WO2018210359A1 - Pyruvate carboxylase having a feedback-resistant mutation, encoding dna, plasmides, and microorganism for the production thereof, and methods for the production of products the biosynthesis of which includes oxaloacetate as precursor - Google Patents

Pyruvate carboxylase having a feedback-resistant mutation, encoding dna, plasmides, and microorganism for the production thereof, and methods for the production of products the biosynthesis of which includes oxaloacetate as precursor Download PDF

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WO2018210359A1
WO2018210359A1 PCT/DE2018/000107 DE2018000107W WO2018210359A1 WO 2018210359 A1 WO2018210359 A1 WO 2018210359A1 DE 2018000107 W DE2018000107 W DE 2018000107W WO 2018210359 A1 WO2018210359 A1 WO 2018210359A1
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pyruvate carboxylase
sequence
production
microorganism
dna
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French (fr)
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Maike KORTMANN
Meike BAUMGART
Michael Bott
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Forschungszentrum Jülich GmbH
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    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • 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
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01001—Pyruvate decarboxylase (4.1.1.1)

Definitions

  • the invention relates to a Pyruvatcarboxylase and a DNA encoding the Pyruvatcarboxylase, a plasmid containing the DNA and a microorganism for production and a process for the preparation of products whose biosynthesis oxaloacetate as a precursor and a chromosome.
  • Corynebacterium glutamicum is used industrially for the production of amino acids, in particular L-glutamate and L-lysine.
  • the intermediate oxalacetate is withdrawn from the citrate cycle, since it serves as a precursor for amino acids or salts of the amino acids of the aspartate family.
  • ATP-dependent formation of oxalacetate from pyruvate and carbon dioxide or HC0 3 " is catalyzed by the enzyme pyruvate carboxylase (Pye) for the industrial microbial production of amino acids of the aspartate family such as L-lysine and the glutamate family such as
  • Pye pyruvate carboxylase
  • L-glutamate is important for a high activity of the pye, and also for the production of other metabolites, which are derived from intermediates of the citrate cycle, a high pye activity is beneficial.
  • the enzymatic activity of the native pye of C. glutamicum is allosterically inhibited, inter alia, by aspartate and has therefore been associated with high intracellular aspartate concentrations.
  • glutamicum DG52-5 leads to a 60% reduction of the lysine titer, while the plasmid-mediated overexpression of the pyc gene in the strain DG52-5 to a leads to a 50% increase in lysine titers.
  • deletion of the pyc gene in the wild-type strain ATCC13032 is shown to reduce Tween 60-induced L-glutamate production by about 50%, while plasmid-based overexpression of the pyc gene increases glutamate production by 700% .
  • the importance of high pye activity for the production of lysine and glutamate was later demonstrated in other publications (Blombach et al., 2007 Applied Microbiology and Biotechnology 76: 615-623, Shirai et al., 2007 Microbial Cell Factories 6: 19, Neuner and Heinzle 201 1 Biotechnology Journal 6: 318-329; Neuner et al., 2013 Journal of Biotechnology 163: 217-224; Guo et al., 2013 Biotechnology Letters 35: 943-950).
  • Cadaverine (1, 5-diaminopentane) has been used to enhance Pyc activity (Nguyen et al 2015 Metabolites 5: 211-231; kind et al., 2010, Metabolism Engineering 12: 341-351).
  • German Patent Application 102012016716.4 discloses a screening method with which improved enzymes can be found.
  • Oxalacetate as a precursor to provide, with which the yield, the titer, the volumetric productivity (g product / liter / hour) or the specific productivity (g product / hour / g cell dry matter) in the production of products derived from oxaloacetate can be increased.
  • the production of amino acids of the aspartate family should be increased, ie L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine and L-methionine.
  • amino acids of the glutamate family such as L-glutamate, L-glutamine, L-arginine or L-proline
  • intermediates such as salts and acids of the citrate cycle, for example, succinate, malate, fumarate, 2-oxoglutarate, citrate or isocitrate, diamines such as 1, 5-diaminopentane or 1, 4-diaminobutane or other products such as itaconate, ectoine, gamma-aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline.
  • the object is achieved by the features specified in the characterizing part of claim 1 and the independent claims.
  • the microorganism, pyruvate carboxylase, the gene coding for pyruvate carboxylase, the plasmid or chromosome containing this gene, as well as the production process the yield of products whose biosynthesis involves oxaloacetate as a precursor can be increased.
  • the production of amino acids of the oxaloacetate / aspartate family can be increased, ie L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine, L-methionine.
  • amino acids of the glutamate family such as L-glutamate, L-glutamine, L-arginine or L-proline
  • intermediates such as salts and acids of the citrate cycle, for example succinate, malate, fumarate or
  • 2-oxoglutarate, citrate or isocitrate, of diamines for example, 1, 5-diaminopentane or 1, 4-diaminobutane or other products such as itaconate, ectoine, gamma
  • glutamicum ATCC 13032 / ysC T3111 comprises an exchange of isoleucine in position 1012 by serine, and a genetically modified gene coding for this pyruvate carboxylase, a plasmid containing this gene, and a The microorganism contains this gene or plasmid, and the manufacturing process solves the tasks set.
  • the ATCC 13032 / ysC T3111 strain is found to increase by 7% in the final L-lysine concentration.
  • a gene of an identity of at least 70% of the gene according to sequence no. 1 coding for a pyruvate carboxylase is provided, which starting from the gene of at least 70% identity of sequence no. 1 in position 3034-3036 exchanges the for lsoleucine-1012 encoding triplets against a triplet encoding serine. Position 3034-3036 therefore encodes serine.
  • the DNA of the invention comprises sequences of 70% to 100% identity.
  • the identity is 80%, 85% to 90%. Particularly preferred are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the gene according to sequence no. 1 is very particularly preferred.
  • nucleic acid sequences which encode polypeptides, includes all sequences that appear possible in accordance with the degeneracy of the genetic code.
  • a vector preferably a plasmid, containing this gene is provided.
  • any empty vector or empty plasmid can be considered as the starting vector or starting plasmid.
  • the plasmid pAN6, as described in the publication of Frunzke et al., Molecular Microbiology 2008, 67: 305-322, can be used, in which the gene of the invention is inserted.
  • a chromosome which contains the DNA according to the invention.
  • the DNA according to the invention should be inserted into the chromosome in such a way that the function of the genes relevant for the viability of the microorganism is not impaired or destroyed.
  • a pyruvate carboxylase according to the invention having at least 90% sequence identity to the pyruvate carboxylase according to sequence no. 2 is obtained, starting from the pyruvate carboxylase of strain ATCC 13032 / ysC T3111 and isoleucine at position 1012 replaced by serine (Pyc l1012S ). In position 1012 of the pyruvate carboxylase is therefore according to the invention serine.
  • Pyruvate carboxylases of 90% to 100% identity to Sequence No. 2 are included in the pyruvate carboxylase of the present invention.
  • the identity is preferably 95%, 96% or 97%, particularly preferably 98% or 99% of the pyruvate carboxylase of sequence No. 2 modified according to the invention.
  • the pyruvate carboxylase according to sequence no. 2 is very particularly preferred.
  • Seq. No. 1 DNA sequence modified according to the invention of the plasmid-based variant, coding for the pyruvate carboxylase modified according to the invention.
  • Seq. No. 2 Amino acid sequence of the pyruvate carboxylase modified according to the invention.
  • Seq. No. 3 DNA sequence of the reference strain ATCC 13032 / ysC T3111 for wild-type
  • ATCC 13032 / ysC T3111 for wild-type pyruvate carboxylase ATCC 13032 / ysC T3111 for wild-type pyruvate carboxylase.
  • Seq. No. 5 DNA sequence of the chromosomal variant DNA-pyc-T3035G-C3039G according to the invention.
  • the expression of the genes according to the invention can be enhanced.
  • stronger promoters can be used, the number of gene copies can be increased, or the ribosome binding site can be changed in order to increase the translation of the messenger RNA.
  • the methods to be used for carrying out these methods are known to the person skilled in the art.
  • a microorganism is the subject of the invention which contains a gene according to the invention or a vector according to the invention.
  • This microorganism is preferably a coryneform bacterium.
  • Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Brevibacterium flavum or Brevibacterium lactofermentum may, for example, be mentioned as coryneform bacteria.
  • Particularly preferred cells according to the invention are those of the genera Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces and Yarrowia, Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum , Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha and Pichia pastoris are particularly preferred. Most preferred cells according to the invention are those of the genus Corynebacterium and Escherich
  • the genetically modified cells may in particular be selected from cells selected from the group consisting of Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium molassecola ATCC17965, Corynebacterium thermoaminogenes FERM BP- 1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC14020, and L-amino acid producing mutants or strains derived therefrom such as the L-lysine producing strains Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463,
  • Escherichia co // - Escherichia coli strains were AJ1 1442 (see JP 56-18596 and US 4,346, 170), Escherichia co // 'strain VL61 1 and Escherichia co //' strain WC196 (see WO-A -96 / 17930).
  • a microorganism containing a pyruvate carboxylase-encoding gene with the genetic modification coding for the exchange of I2012S in the protein is used for the production of oxaloacetate-derived metabolites.
  • the method according to the invention comprises the use of a gene with at least 70% to 100% identity to the gene according to Sequence No. 1.
  • a microorganism having a gene of at least 80% to 90% identity to Sequence No. 1 is used. More preferably, a gene having an identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to Sequence No. 1 is used for the preparation process. The gene according to sequence no. 1 is very particularly preferred.
  • the gene coding for pyruvate carboxylase according to the invention can be used chromosomally or in a vector, preferably a plasmid.
  • the gene is expressed and the pyruvate carboxylase of the present invention having an identity of at least 90% to the pyruvate carboxylase of Sequence No. 2, replacing isoleucine at position 1012 with serine, causes the increased production of oxaloacetate-derived metabolic products.
  • the method of the invention comprises the use of a pyruvate carboxylase having a 90% to 100% identity to the pyruvate carboxylase of Sequence No. 2.
  • the identity of the pyruvate carboxylase used in accordance with the invention is 95%, 96% or 97%, more preferably 98% or 99%, of Sequence No. 2.
  • Very particular preference is given to the use of pyruvate carboxylase according to Sequence No. 2.
  • amplified gene of the invention expressed. The microorganisms thus obtained are fermented.
  • a production organism may preferably be an organism selected from the group consisting of the genera Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces and Yarrowia, where Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha and Pichia pastoris are particularly preferred. Most preferred cells according to the invention are those of the genus Coryne
  • the genetically modified cells or microorganisms can in particular be selected from the group consisting of Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium me- lasscola ATCC 17965, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020, and mutant L-amino acids producing therefrom such as the L-lysine producing strains Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 64
  • Escherichia co // - Escherichia coli strains were AJ1 1442 (see JP 56-18596 and US 4,346,170), Escherichia co // 'strain and Escherichia coli VL61 1.
  • WC196 strain see WO-A-96/17930 be used.
  • amino acids of the aspartate family can be increased, so L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine and L-methionine are produced.
  • amino acids of the glutamate family such as L-glutamate, L-glutamine, L-arginine or L-proline
  • intermediates of the citrate cycle such as succinate, fumarate, malate, citrate, isocitrate or 2 Oxoglutarate
  • diamines such as diaminopentane or diaminobutane or other products such as itaconate, ectoine, gamma-aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline be increased.
  • the product prepared by fermentation and secreted into the culture supernatant is then enriched and isolated.
  • Fig. 1 Growth of C. glutamicum ATCC 13032 / ysC T3111 with chromosomally coded
  • FIG. 2 L-lysine production of the strain C. glutamicum ATCC 13032 / ysC T3111 with chromosome-encoded Pyc I 012S .
  • FIG. 1 shows the growth of the strain C. glutamicum ATCC 13032 / ysC T3111 with the chromosomally coded Pye variant Pyc ' 1012S .
  • the abscissa shows the time in hours (h) and the ordinate the value for backscatter at 620 nm (AU) as a measure of the cell density.
  • the strain C. glutamicum ATCC 13032 / ysC T3111 served as control with native pye , ie with isoleucine at position 1012. All strains were cultured in CGXII minimal medium with 4% (wt / vol) glucose in a BioLector® system at 30 ° C and 1200 cultured for 24 h.
  • Figure 2 shows the L-lysine production of the strain C. glutamicum ATCC 13032 / ysC T3111 with the chromosomally encoded Pye variant Pyc l1012S .
  • the abscissa denotes three independent replicates and the mean of the three experiments and the ordinate the percentage lysine concentration, wherein the lysine concentration of the control strain ATCC 13032 / ysC T3111 (black bars) was set in the three independent replicates in each case as 100% .
  • the L-lysine concentrations for strain ATCC 13032 / ysC T3111 pyc l1012S are shown as shaded bars.
  • a mutation in the pyc gene could be identified, which leads to increased L-lysine production.
  • a plasmid-based Pyc mutant library was prepared by means of error prone PCR, which was then screened for increased fluorescence in strain ATCC1303 / ysC T3111 Apyc using a genetically encoded lysine sensor (pSenLys) and fluorescence-activated cell sorting (FACS) has been. The isolated cells were then propagated and tested for increased lysine formation.
  • FACS fluorescence-activated cell sorting
  • the isolated gene and enzyme variants were genetically characterized, which finally led to the identification of the Pye variant according to the invention, which increases the production of L-lysine, as well as other metabolites derived from oxaloacetate.
  • the mutation found is I1012S.
  • strain C glutamicum ATCC 13032 / ysC T3111 with native chromosomal pyc gene.
  • the strains were grown in 800 ⁇ CGXII minimal medium at 4% (wt / vol).
  • the starting OD at 600 nm was 0.5.
  • the cells of the individual cultures were sedimented and the L-lysine concentration in the supernatant was measured.
  • the measurement was carried out by reversed-phase HPLC with pre-column derivatization of the amino acids via orthophthalaldehyde.
  • As the mobile phase a gradient of 80% solution A (100 mM sodium acetate (pH 7.2) and 20% solution B (100% (vol / vol) methanol) to 20% solution A and 80% solution B was used.
  • the example shows that the investigated Pye variant has a positive effect on lysine production and can be increased by up to 7% if the Pye variant Pyc-M012S is chromosomally encoded instead of the wild-type Pye protein.
  • the invention is not mutations known from the prior art, as Ohnishi et al. (Applied Microbiology and Biotechnology (2002) 58: 217-223), namely the chromosomal introduction of the amino acid exchange proline to serine at position 458 of the pye of C. glutamicum in the C. glutamic tvm Sta mm AHD2, which on the wild type ATCC 13032 and carries two point mutations, Val59Ala in the gene for the homologous dehydrogenase (hom) and Thr31 1 He in the gene for the aspartate kinase (lysC) and from the document US Pat. No.

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Abstract

The invention relates to pyruvate carboxylase and a pyruvate carboxylase-encoding DNA, a plasmid containing said DNA and a microorganism for the production thereof, and to methods for the production of products the biosynthesis of which includes oxaloacetate as a precursor, and to a chromosome. The invention makes a DNA available that codes for a pyruvate carboxylase having an at least 70% sequence identity with sequence no. 1 and where the triplet that codes for isoleucine-1012 is replaced by a serine-encoding triplet in positions 3034-3036. The expression of said gene results in a pyruvate carboxylase by means of which the yield of the reaction of metabolic products derived from oxaloacetate is by 7% higher with respect to the strain containing the native Pyc.

Description

PYRUVATCARBOXYLASE MIT EINER FEEDBACK-RESISTENZ VERURSACHENDE PYRUVATE CARBOXYLASE CAUSED WITH FEEDBACK RESISTANCE
MUTATION, KODIERENDE DNA, PLASMIDE, SOWIE MIGROORGANISMUS ZUR MUTATION, ENCODING DNA, PLASMIDE, AND MIGRO ORGANISM
PRODUKTION UND VERFAHREN ZUR HERSTELLUNG VON PRODUKTEN, DEREN  PRODUCTION AND METHOD FOR THE MANUFACTURE OF PRODUCTS, THEIR
BIOSYNTHESE OXALACETAT ALS VORSTUFE BEINHALTET  BIOSYNTHESIS OXALACETATE INCLUDED AS PRE-STAGE
Die Erfindung betrifft eine Pyruvatcarboxylase und eine für die Pyruvatcarboxylase kodierende DNA, ein Plasmid enthaltend die DNA sowie einen Mikroorganismus zur Produktion und ein Verfahren zur Herstellung von Produkten, deren Biosynthese Oxalacetat als Vorstufe beinhalten und ein Chromosom. Corynebacterium glutamicum wird industriell für die Herstellung von Aminosäuren, insbesondere L-Glutamat und L-Lysin, genutzt. Für die Produktion von L-Lysin wird dem Citrat-Zyklus das Intermediat Oxalacetat entzogen, da es als Vorstufe für Aminosäuren oder Salze der Aminosäuren der Aspartatfamilie dient. Diese sind L-Aspartat, L-Asparagin, L-Lysin, L- Methionin, L-Threonin und L-Isoleucin. Damit der Citrat-Zyklus trotz des Entzugs dieser Intermediate weiter ablaufen kann, muss er durch sogenannte anaplerotische Reaktionen wieder aufgefüllt werden. Bei Wachstum auf Zuckern erfolgt das Auffüllen des Oxalacetat- Pools durch die Carboxylierung von Pyruvat oder Phosphoenolpyruvat zu Oxalacetat. Die ATP-abhängige Bildung von Oxalacetat aus Pyruvat und Kohlenstoffdioxid bzw. HC03 " wird durch das Enzym Pyruvatcarboxylase (Pye) katalysiert. Für die industrielle mikrobielle Pro- duktion von Aminosäuren der Aspartat-Familie wie z.B. L-Lysin und der Glutamat-Familie wie z.B. L-Glutamat ist demnach eine hohe Aktivität der Pye wichtig. Auch für die Produktion anderer Metabolite, die sich aus Intermediaten des Citratzyklus ableiten, ist eine hohe Pye- Aktivität förderlich. The invention relates to a Pyruvatcarboxylase and a DNA encoding the Pyruvatcarboxylase, a plasmid containing the DNA and a microorganism for production and a process for the preparation of products whose biosynthesis oxaloacetate as a precursor and a chromosome. Corynebacterium glutamicum is used industrially for the production of amino acids, in particular L-glutamate and L-lysine. For the production of L-lysine, the intermediate oxalacetate is withdrawn from the citrate cycle, since it serves as a precursor for amino acids or salts of the amino acids of the aspartate family. These are L-aspartate, L-asparagine, L-lysine, L-methionine, L-threonine and L-isoleucine. In order for the citrate cycle to continue despite the withdrawal of these intermediates, it must be replenished by so-called anaplerotic reactions. When grown on sugars, the oxaloacetate pool is replenished by the carboxylation of pyruvate or phosphoenolpyruvate to oxaloacetate. The ATP-dependent formation of oxalacetate from pyruvate and carbon dioxide or HC0 3 " is catalyzed by the enzyme pyruvate carboxylase (Pye) for the industrial microbial production of amino acids of the aspartate family such as L-lysine and the glutamate family such as Thus, for example, L-glutamate is important for a high activity of the pye, and also for the production of other metabolites, which are derived from intermediates of the citrate cycle, a high pye activity is beneficial.
Die enzymatische Aktivität der nativen Pye von C. glutamicum wird allosterisch unter ande- rem durch Aspartat gehemmt und hat daher bei hohen intrazellulären Aspartat-The enzymatic activity of the native pye of C. glutamicum is allosterically inhibited, inter alia, by aspartate and has therefore been associated with high intracellular aspartate concentrations.
Konzentrationen nur eine begrenzte Aktivität. Damit ist auch die Produktion von L-Lysin und anderen, vom Oxalacetat abgeleiteten Produkten, begrenzt, da nur eine begrenzte Menge der Vorläufer-Metabolite bereitgestellt wird. Die bisher beschriebenen Pye-Varianten führen nur zu einer moderaten Erhöhung der L-Lysinproduktion. Die Veröffentlichung von Peters-Wendisch et al.„Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum" im Journal of Molecular Microbiology and Biotechnology (2001 ) 32: 295-300 offenbart, dass die Deletion des pyc- Gens im Stamm C. glutamicum DG52-5 zu einer 60%igen Reduktion des Lysin-Titers führt, während die plasmid-vermittelte Überexpression des pyc-Gens im Stamm DG52-5 zu einem um 50% gesteigerten Lysin-Titer führt. Weiterhin wird gezeigt, dass die Deletion des pyc- Gens im Wildtyp-Stamm ATCC13032 die durch Tween 60 induzierte L-Glutamat-Produktion um etwa 50% reduziert, während die plasmid-basierte Überexpression des pyc-Gens die Glutamat-Bildung um 700% steigert. Die Wichtigkeit einer hohen Pye-Aktivität für die Produk- tion von Lysin und Glutamat wurde später auch noch in anderen Publikationen gezeigt (Blombach et al. 2007 Applied Microbiology and Biotechnology 76: 615-623; Shirai et al. 2007 Microbial Cell Factories 6: 19; Neuner und Heinzle 201 1 Biotechnology Journal 6: 318- 329; Neuner et al. 2013 Journal of Biotechnology 163: 217-224; Guo et al. 2013 Biotechnology Letters 35: 943-950). Auch für die Produktion anderer Metabolite außer Aminosäuren, die Intermediate des Ci- tratzyklus darstellen oder sich von Intermediaten des Citratzyklus ableiten, ist eine hohe Pye- Aktivität von großer Bedeutung. Die Veröffentlichungen von Shohei Okino et al.„An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain" in Appl. Microbiol. Biotechnol. (2008) 81 : 459-464 und Torben Hoefel et al.„Compara- tive reaction engineering studies for succinic acid production from sucrose by metabolically engineered Escherichia coli n fed-batch-operated stirred tank bioreactiors" in Biotechnol. J. 2012, 7, 1277-1287 offenbaren, dass die Expression bzw. Überexpression von Genen, kodierend für eine Pyruvatcarboxylase, zu einer Produktion oder gesteigerten Produktion von Succinat führt. Dies wird auch in weiteren Publikationen gezeigt (Li et al. 2016 Bioresource Technology 218: 217-223; Tajima et al. 2015 Applied and Environmental Microbiology 81 :Concentrations only a limited activity. Thus, the production of L-lysine and other products derived from oxaloacetate is also limited because only a limited amount of the precursor metabolites are provided. The pye variants described so far only lead to a moderate increase in L-lysine production. The publication by Peters-Wendisch et al., "Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum" in the Journal of Molecular Microbiology and Biotechnology (2001) 32: 295-300 discloses that the deletion of the pyc gene in Strain C. glutamicum DG52-5 leads to a 60% reduction of the lysine titer, while the plasmid-mediated overexpression of the pyc gene in the strain DG52-5 to a leads to a 50% increase in lysine titers. Furthermore, deletion of the pyc gene in the wild-type strain ATCC13032 is shown to reduce Tween 60-induced L-glutamate production by about 50%, while plasmid-based overexpression of the pyc gene increases glutamate production by 700% , The importance of high pye activity for the production of lysine and glutamate was later demonstrated in other publications (Blombach et al., 2007 Applied Microbiology and Biotechnology 76: 615-623, Shirai et al., 2007 Microbial Cell Factories 6: 19, Neuner and Heinzle 201 1 Biotechnology Journal 6: 318-329; Neuner et al., 2013 Journal of Biotechnology 163: 217-224; Guo et al., 2013 Biotechnology Letters 35: 943-950). Also, for the production of other metabolites other than amino acids, which are intermediates of the Krebs cycle or derived from intermediates of the Krebs cycle, a high Pye activity is of great importance. The publications of Shohei Okino et al., "An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain" in Appl. Microbiol. Biotechnol. (2008) 81: 459-464 and Torben Hoefel et al., "Comparative reaction engineering studies for succinic acid production from sucrose by metabolically engineered Escherichia coli n fed-batch-operated stirred tank bioreactiors "in Biotechnol. J. 2012, 7, 1277-1287 disclose that expression or overexpression of genes encoding a pyruvate carboxylase results in production or increased production of succinate. This is also shown in other publications (Li et al 2016 Bioresource Technology 218: 217-223, Tajima et al 2015 Applied and Environmental Microbiology 81:
929-937; Litsanov et al. 2012 Applied and Environmental Microbiology 78: 3325-3337; Meng et al. 2016 Microbial Cell Factories 15: 141). Für die Produktion von Malat mit Thermobifida fusca erwies sich eine erhöhte Pye-Aktivität ebenfalls als förderlich (Deng et al. 2016 Biotechnology Progress 31 : 14-20). Ebenfalls wurde für die Produktion von Diaminen, die sich aus Intermediaten des Citratzyklus ableiten, wie z.B. Putrescin (1 ,4-Diaminobutan) oder929-937; Litsanov et al. 2012 Applied and Environmental Microbiology 78: 3325-3337; Meng et al. 2016 Microbial Cell Factories 15: 141). Increased pye activity was also beneficial for the production of malate with Thermobifida fusca (Deng et al 2016 Biotechnology Progress 31: 14-20). Also, for the production of diamines derived from intermediates of the citric acid cycle, e.g. Putrescine (1, 4-diaminobutane) or
Cadaverin (1 ,5-Diaminopentan) eine Verstärkung der Pyc-Aktvität eingesetzt (Nguyen et al. 2015 Metabolites 5: 211-231 ; Kind et al. 2010 Metabolie Engineering 12: 341 -351 ). Cadaverine (1, 5-diaminopentane) has been used to enhance Pyc activity (Nguyen et al 2015 Metabolites 5: 211-231; Kind et al., 2010, Metabolism Engineering 12: 341-351).
Ohnishi et al. (Applied Microbiology and Biotechnology (2002) 58: 217-223) haben die chromosomale Einführung des Aminosäureaustausches Prolin zu Serin an Position 458 der Pye von C. glutamicum in den C. glutamicum-Stemm AHD2 beschrieben, der auf dem Wildtyp ATCC 13032 basiert und zwei Punktmutationen trägt, Val59Ala im Gen für die Homoserin- Dehydrogenase (hom) und Thr31 11 le im Gen für die Aspartatkinase (lysC). Der Stamm AHP- 3 mit der pyc-P458S-Mutation bildete 6% mehr L-Lysin als der Parentalstamm AHD-2. Die Autoren beschreiben, dass es für die Identifizierung der pyc-Mutation keinen bekannten selektierbaren Phänotyp gibt und sie vermutlich nur durch vergleichende Genomanalyse zu finden sei. Darüber hinaus wurde die Pye-Variante Pyc-P458S noch nicht weiter charakterisiert. Ohnishi et al. (Applied Microbiology and Biotechnology (2002) 58: 217-223) have described the chromosomal introduction of the amino acid exchange proline to serine at position 458 of the pye of C. glutamicum into the C. glutamicum stemm AHD2, which is based on the wild type ATCC 13032 and carries two point mutations, Val59Ala in the gene for the homoserine dehydrogenase (hom) and Thr31 11 le in the gene for the aspartate kinase (lysC). Strain AHP-3 with the pyc-P458S mutation formed 6% more L-lysine than the parent strain AHD-2. The authors describe that there is no known selectable phenotype for the identification of the pyc mutation and presumably only through comparative genome analysis be found. In addition, the Pye variant Pyc-P458S has not yet been further characterized.
Die Schrift US 7,300,777 B2 beschreibt einen Organismus, in welchem eine Pyruvatcar- boxylase aus dem C. glutamicum-Stamm NRRL-B1 1474 mit mehreren Mutationen (M1V, E153D, A182S, A206S, H227R, A452G, D1 120E) gegenüber der Pye aus dem C. glutamicum-Stamm ATCC 21523 isoliert wurde. Mindestens eine der genannten Mutationen führt zu einer Pye- Variante, die durch Aspartat in niedrigen Konzentrationen (1-10 mM) bis zu 2,5- fach in ihrer Aktivität stimuliert wird und bei höheren Aspartat-Konzentrationen wieder inhibiert wird bis zu maximal 30% der Aktivität in Abwesenheit von Aspartat. Bei 30 mM Aspartat zeigte die Pye aus Stamm NRRL-B1 1474 noch die gleiche Aktivität wie in Abwesenheit von Aspartat, während die Pye des Stammes ATCC 21523 bei 30 mM Aspartat nur noch 30% der Aktivität besaß, die sie in Abwesenheit von Aspartat zeigte. Die Auswirkung der feed- back-resistenten Pye-Variante aus C. glutamicum NRRL-B1 1474 auf die fermentative Produktion von Aminosäuren, insbesondere L-Lysin und L-Glutamat, wurde in der Schrift US 7,300,777 B2 jedoch nicht offenbart. The document US Pat. No. 7,300,777 B2 describes an organism in which a pyruvate carboxylase from the C. glutamicum strain NRRL-B1 1474 with several mutations (M1V, E153D, A182S, A206S, H227R, A452G, D1 120E) with respect to the Pye from the C. glutamicum strain ATCC 21523 was isolated. At least one of these mutations leads to a pye variant which is stimulated by aspartate in low concentrations (1-10 mM) up to 2.5 times in its activity and is inhibited again at higher aspartate concentrations up to a maximum of 30%. the activity in the absence of aspartate. At 30 mM aspartate, the pye from strain NRRL-B1 1474 still showed the same activity as in the absence of aspartate, while the pye of strain ATCC 21523 at 30 mM aspartate had only 30% of the activity that it showed in the absence of aspartate. However, the effect of the feed-resistant Pye variant from C. glutamicum NRRL-B1 1474 on the fermentative production of amino acids, in particular L-lysine and L-glutamate, has not been disclosed in document US Pat. No. 7,300,777 B2.
Die Deutsche Patentanmeldung 102012016716.4 offenbart ein Screening -Verfahren, mit dem verbesserte Enzyme aufgefunden werden können. German Patent Application 102012016716.4 discloses a screening method with which improved enzymes can be found.
Es ist daher die Aufgabe der Erfindung, einen Mikroorganismus, eine Pyruvatcarboxylase, ein für die Pyruvatcarboxylase kodierendes Gen, ein Plasmid oder Chromosom enthaltend dieses Gen, sowie ein Verfahren zur Herstellung von Produkten, deren Biosynthese It is therefore the object of the invention to provide a microorganism, a Pyruvatcarboxylase, a gene coding for the Pyruvatcarboxylase gene, a plasmid or chromosome containing this gene, and a process for the preparation of products whose biosynthesis
Oxalacetat als Vorstufe beinhaltet, zur Verfügung zu stellen, mit denen die Ausbeute, der Titer, die volumetrische Produktivität (g Produkt/Liter/Stunde) oder die spezifische Produktivität (g Produkt/Stunde/g Zelltrockenmasse) bei der Produktion von aus Oxalacetat abgeleiteten Produkten gesteigert werden kann. Insbesondere soll die Produktion von Aminosäuren der Aspartat-Familie gesteigert werden, also L-Lysin, L-Aspartat, L-Asparagin, L-Threonin, L-Isoleucin und L-Methionin. Weiterhin soll die Produktion von Aminosäuren der Glutamat- Familie, wie L-Glutamat, L-Glutamin, L-Arginin oder L-Prolin, von Intermediaten wie Salzen und Säuren des Citrat-Zyklus beispielsweise Succinat, Malat, Fumarat, 2-Oxoglutarat, Citrat oder Isocitrat, von Diaminen wie zum Beispiel 1 ,5-Diaminopentan oder 1 ,4-Diaminobutan oder auch von weiteren Produkten wie Itaconat, Ectoin, gamma-Aminobutyrat, Butanol, 1 -Propanol, L-Citrullin, L-Ornithin, D-Arginin oder 4-Hydroxyprolin gesteigert werden.  Oxalacetate as a precursor, to provide, with which the yield, the titer, the volumetric productivity (g product / liter / hour) or the specific productivity (g product / hour / g cell dry matter) in the production of products derived from oxaloacetate can be increased. In particular, the production of amino acids of the aspartate family should be increased, ie L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine and L-methionine. Furthermore, the production of amino acids of the glutamate family, such as L-glutamate, L-glutamine, L-arginine or L-proline, intermediates such as salts and acids of the citrate cycle, for example, succinate, malate, fumarate, 2-oxoglutarate, citrate or isocitrate, diamines such as 1, 5-diaminopentane or 1, 4-diaminobutane or other products such as itaconate, ectoine, gamma-aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline.
Ausgehend vom Oberbegriff des Anspruchs 1 und den nebengeordneten Ansprüchen wird die Aufgabe erfindungsgemäß gelöst mit den im kennzeichnenden Teil des Anspruchs 1 und der nebengeordneten Ansprüche angegebenen Merkmalen. Mit dem Mikroorganismus, der Pyruvatcarboxylase, dem für die Pyruvatcarboxylase kodierenden Gen, dem Plasmid oder Chromosom enthaltend dieses Gen, sowie mit dem Herstellungsverfahren kann die Ausbeute von Produkten, deren Biosynthese Oxalacetat als Vorstufe beinhalten, gesteigert werden. Insbesondere kann die Produktion von Aminosäuren der Oxalacetat/Aspartat-Familie gesteigert werden, also L-Lysin, L-Aspartat, L-Asparagin, L- Threonin, L-Isoleucin, L-Methionin. Weiterhin kann die Produktion von Aminosäuren der Glutamat-Familie wie L-Glutamat, L-Glutamin, L-Arginin oder L-Prolin, von Intermediaten wie Salzen und Säuren des Citrat-Zyklus, beispielsweise Succinat, Malat, Fumarat oder Starting from the preamble of claim 1 and the independent claims, the object is achieved by the features specified in the characterizing part of claim 1 and the independent claims. With the microorganism, pyruvate carboxylase, the gene coding for pyruvate carboxylase, the plasmid or chromosome containing this gene, as well as the production process, the yield of products whose biosynthesis involves oxaloacetate as a precursor can be increased. In particular, the production of amino acids of the oxaloacetate / aspartate family can be increased, ie L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine, L-methionine. Furthermore, the production of amino acids of the glutamate family such as L-glutamate, L-glutamine, L-arginine or L-proline, intermediates such as salts and acids of the citrate cycle, for example succinate, malate, fumarate or
2-Oxoglutarat, Citrat oder Isocitrat, von Diaminen beispielsweise, 1 ,5-Diaminopentan oder 1 ,4-Diaminobutan oder auch von weiteren Produkten wie Itaconat, Ectoin, gamma-2-oxoglutarate, citrate or isocitrate, of diamines, for example, 1, 5-diaminopentane or 1, 4-diaminobutane or other products such as itaconate, ectoine, gamma
Aminobutyrat, Butanol, 1-Propanol, L-Citrullin, L-Ornithin, D-Arginin oder 4-Hydroxyprolin gesteigert werden. Vorteilhafte Weiterbildungen der Erfindung sind in den Unteransprüchen angegeben. Aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline be increased. Advantageous developments of the invention are specified in the subclaims.
Im Folgenden wird die Erfindung in ihrer allgemeinen Form beschrieben, ohne dass dies einschränkend auszulegen ist. In the following, the invention is described in its general form without this having to be construed restrictively.
Überraschenderweise wurde gefunden, dass eine gegenüber dem Stamm C. glutamicum ATCC 13032 /ysCT3111 veränderte Pyruvatcarboxylase mit einem Austausch von Isoleucin in Position 1012 durch Serin, und ein für diese Pyruvatcarboxylase kodierendes, genetisch verändertes Gen, ein Plasmid, enthaltend dieses Gen, sowie ein Mikroorganismus enthal- tend dieses Gen oder Plasmid, sowie das Herstellungsverfahren die gestellten Aufgaben löst. Beispielsweise wird für die Produktion von L-Lysin gegenüber dem Stamm ATCC 13032 /ysCT3111 eine um 7% gesteigerte Endkonzentration an L-Lysin festgestellt. It has surprisingly been found that a pyruvate carboxylase modified with respect to the strain C. glutamicum ATCC 13032 / ysC T3111 comprises an exchange of isoleucine in position 1012 by serine, and a genetically modified gene coding for this pyruvate carboxylase, a plasmid containing this gene, and a The microorganism contains this gene or plasmid, and the manufacturing process solves the tasks set. For example, for the production of L-lysine, the ATCC 13032 / ysC T3111 strain is found to increase by 7% in the final L-lysine concentration.
Erfindungsgemäß wird ein Gen einer Identität von mindestens 70% des Gens nach Sequenz Nr.1 , kodierend für eine Pyruvatcarboxylase zur Verfügung gestellt, welches ausgehend von dem Gen einer mindestens 70%igen Identität von Sequenz Nr.1 in Position 3034-3036 einen Austausch des für lsoleucin-1012 kodierenden Tripletts gegen ein Triplett kodierend für Serin besitzt. Position 3034-3036 kodiert daher für Serin. Von der erfindungsgemäßen DNA sind Sequenzen einer 70%igen bis 100%igen Identität umfasst. Vorzugsweise ist die Identität 80%, 85% bis 90%. Besonders bevorzugt 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% oder 99%. Ganz besonders bevorzugt ist das Gen nach Sequenz Nr. 1. According to the invention, a gene of an identity of at least 70% of the gene according to sequence no. 1 coding for a pyruvate carboxylase is provided, which starting from the gene of at least 70% identity of sequence no. 1 in position 3034-3036 exchanges the for lsoleucine-1012 encoding triplets against a triplet encoding serine. Position 3034-3036 therefore encodes serine. The DNA of the invention comprises sequences of 70% to 100% identity. Preferably, the identity is 80%, 85% to 90%. Particularly preferred are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The gene according to sequence no. 1 is very particularly preferred.
Identität wie hierin verwendet, kann durch die Gleichung I (%) = [1-V/X] * 100, definiert werden, worin I Identität bedeutet, X die Gesamtzahl an Nukleobasen der Vergleichssequenz ist und V die Anzahl an unterschiedlichen Nukleobasen der zu betrachtenden Sequenz bezogen auf die Vergleichssequenz ist. Auf jeden Fall sind mit dem Begriff Nukleinsäuresequenzen, welche für Polypeptide kodieren, alle Sequenzen umfasst, die nach Maßgabe der Degeneration des genetischen Codes möglich erscheinen. Identity as used herein may be defined by the equation I (%) = [1-V / X] * 100, where I is identity, X is the total number of nucleobases of the comparison sequence, and V is the number of different nucleobases of the subject to be considered Sequence relative to the comparison sequence. In any case, with the term nucleic acid sequences, which encode polypeptides, includes all sequences that appear possible in accordance with the degeneracy of the genetic code.
Weiterhin wird erfindungsgemäß ein Vektor, vorzugsweise ein Plasmid, enthaltend dieses Gen zur Verfügung gestellt. Dabei kommt grundsätzlich jeder Leervektor oder jedes Leer- plasmid als Ausgangsvektor oder Ausgangsplasmid in Betracht. Beispielsweise kann als Leervektor das Plasmid pAN6, wie es in der Veröffentlichung von Frunzke et al., Molecular Microbiology 2008, 67:305-322 beschrieben wird, eingesetzt werden, bei dem das erfindungsgemäße Gen inseriert ist. Furthermore, according to the invention, a vector, preferably a plasmid, containing this gene is provided. In principle, any empty vector or empty plasmid can be considered as the starting vector or starting plasmid. For example, as an empty vector, the plasmid pAN6, as described in the publication of Frunzke et al., Molecular Microbiology 2008, 67: 305-322, can be used, in which the gene of the invention is inserted.
Erfindungsgemäß wird auch ein Chromosom zur Verfügung gestellt, welches die erfindungs- gemäße DNA enthält. Dabei soll die erfindungsgemäße DNA so in das Chromosom inseriert sein, dass die Funktion der für die Lebensfähigkeit des Mikroorganismus relevanten Gene nicht beeinträchtigt oder zerstört wird. According to the invention, a chromosome is also provided which contains the DNA according to the invention. In this case, the DNA according to the invention should be inserted into the chromosome in such a way that the function of the genes relevant for the viability of the microorganism is not impaired or destroyed.
Durch Expression dieses Gens nach Sequenz Nr. 1 wird eine erfindungsgemäße Pyruvat- carboxylase mit einer mindestens 90%igen Sequenzidentität zu der Pyruvatcarboxylase nach Sequenz Nr. 2 erhalten, bei welcher ausgehend von der Pyruvatcarboxylase des Stammes ATCC 13032 /ysCT3111 das Isoleucin an Position 1012 durch Serin ersetzt ist (Pycl1012S). In Position 1012 der Pyruvatcarboxylase befindet sich daher erfindungsgemäß Serin. Von der erfindungsgemäßen Pyruvatcarboxylase sind Pyruvatcarboxylasen einer 90%igen bis 100%igen Identität zu der Sequenz Nr. 2 umfasst. Vorzugsweise ist die Identität 95%, 96% oder 97%, besonders bevorzugt 98% oder 99% der erfindungsgemäß veränderten Pyruvatcarboxylase von Sequenz Nr.2. Ganz besonders bevorzugt ist die Pyruvatcarboxylase nach Sequenz Nr. 2. By expression of this gene according to sequence no. 1, a pyruvate carboxylase according to the invention having at least 90% sequence identity to the pyruvate carboxylase according to sequence no. 2 is obtained, starting from the pyruvate carboxylase of strain ATCC 13032 / ysC T3111 and isoleucine at position 1012 replaced by serine (Pyc l1012S ). In position 1012 of the pyruvate carboxylase is therefore according to the invention serine. Pyruvate carboxylases of 90% to 100% identity to Sequence No. 2 are included in the pyruvate carboxylase of the present invention. The identity is preferably 95%, 96% or 97%, particularly preferably 98% or 99% of the pyruvate carboxylase of sequence No. 2 modified according to the invention. The pyruvate carboxylase according to sequence no. 2 is very particularly preferred.
Der Ausdruck Identität wie hierin verwendet, kann durch die Gleichung I (%) = [1-V/X] * 100, definiert werden, worin I Identität bedeutet, X die Gesamtzahl an Aminosäuren der Ver- gleichssequenz ist und V die Anzahl an unterschiedlichen Aminosäuren der zu betrachtenden Sequenz bezogen auf die Vergleichssequenz ist. The term identity as used herein can be defined by the equation I (%) = [1-V / X] * 100 where I is identity, X is the total number of amino acids of the comparison sequence and V is the number of different ones Amino acids of the sequence to be considered based on the comparison sequence.
Folgende Sequenzen sind im Sequenzprotokoll gelistet: The following sequences are listed in the sequence listing:
Seq. Nr.1 : Erfindungsgemäß veränderte DNA-Sequenz der Plasmid-basierten Variante, codierend für die erfindungsgemäß veränderte Pyruvatcarboxylase. Seq. Nr. 2: Aminosäuresequenz der erfindungsgemäß veränderten Pyruvatcarboxylase. Seq. No. 1: DNA sequence modified according to the invention of the plasmid-based variant, coding for the pyruvate carboxylase modified according to the invention. Seq. No. 2: Amino acid sequence of the pyruvate carboxylase modified according to the invention.
Seq. Nr. 3: DNA-Sequenz des Referenzstammes ATCC 13032 /ysCT3111 für die Wildform der Seq. No. 3: DNA sequence of the reference strain ATCC 13032 / ysC T3111 for wild-type
Pyruvatcarboxylase. Seq. Nr. 4: Aminosäuresequenz der Pyruvatcarboxylase des Referenzstammes Pyruvate. Seq. No. 4: Amino acid sequence of the pyruvate carboxylase of the reference strain
ATCC 13032 /ysCT3111 für die Wildform der Pyruvatcarboxylase. ATCC 13032 / ysC T3111 for wild-type pyruvate carboxylase.
Seq. Nr. 5: DNA-Sequenz der erfindungsgemäßen chromosomalen Variante DNA-pyc- T3035G-C3039G. In einer vorteilhaften Weiterbildung der Erfindung kann die Expression der erfindungsgemäßen Gene verstärkt werden. Dazu können beispielhaft aber nicht beschränkend stärkere Promotoren verwendet werden, die Anzahl der Genkopien kann erhöht werden oder es kann die Ribosomenbindestelle verändert werden, um die Translation der Boten-RNA zu steigern. Die für die Durchführung dieser Methoden zu verwendenden Verfahren sind dem Fachmann bekannt. Seq. No. 5: DNA sequence of the chromosomal variant DNA-pyc-T3035G-C3039G according to the invention. In an advantageous embodiment of the invention, the expression of the genes according to the invention can be enhanced. For this purpose, by way of example, but not limitation, stronger promoters can be used, the number of gene copies can be increased, or the ribosome binding site can be changed in order to increase the translation of the messenger RNA. The methods to be used for carrying out these methods are known to the person skilled in the art.
Weiterhin ist ein Mikroorganismus Gegenstand der Erfindung, welcher ein erfindungsgemäßes Gen oder einen erfindungsgemäßen Vektor enthält. Dieser Mikroorganismus ist vorzugsweise ein coryneformes Bakterium. Als coryneforme Bakterien können beispielsweise Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoa- cidophilum, Corynebacterium melassecola, Corynbacterium thermoaminogenes, Corynebacterium efficiens, Brevibacterium flavum oder Brevibacterium lactofermentum genannt werden. Furthermore, a microorganism is the subject of the invention which contains a gene according to the invention or a vector according to the invention. This microorganism is preferably a coryneform bacterium. Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Brevibacterium flavum or Brevibacterium lactofermentum may, for example, be mentioned as coryneform bacteria.
Erfindungsgemäß besonders bevorzugte Zellen sind diejenigen der Gattungen Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces und Yarrowia, wobei Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipoly- tica, Methylobacterium extorquens, Ralstonia eutropha und Pichia pastoris besonders bevor- zugt sind. Erfindungsgemäß am meisten bevorzugte Zellen sind solche der Gattung Corynebacterium und Escherichia, wobei Corynebacterium glutamicum und Escherichia coli ganz besonders bevorzugte Bakterienstämme sind.  Particularly preferred cells according to the invention are those of the genera Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces and Yarrowia, Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum , Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha and Pichia pastoris are particularly preferred. Most preferred cells according to the invention are those of the genus Corynebacterium and Escherichia, with Corynebacterium glutamicum and Escherichia coli being very particularly preferred bacterial strains.
Insbesondere in dem Fall, in dem das Produkt L-Lysin ist, können sich die gentechnisch veränderten Zellen insbesondere von Zellen ausgewählt aus der Gruppe bestehend aus Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium melassecola ATCC17965, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 und Brevibacterium divaricatum ATCC14020, und daraus hergestellte L-Aminosäuren produzierende Mutanten bzw. Stämme wie beispielsweise die L-Lysin produzierenden Stämme Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM- P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 und Corynebacterium glutamicum DSM 5715 oder wie beispielsweise der L-Methionin produzierende Stamm Corynebacterium glutamicum ATCC21608 ableiten. Als Beispiele geeigneter Escherichia co//-Stämme seien Escherichia coli AJ1 1442 (siehe JP 56-18596 und US 4,346, 170), Escherichia co//'-Stamm VL61 1 und Escherichia co//'-Stamm WC196 (siehe WO-A-96/17930) genannt. In particular, in the case where the product is L-lysine, the genetically modified cells may in particular be selected from cells selected from the group consisting of Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium molassecola ATCC17965, Corynebacterium thermoaminogenes FERM BP- 1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC14020, and L-amino acid producing mutants or strains derived therefrom such as the L-lysine producing strains Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM 5715 or such as Derive the L-methionine producing strain Corynebacterium glutamicum ATCC21608. As examples of suitable Escherichia co // - Escherichia coli strains were AJ1 1442 (see JP 56-18596 and US 4,346, 170), Escherichia co // 'strain VL61 1 and Escherichia co //' strain WC196 (see WO-A -96 / 17930).
Erfindungsgemäß wird ein Verfahren zur Herstellung von Produkten, deren Biosynthese Oxalacetat als Vorstufe beinhaltet, zur Verfügung gestellt. According to the invention, a process for the preparation of products whose biosynthesis involves oxaloacetate as a precursor is provided.
Dazu wird ein Mikroorganismus, enthaltend ein für die Pyruvatcarboxylase codierendes Gen mit der genetischen Veränderung, kodierend für den Austausch I2012S im Protein, für die Produktion von aus Oxalacetat abgeleiteten Stoffwechselprodukten verwendet. For this purpose, a microorganism containing a pyruvate carboxylase-encoding gene with the genetic modification coding for the exchange of I2012S in the protein is used for the production of oxaloacetate-derived metabolites.
Hierzu wird ein Gen mit einer mindestens 70%igen Identität zu der Sequenz Nr.1 , bei der in den Positionen 3034-3036 das für Isoleucin kodierende Triplett gegen ein Triplett kodierend für Serin ausgetauscht ist, in den Mikroorganismus eingeführt. For this purpose, a gene having an identity of at least 70% to the sequence No. 1, in which the isoleucine-encoding triplet is exchanged for a triplet coding for serine in positions 3034-3036, is introduced into the microorganism.
Von dem erfindungsgemäßen Verfahren ist die Verwendung eines Gens mit einer mindestens 70%igen bis 100%igen Identität zu dem Gen nach Sequenz Nr.1 umfasst. The method according to the invention comprises the use of a gene with at least 70% to 100% identity to the gene according to Sequence No. 1.
Vorzugsweise wird ein Mikroorganismus mit einem Gen einer Identität von mindestens 80% bis 90% zu Sequenz Nr. 1 verwendet. Besonders bevorzugt wird ein Gen mit einer Identität von 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% oder 99% zu Sequenz Nr. 1 für das Herstellungsverfahren verwendet. Ganz besonders bevorzugt ist das Gen nach Sequenz Nr. 1. Preferably, a microorganism having a gene of at least 80% to 90% identity to Sequence No. 1 is used. More preferably, a gene having an identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to Sequence No. 1 is used for the preparation process. The gene according to sequence no. 1 is very particularly preferred.
Das erfindungsgemäß eingesetzte für die Pyruvatcarboxylase kodierende Gen kann dabei chromosomal oder in einem Vektor, vorzugsweise einem Plasmid, verwendet werden. Das Gen wird exprimiert und die erfindungsgemäße Pyruvatcarboxylase mit einer Identität von mindestens 90% zu der Pyruvatcarboxylase nach Sequenz Nr. 2, bei der in Position 1012 Isoleucin durch Serin ersetzt ist, bewirkt die erhöhte Produktion von Oxalacetat abgeleiteten Stoffwechselprodukten. The gene coding for pyruvate carboxylase according to the invention can be used chromosomally or in a vector, preferably a plasmid. The gene is expressed and the pyruvate carboxylase of the present invention having an identity of at least 90% to the pyruvate carboxylase of Sequence No. 2, replacing isoleucine at position 1012 with serine, causes the increased production of oxaloacetate-derived metabolic products.
Von dem erfindungsgemäßen Verfahren ist die Verwendung einer Pyruvatcarboxylase mit einer 90%igen bis 100%igen Identität zu der Pyruvatcarboxylase nach Sequenz Nr. 2 umfasst. Vorzugsweise ist die Identität der erfindungsgemäß verwendeten Pyruvatcarboxylase 95%, 96% oder 97%, besonders bevorzugt 98% oder 99% zu Sequenz Nr. 2. Ganz besonders bevorzugt ist die Verwendung der Pyruvatcarboxylase nach Sequenz Nr. 2. In einer bevorzugten Ausführungsform wird das erfindungsgemäße Gen verstärkt exprimiert. Die so erhaltenen Mikroorganismen werden fermentiert. The method of the invention comprises the use of a pyruvate carboxylase having a 90% to 100% identity to the pyruvate carboxylase of Sequence No. 2. Preferably, the identity of the pyruvate carboxylase used in accordance with the invention is 95%, 96% or 97%, more preferably 98% or 99%, of Sequence No. 2. Very particular preference is given to the use of pyruvate carboxylase according to Sequence No. 2. In a preferred embodiment amplified gene of the invention expressed. The microorganisms thus obtained are fermented.
Als Produktionsorganismus kann vorzugsweise ein Organismus aus der Gruppe bestehend aus den Gattungen Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces und Yarrowia, wobei Corynebacterium glutamicum, Cory- nebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha und Pichia pastoris besonders bevorzugt sind, verwendet werden. Erfindungsgemäß am meisten bevorzugte Zellen sind solche der Gattung Corynebacterium und Escherichia, wobei Corynebacterium glutamicum und Escherichia coli ganz besonders bevorzugte Bakterienstämme sind. As a production organism may preferably be an organism selected from the group consisting of the genera Corynebacterium, Brevibacterium, Escherichia, Bacillus, Lactobacillus, Lactococcus, Zymomonas, Methylobacterium, Ralstonia, Clostridium, Candida, Pichia, Kluyveromyces, Saccharomyces and Yarrowia, where Corynebacterium glutamicum, Corynebacterium efficiens, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida blankii, Candida rugosa, Zymomonas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha and Pichia pastoris are particularly preferred. Most preferred cells according to the invention are those of the genus Corynebacterium and Escherichia, with Corynebacterium glutamicum and Escherichia coli being very particularly preferred bacterial strains.
Insbesondere in dem Fall, in dem der Metabolit L-Lysin ist, können die gentechnisch veränderten Zellen bzw. Mikroorganismen insbesondere von Zellen ausgewählt aus der Gruppe bestehend aus Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutami- cum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium me- lassecola ATCC 17965, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 und Brevibacterium divaricatum ATCC 14020, und daraus hergestellte L-Aminosäuren produzierende Mutanten bzw. Stämme wie beispielsweise die L-Lysin produzierenden Stämme Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 und Corynebacterium glutamicum DSM 5715 oder wie beispielsweise der L-Methionin produzierende Stamm Corynebacterium glutamicum ATCC21608 ableiten. Als Beispiele geeigneter Escherichia co//-Stämme seien Escherichia coli AJ1 1442 (siehe JP 56-18596 und US 4,346,170), Escherichia co//'-Stamm VL61 1 und Escherichia coli- Stamm WC196 (siehe WO-A-96/17930) eingesetzt werden. In particular, in the case in which the metabolite is L-lysine, the genetically modified cells or microorganisms can in particular be selected from the group consisting of Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium me- lasscola ATCC 17965, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020, and mutant L-amino acids producing therefrom such as the L-lysine producing strains Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM 5715 or as for example the L-methionine producing strain Corynebacterium glutamicum ATCC21608. As examples of suitable Escherichia co // - Escherichia coli strains were AJ1 1442 (see JP 56-18596 and US 4,346,170), Escherichia co // 'strain and Escherichia coli VL61 1. WC196 strain (see WO-A-96/17930) be used.
Mit dem erfindungsgemäßen Verfahren können insbesondere Aminosäuren der Aspartat- Familie gesteigert werden, also L-Lysin, L-Aspartat, L-Asparagin, L-Threonin, L-Isoleucin und L-Methionin hergestellt werden. Weiterhin kann die Produktion von Aminosäuren der Gluta- mat-Familie, wie L-Glutamat, L-Glutamin, L-Arginin oder L-Prolin, von Intermediaten des Citrat-Zyklus, wie z.B. Succinat, Fumarat, Malat, Citrat, Isocitrat oder 2-Oxoglutarat, von Diaminen z.B. Diaminopentan oder Diaminobutan oder auch anderen Produkten wie Itaco- nat, Ectoin, gamma-Aminobutyrat, Butanol, 1-Propanol, L-Citrullin, L-Ornithin, D-Arginin oder 4-Hydroxyprolin gesteigert werden. With the method according to the invention in particular amino acids of the aspartate family can be increased, so L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine and L-methionine are produced. Furthermore, the production of amino acids of the glutamate family, such as L-glutamate, L-glutamine, L-arginine or L-proline, intermediates of the citrate cycle, such as succinate, fumarate, malate, citrate, isocitrate or 2 Oxoglutarate, diamines such as diaminopentane or diaminobutane or other products such as itaconate, ectoine, gamma-aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline be increased.
Das durch Fermentation hergestellte und in den Kulturüberstand sekretierte Produkt wird dann angereichert und isoliert. The product prepared by fermentation and secreted into the culture supernatant is then enriched and isolated.
Im Folgenden werden experimentelle Ergebnisse anhand von Figuren dargestellt, die die Erfindung exemplarisch wiedergeben. In the following, experimental results are presented with reference to figures, which represent the invention by way of example.
Es zeigt: It shows:
Fig. 1 : Wachstum von C. glutamicum ATCC 13032 /ysCT3111 mit chromosomal codierter Fig. 1: Growth of C. glutamicum ATCC 13032 / ysC T3111 with chromosomally coded
Pye110125 Pye 110125
Fig. 2: L-Lysin-Produktion des* Stammes C. glutamicum ATCC 13032 /ysCT3111 mit chromoso- mal codierter Pycl 012S. FIG. 2: L-lysine production of the strain C. glutamicum ATCC 13032 / ysC T3111 with chromosome-encoded Pyc I 012S .
In Figur 1 wird das Wachstum des Stammes C. glutamicum ATCC 13032 /ysCT3111 mit der chromosomal codierten Pye-Variante Pyc'1012S dargestellt. In Figur 1 zeigt die Abszisse die Zeit in Stunden (h) und die Ordinate den Wert für Backscatter bei 620 nm (A.U.) als Maß der Zelldichte. Als Kontrolle diente der Stamm C. glutamicum ATCC 13032 /ysCT3111 mit nativer Pye, also mit Isoleucin an Position 1012. Alle Stämme wurden in CGXII Minimalmedium mit 4% (wt/vol) Glucose in einem BioLector® System bei 30 °C und 1200 rpm für 24 h kultiviert. FIG. 1 shows the growth of the strain C. glutamicum ATCC 13032 / ysC T3111 with the chromosomally coded Pye variant Pyc ' 1012S . In FIG. 1 the abscissa shows the time in hours (h) and the ordinate the value for backscatter at 620 nm (AU) as a measure of the cell density. The strain C. glutamicum ATCC 13032 / ysC T3111 served as control with native pye , ie with isoleucine at position 1012. All strains were cultured in CGXII minimal medium with 4% (wt / vol) glucose in a BioLector® system at 30 ° C and 1200 cultured for 24 h.
Figur 2 zeigt die L-Lysin-Produktion des Stammes C. glutamicum ATCC 13032 /ysCT3111 mit der chromosomal codierten Pye-Variante Pycl1012S. Darin bezeichnet die Abszisse drei unabhängige Replikate sowie den Mittelwert aus den drei Experimenten und die Ordinate die prozentuale Lysin-Konzentration, wobei die Lysin-Konzentration des Kontrollstammes ATCC 13032 /ysCT3111 (schwarze Balken) in den drei unabhängigen Replikaten jeweils als 100% gesetzt wurde. Die L-Lysin Konzentrationen für den Stamm ATCC 13032 /ysCT3111 pycl1012S sind als schraffierte Balken dargestellt. Nach Kultivierung der Zellen in einem BioLector® System für 24 h (s. Fig. 1 ) wurden die Zellen geerntet und die L-Lysin Konzentration im Überstand der jeweiligen Kulturen via reversed-phase HPLC mit ortho-Phthaldialdehyd-Figure 2 shows the L-lysine production of the strain C. glutamicum ATCC 13032 / ysC T3111 with the chromosomally encoded Pye variant Pyc l1012S . Therein, the abscissa denotes three independent replicates and the mean of the three experiments and the ordinate the percentage lysine concentration, wherein the lysine concentration of the control strain ATCC 13032 / ysC T3111 (black bars) was set in the three independent replicates in each case as 100% , The L-lysine concentrations for strain ATCC 13032 / ysC T3111 pyc l1012S are shown as shaded bars. After culturing the cells in a BioLector® system for 24 h (see Fig. 1), the cells were harvested and the L-lysine concentration in the supernatant of the respective cultures via reversed-phase HPLC with ortho-Phthaldialdehyd-
Derivatisierung bestimmt. In jedem der drei Replikate zeigt der Stamm mit der mutierten Pye- Variante Pyc-M 012S einen höheren Lysin-Titer als der Vergleichsstamm mit nativer Pye. Es ist eine Steigerung der finalen L-Lysin-Konzentration um durchschnittlich 7% gegenüber C. glutamicum ATCC 13032 /ysCT3111 zu beobachten. Derivatization determined. In each of the three replicates, the strain with the mutated Pye variant Pyc-M 012S shows a higher lysine titer than the native Pye reference strain. It an increase of the final L-lysine concentration by an average of 7% compared to C. glutamicum ATCC 13032 / ysC T3111 is observed.
Im Rahmen der Erfindung konnte eine Mutation im pyc-Gen identifiziert werden, die zu einer erhöhten L-Lysin-Produktion führt. Es wurde zunächst mittels error prone-PCR eine Plasmid- basierte Pyc-Mutanten-Bibliothek erstellt, die anschließend im Stamm ATCC1303 /ysCT3111 Apyc mit Hilfe eines genetisch kodierten Lysin-Sensors (pSenLys) und Fluoreszenzaktivierter Zellsortierung (FACS) zunächst auf erhöhte Fluoreszenz gescreent wurde. Die isolierten Zellen wurden anschließend vermehrt und auf erhöhte Lysinbildung getestet. Diese Methoden sind dem Fachmann bekannt (vgl. Binder et al., Genome Biol. 2012, 13:R40). Die dabei isolierten Gen- und Enzymvarianten wurden genetisch charakterisiert, was schließlich zur Identifizierung der erfindungsgemäßen Pye-Variante führte, welche die Produktion von L- Lysin, sowie anderen, von Oxalacetat abgeleiteten Metaboliten, steigert. Die gefundene Mutation lautet I1012S. In the context of the invention, a mutation in the pyc gene could be identified, which leads to increased L-lysine production. Initially, a plasmid-based Pyc mutant library was prepared by means of error prone PCR, which was then screened for increased fluorescence in strain ATCC1303 / ysC T3111 Apyc using a genetically encoded lysine sensor (pSenLys) and fluorescence-activated cell sorting (FACS) has been. The isolated cells were then propagated and tested for increased lysine formation. These methods are known to the person skilled in the art (compare Binder et al., Genome Biol. 2012, 13: R40). The isolated gene and enzyme variants were genetically characterized, which finally led to the identification of the Pye variant according to the invention, which increases the production of L-lysine, as well as other metabolites derived from oxaloacetate. The mutation found is I1012S.
1. Chromosomal kodierte Pye- ariante Pvc-I1Q12S: 1. Chromosomally encoded Pyeariante Pvc-I1Q12S:
Messung der L-Lysin-Produktion von C. glutamicum ATCC 13032 /ysCT3111 mit der chromosomal kodierten Pye-Variante Pyc-M 012S: Measurement of L-lysine production of C. glutamicum ATCC 13032 / ysC T3111 with the chromosomally encoded Pye variant Pyc-M 012S:
Der Austausch des Isoleucin-Codons 1012 im chromosomalen pyc-Gen durch ein Serin- Codon erfolgte mittels zweifacher homologer Rekombination unter Verwendung des Vektors pK19mobsacB nach dem Fachmann bekannten Verfahren (Schäfer et al. 1994 Gene 145:69-73; Hochheim et al. 2017 Biotechnol Lett 39:283-288). Verglichen wurde die The replacement of the isoleucine codon 1012 in the chromosomal pyc gene by a serine codon was carried out by means of double homologous recombination using the vector pK19mobsacB according to methods known to those skilled in the art (Schäfer et al., 1994, Gene 145: 69-73, Hochheim et al Biotechnol Lett 39: 283-288). Was compared
Produktion mit dem Stamm C. glutamicum ATCC 13032 /ysCT3111 mit nativem chromosomalem pyc-Gen. Die Stämme wurden in 800 μΙ CGXII-Minimalmedium mit 4% (wt/vol)Production with strain C. glutamicum ATCC 13032 / ysC T3111 with native chromosomal pyc gene. The strains were grown in 800 μΙ CGXII minimal medium at 4% (wt / vol).
Glucose für 24h in einem Biolector-System bei 30°C und 1200 rpm kultiviert (Fig. 1 ). Die Start-OD bei 600 nm betrug 0,5. Nach 24 h wurden die Zellen der einzelnen Kulturen se- dimentiert und die L-Lysin-Konzentration im Überstand gemessen. Die Messung erfolgte über reversed phase HPLC mit einer Vorsäulenderivatisierung der Aminosäuren über or- tho-Phthaldialdehyd. Als mobile Phase wurde ein Gradient von 80% Lösung A (100 mM Natrium-Acetat (pH 7,2) und 20% Lösung B (100% (vol/vol) Methanol) zu 20% Lösung A und 80% Lösung B verwendet. Das Beispiel zeigt, dass die untersuchte Pye-Variante einen positiven Effekt auf die Lysin-Produktion hat und diese bis zu 7% gesteigert werden kann, wenn die Pye- Variante Pyc-M012S anstelle des wildtypischen Pye-Proteins chromosomal codiert ist. Disclaimer: Glucose cultured for 24 h in a Biolector system at 30 ° C and 1200 rpm (Fig. 1). The starting OD at 600 nm was 0.5. After 24 h, the cells of the individual cultures were sedimented and the L-lysine concentration in the supernatant was measured. The measurement was carried out by reversed-phase HPLC with pre-column derivatization of the amino acids via orthophthalaldehyde. As the mobile phase, a gradient of 80% solution A (100 mM sodium acetate (pH 7.2) and 20% solution B (100% (vol / vol) methanol) to 20% solution A and 80% solution B was used. The example shows that the investigated Pye variant has a positive effect on lysine production and can be increased by up to 7% if the Pye variant Pyc-M012S is chromosomally encoded instead of the wild-type Pye protein. Disclaimer:
Gegenstand der Erfindung sind nicht Mutationen, die aus nachstehendem Stand der Technik bekannt sind, wie Ohnishi et al. (Applied Microbiology and Biotechnology (2002) 58: 217- 223), nämlich die chromosomale Einführung des Aminosäureaustausches Prolin zu Serin an Position 458 der Pye von C. glutamicum in den C. glutamic tvm-Sta mm AHD2, der auf dem Wildtyp ATCC 13032 basiert und zwei Punktmutationen trägt, Val59Ala im Gen für die Ho- moserin-Dehydrogenase (hom) und Thr31 1 He im Gen für die Aspartatkinase (lysC) und aus der Schrift US 7,300,777 B2, mit einem Organismus, in welchem eine Pyruvatcarboxylase aus dem C. glutamicum-Stamm NRRL-B1 1474 mit mehreren Mutationen (M1V, E153D, A182S, A206S, H227R, A452G, D1 120E) gegenüber der Pye aus dem C. glutamicum- Stamm ATCC 21523 isoliert wurde.  The invention is not mutations known from the prior art, as Ohnishi et al. (Applied Microbiology and Biotechnology (2002) 58: 217-223), namely the chromosomal introduction of the amino acid exchange proline to serine at position 458 of the pye of C. glutamicum in the C. glutamic tvm Sta mm AHD2, which on the wild type ATCC 13032 and carries two point mutations, Val59Ala in the gene for the homologous dehydrogenase (hom) and Thr31 1 He in the gene for the aspartate kinase (lysC) and from the document US Pat. No. 7,300,777 B2, with an organism in which a pyruvate carboxylase from the C glutamicum strain NRRL-B1 1474 with several mutations (M1V, E153D, A182S, A206S, H227R, A452G, D1 120E) to the pye from the C. glutamicum strain ATCC 21523.

Claims

P a t e n t a n s p r ü c h e Patent claims
1. DNA-Sequenz mit einer mindestens 70%igen Identität zu der Sequenz Nr.1 , wobei das Triplett in Position 3034-3036 für Serin kodiert. A DNA sequence having at least 70% identity to Sequence No. 1, said triplet encoding serine at position 3034-3036.
2. DNA-Sequenz nach Anspruch 1 , 2. DNA sequence according to claim 1,
dadurch gekennzeichnet,  characterized,
dass die DNA eine Identität von mindestens 80% zu der Sequenz Nr. 1 besitzt.  the DNA has an identity of at least 80% to the sequence No. 1.
3. DNA-Sequenz nach einem der Ansprüche 1 , 3. DNA sequence according to one of claims 1,
dadurch gekennzeichnet,  characterized,
dass sie eine DNA nach Sequenz Nr.1 ist.  that it is a DNA according to sequence no.1.
4. Pyruvatcarboxylase mit einer mindestens 90%igen Identität zu der Pyruvatcarboxylase nach Sequenz Nr. 2, bei der sich in Position 1012 Serin befindet. 4. Pyruvate carboxylase with at least 90% identity to the pyruvate carboxylase of Sequence No. 2, in which position 1012 is serine.
5. Pyruvatcarboxylase nach Anspruch 4, 5. pyruvate carboxylase according to claim 4,
dadurch gekennzeichnet,  characterized,
dass die Pyruvatcarboxylase eine Identität von mindestens 95% zu der Pyruvatcarboxylase nach Sequenz Nr. 2 besitzt.  the pyruvate carboxylase has an identity of at least 95% to the pyruvate carboxylase according to sequence no.
6. Pyruvatcarboxylase nach Anspruch 4 oder 5, 6. pyruvate carboxylase according to claim 4 or 5,
dadurch gekennzeichnet,  characterized,
dass sie eine Pyruvatcarboxylase nach Sequenz Nr. 2 ist.  that it is a pyruvate carboxylase according to sequence no.
7. Vektor, 7. vector,
dadurch gekennzeichnet,  characterized,
dass er eine DNA-Sequenz nach einem der Ansprüche 1 bis 3 enthält.  that it contains a DNA sequence according to one of claims 1 to 3.
8. Vektor nach Anspruch 7, 8. Vector according to claim 7,
dadurch gekennzeichnet,  characterized,
dass er ein Plasmid ist.  that he is a plasmid.
9. Mikroorganismus, 9. microorganism,
dadurch gekennzeichnet,  characterized,
dass er eine DNA nach einem der Ansprüche 1 bis 3 enthält. that it contains a DNA according to any one of claims 1 to 3.
10. Mikroorganismus nach Anspruch 9, 10. microorganism according to claim 9,
dadurch gekennzeichnet,  characterized,
dass er einen Vektor nach einem der Ansprüche 7 oder 8 enthält.  that it contains a vector according to one of claims 7 or 8.
1 1 . Mikroorganismus nach Anspruch 9 oder 10, 1 1. Microorganism according to claim 9 or 10,
dadurch gekennzeichnet,  characterized,
dass er ein Mikroorganismus aus der Gruppe bestehend aus den Gattungen Coryne- bacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacteri- um, Ralstonia, Vibrio und Clostridium ist.  that it is a microorganism selected from the group consisting of the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Vibrio and Clostridium.
12. Verfahren zur Herstellung von Produkten, deren Biosynthese Oxalacetat als Vorstufe beinhaltet, 12. Process for the preparation of products whose biosynthesis involves oxaloacetate as precursor
dadurch gekennzeichnet,  characterized,
dass ein Mikroorganismus, enthaltend eine DNA nach den Ansprüchen 1 bis 3, der eine Pyruvatcarboxylase mit einer Sequenz nach einem der Ansprüche 4 bis 6 bildet, fermentiert wird, eine Anreicherung des Produktes im Medium oder in den Zellen durchgeführt wird.  in that a microorganism containing a DNA according to claims 1 to 3, which forms a pyruvate carboxylase having a sequence according to any one of claims 4 to 6, is fermented, an enrichment of the product in the medium or in the cells is carried out.
13. Verfahren nach Anspruch 12, 13. The method according to claim 12,
dadurch gekennzeichnet,  characterized,
dass das Produkt isoliert wird.  that the product is isolated.
14. Verfahren nach Anspruch 12 oder 13, 14. The method according to claim 12 or 13,
dadurch gekennzeichnet,  characterized,
dass als Mikroorganismus eine Spezies aus der Gruppe bestehend aus den Gattungen Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methyl- obacterium, Ralstonia, Vibrio und Clostridium eingesetzt wird.  in that a species from the group consisting of the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluyveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Vibrio and Clostridium is used as the microorganism.
15. Verfahren nach einem der Ansprüche 12 bis 14, 15. The method according to any one of claims 12 to 14,
dadurch gekennzeichnet,  characterized,
dass eine Aminosäure der Aspartat-Familie hergestellt wird.  that an amino acid of the aspartate family is produced.
16. Verfahren nach Anspruch 15, 16. The method according to claim 15,
dadurch gekennzeichnet,  characterized,
dass L-Lysin, L-Aspartat, L-Asparagin, L-Threonin, L-Isoleucin oder L-Methionin hergestellt wird. that L-lysine, L-aspartate, L-asparagine, L-threonine, L-isoleucine or L-methionine is produced.
17. Verfahren nach einem der Ansprüche 12 bis 14, 17. The method according to any one of claims 12 to 14,
dadurch gekennzeichnet,  characterized,
dass Stoffe aus der Glutamat-Familie der Aminosäuren wie L-Glutamat, Glutamin L- Arginin, L-Prolin, L-Citrullin oder L-Ornithin, Intermediate des Citrat-Zyklus, wie Malat, Fumarat, Succinat, Malat, Fumarat Citrat, Isocitrat oder 2-Oxoglutarat, Diamine wie Di- aminopentan oder Diaminobutan sowie weitere aus Oxalacetat hergestellte Stoffwechselprodukte, wie Itaconat, Ectoin, gamma-Aminobutyrat, Butanol, 1-Propanol, L- Citrullin, L-Ornithin, D-Arginin oder 4-Hydroxyprolin hergestellt werden.  that substances from the glutamate family of amino acids such as L-glutamate, glutamine L-arginine, L-proline, L-citrulline or L-ornithine, intermediates of the citrate cycle, such as malate, fumarate, succinate, malate, fumarate citrate, isocitrate or 2-oxoglutarate, diamines such as diaminopentane or diaminobutane and other metabolic products prepared from oxalacetate, such as itaconate, ectoine, gamma-aminobutyrate, butanol, 1-propanol, L-citrulline, L-ornithine, D-arginine or 4-hydroxyproline become.
18. Verfahren nach einem der Ansprüche 12 bis 17, 18. The method according to any one of claims 12 to 17,
dadurch gekennzeichnet,  characterized,
dass die Gene nach den Ansprüchen 1 bis 3 verstärkt exprimiert werden.  in that the genes according to claims 1 to 3 are expressed more intensively.
19. Chromosom, enthaltend eine DNA-Sequenz nach einem der Ansprüche 1 bis 3. 19. Chromosome containing a DNA sequence according to one of claims 1 to 3.
PCT/DE2018/000107 2017-05-18 2018-04-17 Pyruvate carboxylase having a feedback-resistant mutation, encoding dna, plasmides, and microorganism for the production thereof, and methods for the production of products the biosynthesis of which includes oxaloacetate as precursor WO2018210359A1 (en)

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