WO2018210359A1 - Pyruvate carboxylase présentant une mutation à l'origine d'une résistance de rétraction, adn codant, plasmides, et micro-organisme de production et procédé de préparation de produits dont la biosynthèse comprend l'oxalacétate comme précurseur - Google Patents

Pyruvate carboxylase présentant une mutation à l'origine d'une résistance de rétraction, adn codant, plasmides, et micro-organisme de production et procédé de préparation de produits dont la biosynthèse comprend l'oxalacétate comme précurseur 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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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

La présente invention concerne une pyruvate carboxylase et l'ADN codant pour la pyruvate carboxylase, un plasmide contenant l'ADN, et un micro-organisme de production et un procédé pour la préparation de produits dont la biosynthèse comprend l'oxalacétate comme précurseur et un chromosome. Selon l'invention, un ADN codant pour une pyruvate carboxylase est fourni, lequel a une séquence qui est au moins à 70% identique à la séquence Nr. 1 dans laquelle le triplet, codant pour isoleucine-1012, en positions 3034-3036, est remplacé par un triplet codant pour sérine. L'expression de ce gène permet d'obtenir une pyruvate carboxylase qui permet d'obtenir des métabolites dérivés de l'oxalacétate avec un rendement supérieur de 7% à celui de la souche de la Pyc native.
PCT/DE2018/000107 2017-05-18 2018-04-17 Pyruvate carboxylase présentant une mutation à l'origine d'une résistance de rétraction, adn codant, plasmides, et micro-organisme de production et procédé de préparation de produits dont la biosynthèse comprend l'oxalacétate comme précurseur WO2018210359A1 (fr)

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DE102017004750.2A DE102017004750A1 (de) 2017-05-18 2017-05-18 Pyruvvatcarboxylase und für die Pyrovatcarboxylase kodierende DNA, Plasmid enthaltend die DNA, sowie Mikroorganismen zur Produktion und Verfahren zur Herstellung von Produkten, deren Biosynthese Oxalacetat als Vorstufe beeinhaltet und Chromsom

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DK2194122T3 (en) * 1998-04-13 2016-02-15 Univ Georgia Overexpression of pyruvate carboxylase increased the production of biochemicals derived from oxaloacetate, in microbial cells
US6171833B1 (en) * 1998-12-23 2001-01-09 Massachusetts Institute Of Technology Pyruvate carboxylase from corynebacterium glutamicum

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US4346170A (en) 1979-07-23 1982-08-24 Ajinomoto Company, Incorporated Method for producing L-lysine by fermentation
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