzymes for the production of amino acids (proteins) in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or a Gene or allele used, which codes for a corresponding enzyme with a reduced activity or expressed the corresponding gene (protein) expressed reduced and, where appropriate, combines these measures, or even completely deleted the gene.
• the microorganisms which are the subject of the present invention can produce L-valine or else other L-amino acids which are formed from pyruvate, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
• L-valine or else other L-amino acids which are formed from pyruvate, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
• These are representatives of coryneform bacteria, in particular of the genus Corynebacterium.
• the species Corynebacterium glutamicum is to be mentioned, which is known in the art for its ability to produce L-amino acids.
• Suitable starting strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the known wild-type strains
• thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020
• coryneform bacteria produce the L-amino acids valine, leucine and isoleucine in an improved manner after reduction or elimination of the gene coding for the alanine transaminase.
• the nucleotide sequence of the alanine transaminase gene is inevitably known by the generation of the complete genome sequence of C. glutamicum (Kalinowski et al., 2003, J. Biotechnol., 104: 5-25; Ikeda M., and Nakagawa S. 2003 Appl Microbiol Biotechnol 62: 99-109), but without the assignment of an open reading frame to the
• Alanine transaminase is known.
• the open reading frame coding for alanine transaminase described and identified below in the example bears the number NCgl2747 and is deposited in the publicly accessible database of the "National Institutes of Health" (http://www.ncbi.nlm.nih .gov), as well as Cgl2844 in the publicly available "DNA Data Bank of Japan” (http://gib.genes.nig.ac.jp).
• the alanine transaminase gene described under these numbers is preferably used as starting point of the invention. Furthermore, alleles of the alanine transaminase gene can be used, resulting for example from the degeneracy of the genetic code or by functionally neutral sense mutations (sense mutations) or by deletion or insertion of nucleotides. In order to achieve an attenuation, either the expression of the alanine transaminase gene or the catalytic properties of the enzyme protein can be reduced. Also, the catalytic property of the enzyme protein can be changed with regard to its substrate specificity. If necessary, both measures can be combined.
• the attenuation of gene expression can be achieved by suitable culture guidance or by genetic modification (mutation) of the signal structures of gene expression.
• Signaling structures of gene expression include repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon, and terminators. Information on this is the expert z.
• Boyd and Murphy J. Bacteriol 1988: 170: 5949
• Voskuil and Chambliss Nucleic Acids Res. 1998 26: 3548, Jensen and Hammer (Biotechnol 58: 191)
• Patek et al. Molecular biology
• Mutations include transitions, transversions, insertions and deletions, as well as directed evolutionary methods. Instructions for the Generation of such mutations and proteins belong to the state of the art and can be known textbooks (R. Knippers "Molecular Genetics", 8th edition, 2001, Georg Thieme Verlag, Stuttgart, Germany), or review articles (N. Pokala 2001, J. Struct. Biol.
• the attenuated expression of the genes or the mutated genes is carried out according to customary methods of gene exchange by replacing the native chromosomal gene by the mutated gene, as described, for example, in Morbach et al. (Microbiol Biotechnol 1996, 45: 612-620).
• the deletion of the gene is carried out as in Scharzer and Pühler (Biotechnology 1990, 9: 84-87), as well as Schwarz et al. (Appl., Environ., Microbio., 1994, 60: 756-759).
• the transformation of the desired strain with the vector for gene replacement or deletion is carried out by conjugation or electroporation of the parent strain.
• the method of conjugation is described by Schwarz et al. (Appl., Environ., Microbio., 1994, 60: 756-759). Methods for transformation are, for example, in Tauch et al. (FEMS Microbiological Letters (1994) 123: 343-347).
• the alanine transaminase gene can be deleted or its allele exchanged into C. glutamicum.
• L-valine Furthermore, it may be beneficial for the production of L-valine, in addition to mitigation or deletion. on the alanine transaminase activity of one or more of the genes selected from the group
• the ilvBN genes encoding feedback-resistant acetohydroxy acid synthase
• L-valine in addition to the reduction of alanine - transaminase activity, one or more of the genes selected from the group
• panBCD genes coding for pantothenate synthesis
• L-valine in addition to the attenuation of alanine transaminase, it may be advantageous for the production of L-valine to eliminate undesirable side reactions which lead to leucine, for example (Nakayama: “Breeding of Amino Acid Producing Microorganisms", in: Overproduction). Duction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.) / Academic Press, London, UK, 1982).
• microorganisms produced according to the invention can be cultivated continuously or discontinuously in the batch process (batch culturing) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing valine.
• the culture medium to be used must suitably satisfy the requirements of the respective microorganisms. Descriptions of culture media of various microorganisms are given in the manual "Manual of Methods for
• sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, masseclose, Starch and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols, such as, for example, glycerol and ethanol and organic acids, such as acetic acid, can be used the. 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 ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
• the nitrogen sources can be used singly or as a mixture.
• As a source of phosphorus it is possible to use potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
• the culture medium must also contain salts of metals, such as. As magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins can be used in addition to the above-mentioned substances.
• the said feedstocks can be added to the culture in the form of a one-time batch or fed in a suitable manner during the cooling.
• the medium may have suitable selective substances, eg. As antibiotics, are added.
• oxygen or oxygen-containing gas mixtures such as. As air, registered in the culture.
• the temperature of the culture is normally from 20 0 C to 45 ° C and preferably 25 ° C to 40 0 C. The culture is continued until a maxi- raura has formed to L-valine. This goal is usually reached within 10 to 160 hours.
• the indicated primers were synthesized by MWG Biotech AG (Anzinger Str 7a, D-85560 Ebersberg) and the PCR reaction was carried out according to standard protocols (Innis et al., PCR Protocols, A Guide to Methods and Applications, 1990. Academic Press ). With the primers, a DNA fragment of about 1.3 kb was obtained, which codes for the alanine transaminase.
• the primers contain the restriction enzyme Bsal site cleaved in the above nucleotide sequences.
• the amplified DNA fragment of about 1.3 kb was identified in 0.8% agarose gel and isolated from the gel using existing methods (QIAquik Gel Extraction Kit, Quiagen, Hilden). The ligation of the fragment was carried out with the SureCloning Kit (Amersham, UK) in the expression vector pASK-IBA-3C (IBA, Gttingen). The ligation mixture was used to transform E. coli DH5 (Grant et al. , 1990. Proceedings of the United States of America of the United States of America, 87: 4645-4649). The selection for plasmid-containing strains was carried out by plating the transformation mixture to 25 mg per liter of chloramphenicol-containing LB plates.
• the resulting plasmids were characterized by restriction digestion and gel electrophoretic analysis.
• the resulting plasmid was named pASK-IBA-3Corf234. It is indicated in FIG.
• E. coli DH5 with pASK-IBA 3Corf234 was performed at 30 0 C in 100 ml LB with 25 mg were grown to an optical density of 0.5 per liter of chloramphenicol. Then, 0.01 ml of an anhydrotetracycline solution containing 2 mg of anhydrotetracycline per milliliter of dimethylformamide was added. The culture was further incubated at 30 ° C. for 3 hours. The cells were then passed through 12 minutes at 4 0 C and 5000 revolutions per minute
• StrepTactin affinity columns manufacturer IBA (IBA, Göttingen, Germany) were filled with 1 ml bed volume StrepTactin-Sepharose. After equilibration of the columns with washing buffer from the manufacturer IBA, 1 ml of the crude extract was added to the Sepharose. After passing through the extract, the affinity column was washed five times with 1 ml of washing buffer. Elution of the alanine transaminase protein was performed with elution buffer consisting of 100 mM Tris, 1 mM EDTA, 2.5 mM desthiobiotin, pH 8. The elution fractions were aliquoted, frozen at -20 0 C and used directly in the enzyme test.
• IBA IBA, Göttingen, Germany
• the reaction batch of the enzyme assay contained in a total volume of 1 ml: 0.2 ml 0.25 M Tris / HCl, pH 8, 0.005 ml alanine transaminase protein and 0.1 ml 2.5 mM pyridoxal phosphate, and 0.1 ml 40 mM pyruvate and 0.1 ml of 0.5 M L-glutamate, or 0.1 ml of 40 mM pyruvate and 0.1 ml of 0.5 M aspartate, or 0.1 ml of 40 mM pyruvate and 0.1 ml of 0.5 M ⁇ -amino-butyrate, or 0.1 ml of 40 mM pyruvate and 0.1 ml of 0.5 M L-glutamate without alanine transaminase protein.
• the enzyme test was carried out at 30 ° C. in a thermocycler 5436 from Eppendorf (Hamburg). The reaction was started by adding the protein. By adding 30 ⁇ l of a stop reagent (6.7% (v / v) perchloric acid (70%), 40% (v / v) ethanol (95%) in water) to 50 ⁇ l each of the test mixture, the enzyme was zymtest stopped. In order to prepare the samples for the detection of the amino acids formed by reversed-phase HPLC, 20 ⁇ l of a neutralization buffer (20 mM Tris, 2.3 M di-potassium carbonate, pH 8) were added.
• a neutralization buffer (20 mM Tris, 2.3 M di-potassium carbonate, pH 8) were added.
• the precipitated by the neutralization of perchloric acid was centrifuged off (13000 rpm, 10 min) and the supernatant in different dilutions was used for the quantification by means of HPLC. This was done after automated derivatization with o-phthaldialdehyde as described (Hara et al., 1985, Analytica Chimica Acta 172: 167-173). As shown in Table 1, the isolated protein catalyzes the L-glutamate, L-aspartate and ce-aminobutyrate-dependent amination of pyruvate to alanine.
• the indicated primers were synthesized by MWG Biotech AG (Anzinger Str 7a, D-85560 Ebersberg) and the PCR reaction was carried out according to standard protocols (Innis et al., PCR Protocols, A Guide to Methods and Applications, 1990. Academic Press ). The primers were used to amplify two DNA fragments of about 400 bp flanking the alanine transaminase gene.
• the primers Del234_1 and Del234_4 additionally contain the restriction enzyme BamHI site, which is indicated in brackets above in the nucleotide sequences.
• the amplified DNA fragments of about 400 bp were identified in 0.8% agarose gel and isolated from the gel according to existing methods (QIAquik Gel Extraction Kit, Quiagen, Hilden). With the aid of a second PCR reaction in which the two previously amplified DNA fragments were used as template DNA (Link et al., 1997, J. Bacteriol 179: 6228-6237), An approximately 800 bp fragment was amplified. This fragment contains both the alanine transaminase flanking DNA regions. The amplified DNA fragment of about 800 bp was identified in 0.8% agarose gel and isolated from the gel using existing methods (QIAquik Gel Extraction Kit, Quiagen, Hilden).
• the ligation of the fragment was carried out with the SureCloning Kit (Amersham, UK) into the deletion vector pK19mobsacB (Schäfer et al., 1994, Gene 145: 69-73).
• the ligation reaction was used to transform E. coli DH5 (Grant et al., 1990. Proceedings of the United States of America of the United States of America, 87: 4645-4649).
• the selection of plasmid-containing strains was carried out by plating the transformation mixture on LB plates containing 50 mg per liter kanamycin.
• plasmids obtained were characterized by restriction digestion and gel electrophoretic analysis.
• the resulting plasmid was named pkl9mobsacB orf234. It is indicated in FIG.
• the plasmid pkl9mobsacB-orf234 was used to transform the strain 13032 ⁇ panBC to kanamycin resistance.
• the strain is described in EP1155139B1, and the transformation technique in Kirchner et al. J. Biotechnol. 2003, 104: 287-99.
• the deletion of the gene for alanine transaminase was carried out according to the protocol for the deletion of genes in Corynebacterium glutamicum according to Shufer et al. , 1994, Gene 145: 69-73, performed by two consecutive homologous recombinations. With the aid of the PCR reaction, the deletion of the gene for the alanine transaminase was confirmed.
• the following primers were used:
• the indicated primers were synthesized by MWG Biotech and the PCR reaction was carried out according to standard protocols (Innis et al., PCR Protocols, A Guide to Methods and Applications, 1990. Academic Press). Amplification of a 1 kb DNA fragment confirmed the deletion of the alanine transaminase gene.
• the obtained strain was designated strain 13032 ⁇ panBC ⁇ alaT.
• Example 5 Reduction of alanine formation and increase of L-valine formation by deletion of alanine transaminase
• the strain 13032 ⁇ panBC ⁇ alaT and the control strain was 13032 ⁇ panBC in the medium CgIII (Menkel et al, 1989, Appl Environ Microbiol. 55: 684-8...) Grown at 3O 0 C.
• the medium CGXII was inoculated with an optical density of 1.
• the medium CGXII contains per liter: 20 g (NH 4 ) 2 SO 4 , 5 g urea, 1 g KH 2 PO 4 ,

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