Classifications

• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
  • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)

Definitions

• the invention provides a process for the production of L- amino acids, especially -lysine, by fermentation using coryneform bacteria in which the mqo gene, which codes for malate quinone oxidoreductase, has been attenuated.
• L-amino acids especially L-lysine
• L-lysine are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and, very especially, in the feeding of animals .
• EP-A-1038969 it is described that an improvement in the production of L-amino acids by fermentation can be achieved by enhancement, especially overexpression, of the mqo gene.
• the inventors have set themselves the object of providing novel bases for improved processes for the production of L- amino acids, especially L-lysine, by fermentation using coryneform bacteria.
• L-amino acids or amino acids are mentioned hereinbelow, they are to be understood as meaning one or more amino acids, including their salts, selected from the group 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.
• L-lysine or lysine is mentioned hereinbelow, it is to be understood as meaning. not only the bases but also the salts, such as, for example, lysine monohydrochloride or lysine sulfate.
• the invention provides a process for the production of L- amino acids by fermentation using coryneform bacteria in which at least the nucleotide sequence coding for malate quinone oxidoreductase (mqo gene) is attenuated, especially excluded or expressed at a low level.
• This invention also provides a process for the production of L-amino acids by fermentation in which the following steps are carried out:
• the strains used preferably produce L-amino acids, especially L-lysine, even before attenuation of the mqo gene.
• Attenuation or “attenuate” in this context describes the diminution or exclusion of the intracellular activity of one or more enzymes (proteins) in a microorganism that are coded for by the corresponding DNA, by, for example, using a weak promoter or using a gene or allele that codes for a corresponding enzyme having a low level of activity, or by inactivating the corresponding gene or enzyme (protein) , and optionally by combining those measures .
• the microorganisms provided by the present invention are able to produce amino acids from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol . They may be representatives of coryneform bacteria, especially of the genus Corynebacterium. In the case of the genus Corynebacterium, special mention may be made of the species Corynebacterium glutamicum, which is known to those skilled in the art for its ability to produce L-amino acids.
• Suitable strains of the genus Corynebacterium, especially of the species Corynebacterium glutamicum, are especially the known wild-type strains
• malate quinone oxidoreductase The sequences described in the mentioned references coding for malate quinone oxidoreductase can be used according to the invention. It is also possible to use alleles of malate quinone oxidoreductase, which are formed from the degeneracy of the genetic code or by sense mutations that are neutral in terms of function.
• Gene expression can be diminished by carrying out the culturing in a suitable manner or by genetic alteration (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 person skilled in the art will find information thereon, for example, in patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al .
• the invention provides the allele 672, shown in SEQ ID No. 3, of the mqo gene, which allele carries the nucleotide adenine instead of the nucleotide guanine at position 672 of the DNA sequence (see SEQ ID No. 1) , which leads to substitution of the TGG codon coding for the amino acid tryptophan-224 (see SEQ ID No. 2) by an opal (TGA) stop codon.
• the invention also provides the allele 1230, shown in SEQ ID No. 4, of the mqo gene, which allele carries the nucleotide adenine instead of the nucleotide guanine at position 672 of the DNA sequence (see SEQ ID No. 1), which leads to substitution of the tgg codon coding for the amino acid tryptophan-224 (see SEQ ID No. 2) by an opal stop codon and which additionally carries a nucleotide substitution at position 1230 of cytosine to thymine.
• a central portion of the coding region of the gene in question is cloned into a plasmid vector which is able to replicate in a host (typically E. coli) , but not in C. glutamicum.
• Suitable vectors are, for example, pSUP301 (Simon et al . , Bio/Technology 1, 784-791 (1983)), pKl ⁇ mob or pKl9mob (Schafer et al . , Gene 145, 69-73 (1994)), pKl ⁇ mobsacB or pKl9mobsacB (Jager et al .
• the plasmid vector containing the central portion of the coding region of the gene is then transferred to the desired strain of C. glutamicum by conjugation or transformation.
• the method of conjugation is described, for example, in Schafer et al . (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods of transformation are described, for example, in Thierbach et al . (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al .
• a mutation such as, for example, a deletion, insertion or base substitution
• the allele that is produced is in turn cloned into a vector that is not replicative for C. glutamicum, and the latter is then transferred to the desired host of C. glutamicum by transformation or conjugation.
• homologous recombination by means of a first cross-over occurrence effecting integration and by means of a suitable second cross-over occurrence effecting an excision in the target gene or in the target sequence, incorporation of the mutation or of the allele is achieved.
• That method has been used, for example, by Peters-Wendisch et al . (Microbiology 144, 915-927 (1998)) to exclude the pyc gene of C. glutamicum by means of a deletion. That method has been used by Schafer et al . (Gene 145: 69-73 (1994) ) , for example, in order to incorporate a deletion into the hom-thrB gene region. In the same way, a deletion has been introduced into the cgl gene region of C. glutamicum by Schafer et al . (Journal of Bacteriology 176: 7309-7319 (1994)).
• a deletion, insertion or a base substitution can thus be incorporated into the mqo gene.
• L-amino acids in addition to attenuating the mqo gene, to enhance, especially to overexpress, one or more enzymes of the biosynthesis pathway in question, of glycolysis, of the anaplerotic pathway, of the citric acid cycle, of the pentose phosphate cycle, of amino acid export, and optionally regulatory proteins.
• enhancement or “enhance” in this context describes an increase in the intracellular activity of one or more enzymes or proteins in a microorganism that are coded for by the corresponding DNA, by, for example, increasing the number of copies of the gene or genes, using a strong promoter or a gene or allele that codes for a corresponding enzyme or protein having a high level of activity, and optionally by combining those measures.
• amino acids especially L-lysine
• attenuating the mqo gene at the same time to attenuate, especially to diminish the expression of, one or more genes selected from the group
• microorganisms produced according to the invention also form part of the invention and can be cultivated, for the purposes of the production of L-amino acids, continuously or discontinuously by the batch process or by the fed batch or repeated fed batch process.
• a summary of known cultivation methods is described in the textbook of Chmiel (Bioreatechnik 1. Einf ⁇ hrung in die
• the culture medium to be used must meet the requirements of the strains in question in a suitable manner. Descriptions of culture media for various microorganisms are to be found in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C. , USA, 1981).
• carbon source sugars and carbohydrates such as, for example, glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut oil, 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, for example, acetic acid. Those substances may be used individually or in the form of a mixture.
• oils and fats such as, for example, soybean oil, sunflower oil, groundnut oil and coconut oil
• fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid
• alcohols such as, for example, glycerol and ethanol
• organic acids such as, for example, acetic acid.
• Those substances may be used individually or in the form of a mixture.
• nitrogen source organic nitrogen- containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
• organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea
• inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
• the nitrogen sources may be used individually or in the form of a mixture.
• the culture medium must also contain salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth.
• essential growth substances such as amino acids and vitamins, may be used in addition to the above-mentioned substances.
• Suitable precursors may also be added to the culture medium. The mentioned substances may be added to the culture in the form of a single batch, or they may be fed in in a suitable manner during the cultivation.
• basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds, such as phosphoric acid or sulfuric acid
• acid compounds such as phosphoric acid or sulfuric acid
• anti-foams such as, for example, fatty acid polyglycol esters
• suitable substances having a selective action such as, for example, antibiotics
• oxygen or gas mixtures containing oxygen such as, for example, air, are introduced into the culture.
• the temperature of the culture is normally from 20°C to 45°C and preferably from 25°C to 40°C. The culture is continued until the maximum amount of the desired product has formed. That aim is normally achieved within a period of from 10 hours to 160 hours.
• chromosomal DNA was isolated by the method of Eikmanns et al . (Microbiology 140: 1817-1828 (1994) ) .
• the following oligonucleotides were chosen for the polymerase chain reaction (see SEQ ID No. 5 and SEQ ID No . 6 ) :
• primers were chosen here so that the amplified fragment contains the incomplete gene, starting with the native riboso e binding site without the promoter region, and the front region of the mqo gene. Furthermore, the primer mqo_oPl contains the sequence for the cleavage site of the restriction endonuclease BamHI, and the primer mqo_hind the cleavage site of the restriction endonuclease Hindlll, which are marked by underlining in the nucleotide sequence shown above.
• the primers shown were synthesized by MWG-Biotech AG.
• PCR reaction was carried out by the standard PCR method of Innis et al . (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Pwo-Polymerase from Roche Diagnostics GmbH (Mannheim, Germany) .
• the primers allow amplification of a DNA fragment 468 bp in size, which carries the incomplete mqo gene, including the native ribosome binding site.
• the mqo fragment 468 bp in size was cleaved with the restriction endonucleases BamHI and Hindlll and then isolated from the agarose gel with the QiaExll Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
• the IPTG-inducible expression vector pXK99Emob was constructed according to the prior art.
• the vector is based on the Escherichia coli expression vector pTRC99A (Amann et al., Gene 69: 301-315 (1988)) and contains the trc promoter, which can be induced by addition of the lactose derivative IPTG (isopropyl /?-D-thiogalactopyranoside) , the termination regions Tl and T2 , the replication origin ColEl from E. Coli, the lacl q gene (repressor of the lac operon from E.coli), a multiple cloning site (mcs) (Norrander, J.M. et al.
• the vector pXK99Emob is quite specifically suitable for regulating the expression of a gene, in particular effecting attenuated expression in coryneform bacteria.
• the vector pXK99Emob is an E. coli expression vector and can be employed in E. coli for enhanced expression of a gene. Since the vector cannot replicate independently in coryneform bacteria, this is retained in the cell only if it is integrated into the chromosome.
• the peculiarity of this vector here is the use for regulated expression of a gene after cloning of a gene section from the front region of the corresponding gene in the vector containing the start codon and the native ribosome binding site, and subsequent integration of the vector into coryneform bacteria, in particular C.
• Gene expression is regulated by addition of metered amounts of IPTG to the nutrient medium. Amounts of 0.5 ⁇ M up to 10 ⁇ M IPTG have the effect of very weak expression of the corresponding gene, and amounts of 10 ⁇ M up to 100 ⁇ M have the effect of a slightly attenuated to normal expression of the corresponding gene.
• the E. coli expression vector pXK99Emob constructed was transferred by means of electroporation (Tauch et al . 1994, FEMS Microbiol Letters, 123: 343-347) into E. coli DH5 ⁇ mcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649). Selection of the transformants was carried out 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 was isolated from a transformant by conventional methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 - 927) , cleaved with the restriction endonuclease Ncol, and the plasmid was checked by subsequent agarose gel electrophoresis .
• the plasmid construct obtained in this way was called pXK99Emob ( Figure 1) .
• the strain obtained by electroporation of the plasmid pXK99Emob in the E. coli strain DH5 ⁇ mcr was called E. coli DH5alphamcr/pXK99Emob.
• the E. coli expression vector pXK99Emob described in Example 1.2 was used as the vector.
• DNA of this plasmid was cleaved completely with the restriction enzymes BamHI and Hindlll and then dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) .
• the mqo fragment approx. 458 bp in size described in 1.1, obtained by means of PCR and cleaved with the restriction endonucleases BamHI and Hindlll was mixed with the prepared vector pXK99Emob and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04) .
• T4 DNA ligase Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04
• DH5ocmcr (Hanahan, In: DNA cloning. A Practical Approach. Vol. I, IRL-Press, Oxford, Washington DC, USA). Selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kanamycin. After incubation overnight at 37 a C, recombinant individual clones were selected. Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No.
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• the vector pXK99Emobmqo mentioned in Example 1 was electroporated by the electroporation method of Tauch et al.,(1989 FEMS Microbiology Letters 123: 343-347) in the strain C. glutamicum DSM5715.
• the vector cannot replicate independently in DSM5715 and is retained in the cell only if it has integrated into the chromosome.
• Selection of clones with integrated pXK99Emobmqo was 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, New York, 1989) , which had been supplemented with 15 mg/1 kanamycin and IPTG (ImM) .
• DSM5715 A selected kanamycin-resistant clone which has the Plasmid pXK99Emobmqo, mentioned in Example 1, inserted in the chromosomal mqo-gene of DSM5715, was called DSM5715: :pXK99Emobmqo.
• the C. glutamicum strain DSM5715 : :pXK99Emobmqo obtained in Example 2 was cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant was determined. " By addition of IPTG, attenuated expression of the mqo gene occurs, regulated by the trc promoter.
• the strain was first incubated on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/1) and IPTG (10 ⁇ M) ) for 24 hours at 33 a C.
• a preculture was seeded (10 ml medium in a 100 ml conical flask) .
• the complete medium Cg III was used as the medium for the preculture.
• Kanamycin (25 mg/1) and IPTG (10 ⁇ M) were added to this.
• the preculture was incubated for 16 hours at 33 B C at 240 rpm on a shaking machine.
• the OD (660 nm) of the preculture was 0.5.
• 500 ⁇ l of this preculture were transinoculated into a main culture.
• the IPTG concentration in the main culture was approx. 0.5 ⁇ M.
• Medium MM was 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 was carried out in a 10 ml volume in a 100 ml conical flask with baffles. Kanamycin (25 mg/1) was added. Culturing was carried out at 33 a C and 80% atmospheric humidity.
• the OD was determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Kunststoff) .
• the amount of lysine formed was 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 pXK99Emob
• Figure 2 Map of the plasmid pXK99Emobmqo.
• Kan Kanamycin resistance gene aph(3 ' ) -Ha from Escherichia coli
• the microorganism identified under L above was accompanied by:
• This International Depositary Authority accepts the microorganism identified under L above, which was received by it on 2002-02- 15 (Date of the original deposit)'.
• microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

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