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  • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
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  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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  • 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
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

• This invention relates to a polypeptide having phosphoenolpyruvate carboxylase activity which does not require acetyl coenzyme A for activation and is desensitized to feedback inhibition by aspartic acid, and to genes coding for this polypeptide.
• the invention also relates to recombinant DNA molecules containing these genes, to bacteria transformed with these genes, and to methods of producing amino acids using the transformed bacteria.
• Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) is an enzyme which is found in almost all bacteria and all plants. PEP carboxylase catalyzes the condensation reaction between the three carbon glycolytic intermediate PEP and carbon dioxide resulting in the formation of the four carbon oxaloacetate (OAA), a metabolic intermediate common to the tricarboxylic acid (TCA) cycle and to L- aspartic acid biosynthesis.
• OAA oxaloacetate
• TCA a metabolic intermediate common to the tricarboxylic acid
• the TCA cycle requires continuous replenishment of C 4 molecules in order to replace the intermediates withdrawn for amino acid biosynthesis, and by playing an anaplerotic role in supplying OAA to the TCA cycle, the biotin-independent PEP carboxylase aids in fulfilling this function.
• OAA is a very important substrate for the production of cell metabolites such as amino acids, especially the glutamate family, i.e. , glutamate, arginine and proline, and the aspartate family, . e. , aspartate, lysine, methionine, threonine and isoleucine.
• glutamate family i.e. , glutamate, arginine and proline
• the aspartate family . e. , aspartate, lysine, methionine, threonine and isoleucine.
• PEP carboxylase plays an important role in supplying organic acids by metabolic processes. For example, fermentive production of succinic acid from glucose by
• PEP carboxylase also plays an important role in the production of amino acids which are formed from glutamate and aspartate.
• the amino acid is a compound which universally exists in cells as components of proteins. However, for the sake of economic energy metabolism and substance metabolism, its production is strictly controlled. This control is principally feedback control, in which the final product of a metabolic pathway inhibits the activity of an enzyme which catalyzes an earlier step of the pathway. PEP carboxylase also undergoes various regulations in expression of its activity. For example, in the case of PEP carboxylase of microorganisms belonging to the genus Brevibacterium, Corynebacterium or the genus Escherichia, PEP carboxylase activity is inhibited by aspartic acid. See e.g., Mori, M., et al, J. Biochem.
• acetyl coenzyme A is an allosteric activator of PEP carboxylase from Brevibacterium flavum and Escherichia coli, for example. See Mori, M., et al,
• the anaplerotic enzyme PEP carboxylase is critical to the maintenance of an optimal pool of OAA, and consequently determines the biosynthetic levels of amino acids deriving from OAA, one way of improving amino acid production by fermentation would be to manipulate the corresponding ppc gene.
• the amplification of the ppc gene from Brevibacterium lactofermentum has been shown to improve the production of proline and threonine. See Sano, K., et al, Agric. Biol Chem. 57:597-599 (1987).
• U.S. Patent No. 4,757,009 discloses a process for producing an amino acid by fermentation which comprises cultivating in a culture medium a Corynebacterium or Brevibacterium strain carrying a recombinant DNA molecule comprising a plasmid having operationally inserted therein a gene coding for PEP carboxylase, wherein the gene is a chromosomal gene isolated from a Corynebacterium or a Brevibacterium strain carrying a PEP carboxylase gene and has a chromosomal gene coding for an amino acid, and isolating the amino acid from the culture medium.
• the Corynebacterium or Brevibacterium strain from which the gene coding for PEP carboxylase is isolated is a strain which exhibits weakened feedback inhibition by aspartic acid.
• European Patent No. 358,940 (Bachmann et al; Degussa Aktiengesellschaft) discloses a plasmid pDM6 that is introduced into Corynebacterium glutamicum DM58-1, which is deposited at the Deutsche Sammlung von Mikroorganismen (DSM) under DSM 4697, wherein the plasmid contains a genetic sequence comprising information coding for the production of a protein having PEP carboxylase activity.
• the ppc gene is isolated from a genomic bank of Corynebacterium glutamicum ATCC 13032, and the PEP carboxylase is not stimulated by acetyl-CoA.
• Also disclosed is a method of producing L-lysine, L-threonine, and L-isoleucine by fermentation which comprises culturing in an appropriate medium a host bacterium belonging to the genus Corynebacterium or Brevibacterium which contains plasmid pDM6, and recovering the L-amino acid from the medium.
• U.S. Patent No. 5,876,983 discloses a method of producing an amino acid which comprises selecting a microorganism of the genus Escherichia containing a DNA sequence encoding a mutant PEP carboxylase desensitized to feedback inhibition by aspartic acid by growing Escherichia microorganisms in the presence of a wild-type PEP carboxylase inhibitor selected from the group consisting of 3-bromopyruvate, aspartic acid- ⁇ -hydrazide and DL-threo- ⁇ -hydroxyaspartic acid; culturing a microorganism of the genus Escherichia or coryneform bacteria transformed with the DNA sequence encoding a mutant PEP carboxylase in a suitable medium; and separating from the medium an amino acid selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-gluta
• a PEP carboxylase that is not substantially regulated by acetyl-CoA or aspartic acid could improve carbon flow from the three carbon intermediate PEP to the four carbon intermediate OAA. The improved flow could contribute to compounds derived from OAA and increase amino acid biosynthesis.
• the present invention relates to a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the gene is capable of being expressed in a host microorganism, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the present invention also relates to a recombinant DNA molecule comprising a plasmid and a gene encoding a polypeptide having PEP carboxylase activity operationally inserted therein, wherein the recombinant DNA molecule is capable of propagating and the gene is capable of being expressed in a host microorganism comprising the genus Escherichia, Corynebacterium and
• polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the present invention further relates to a host microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium transformed with a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the gene is derived from a plant belonging to the class Monocotyledonae or Dicotyledonae or from a microorganism belonging to the genus Corynebacterium or Brevibacterium, wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid, and wherein the host microorganism transformed with the DNA fragment expresses the gene.
• a method of producing an amino acid by fermentation comprises cultivating a host microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium in a suitable medium and isolating from the culture medium an amino acid, wherein the host microorganism is transformed with a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the host microorganism expresses the gene, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the present invention relates to a method of selecting a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid, to a method of increasing the rate of conversion of PEP to OAA, to a method of recycling carbon in a fermentation process, to a method of assimilating carbon in a fermentation process which does not require biotin, to a method of increasing the production of organic acids in a fermentation process, and to a method of increasing the production of amino acids in a fermentation process.
• Figure 1 is a diagram of a strategy for gene replacement.
• Activator includes both a substance necessary for the polypeptide to become active in the first place, as well as a substance which merely accentuates activity.
• amino acids refer to the naturally occurring L amino acids (alanine, arginine, aspartic acid, asparagine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine).
• Chimeric gene refers to a gene comprising heterogeneous regulatory and coding sequences. It is a hybrid gene produced by recombinant DNA technology.
• DNA fragment refers to a fraction of a deoxyribonucleic acid molecule.
• “Expression,” as used herein, is intended to mean the production of the protein product encoded by a gene.
• Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding region. It is a discrete chromosomal region comprising regulatory DNA sequences responsible for the control of expression, i.e., transcription and translation, and for a coding sequence which is transcribed and translated to give a distinct polypeptide.
• "Host microorganism” means the microorganism that is transformed with the introduced genetic material.
• “Inhibition” includes both the reduction of activity of the polypeptide and the complete lack of activity as well.
• “Isolated” as used herein means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
• Polypeptide or “protein” as used herein refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide.
• a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence. It may also be generated in any manner, including chemical synthesis or expression of a recombinant expression system.
• regulatory sequences refer to nucleotide sequences located upstream (5'), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell.
• Synthetic DNA refers to a nucleic acid molecule produced in whole or in part by chemical synthesis methods.
• Transformation herein refers to the transfer of a foreign gene into a host cell either as part of the host cell genomic DNA or as an independent molecule, and its genetically stable inheritance.
• a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the gene is capable of being expressed in a host microorganism, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the ppc gene which encodes the enzyme PEP carboxylase, may be any one provided that it is a gene encoding for the PEP carboxylase of a plant belonging to the class Monocotyledonae or Dicotyledonae or of a microorganism belonging to the genus Brevibacterium or Corynebacterium, and provided the expressed polypeptide does not require acetyl-CoA for activation and is substantially desensitized to feedback inhibition by aspartic acid.
• the ppc gene is preferably determined for its base sequence and cloned.
• a DNA fragment containing the gene can be amplified and isolated by using the PCR method and the like, followed by using a suitable vector to achieve cloning.
• Preferred donors of the ppc gene are strains which exhibit weakened feedback inhibition by aspartic acid. Such strains are recognized as being resistant to aspartic acid-antagonistic inhibitors.
• PEP carboxylase is a key enzyme of photosynthesis in C 4 plants. It is specifically localized in the cytosol of mesophyll cells and is regulated by a phosphorylation/dephosphoylation process. See Giglioli-Guivarc'h, N., et al,
• the DNA fragment containing a gene encoding a polypeptide having PEP carboxylase activity is derived from a plant belonging to the class Monocotyledonae or Dicotyledonae.
• the DNA fragment is derived from an alfalfa plant.
• the DNA fragment is derived from a Medicago sativa strain.
• ppc nucleotide sequence from Medicago sativa is known (Pathirana, S., et al, Plant Molecular Biology 20:437-450 (1992)) and provided in SEQ ID NO: 1 , and the amino acid sequence of the native PEP carboxylase encoded thereby is provided in SEQ ID NO:2. Since these sequences are known, primers may be designed and synthesized based on the nucleotide sequences, and then the genes may be obtained by PCR, using the messenger RNA as a template.
• Post-translational regulation of plant PEP carboxylase is achieved, for example, through phosphorylation of the protein. See Jiao, J.A., et al, Arch. Biochem. Biophys. 269:526-535 (1989); Duff, S.M., et al, Eur. J. Biochem.
• the DNA fragment containing a gene encoding a polypeptide having PEP carboxylase activity which is derived from a plant belonging to the class Monocotyledonae or Dicotyledonae is modified by one or more nucleotide substitutions, deletions and/or insertions. Most preferably, the modification comprises deleting the nucleotides encoding the amino acid sequence: Met - Ala - Ser - He - Asp - Ala - Gin - Leu - Arg.
• the DNA fragment containing a gene encoding a polypeptide having PEP carboxylase activity is derived from a microorganism belonging to the genus Brevibacterium or Corynebacterium.
• the DNA fragment is derived from a Corynebacterium glutamicum strain.
• the native ppc nucleotide sequence of Corynebacterium glutamicum is shown in SEQ ID NO:3.
• the number of amino acids in the active PEP carboxylase molecule of the present invention may vary, and all amino acid sequences derived from an alfalfa plant or a Corynebacterium strain that have PEP carboxylase activity and the desired de-regulatory characteristics are contemplated as being included in the present invention.
• Polypeptide sequences which differ from each other only by conservative substitutions are included as well. Such conservative substitutions consist of a substitution of one amino acid at a given position in the sequence for another amino acid of the same class.
• One or more non-conservative amino acid substitutions, deletions and/or insertions, located at positions of the sequence that do not alter the polypeptide to the extent that the biological activity of the polypeptide is destroyed, are also included.
• Modifications to the sequence such as deletions, insertions, and/or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting PEP carboxylase protein molecule are also contemplated.
• an alteration in the gene sequence which reflects the degeneracy of the genetic code, or which results in the production of a chemically equivalent amino acid at a given site are contemplated. It is therefore understood that the invention encompasses more than the specific exemplary sequences.
• Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
• the DNA fragment containing a gene encoding a polypeptide having PEP carboxylase activity is a chimeric gene comprising an incomplete PEP carboxylase nucleotide sequence derived from a microorganism belonging to the genus Brevibacterium or Corynebacterium and an incomplete PEP carboxylase nucleotide sequence derived from a plant belonging to the class Monocotyledonae or Dicotyledonae.
• the two incomplete sequences form a complete chimeric ppc gene capable of expressing a polypeptide having PEP carboxylase activity in which the polypeptide does not require acetyl coenzyme A for activation and is desensitized to feedback inhibition by aspartic acid.
• one incomplete PEP carboxylase nucleotide sequence is derived from a microorganism belonging to the genus Corynebacterium, and the other incomplete PEP carboxylase nucleotide sequence is derived from an alfalfa plant. Most preferably, one incomplete PEP carboxylase nucleotide sequence is derived from a Corynebacterium glutamicum strain, and the other incomplete PEP carboxylase nucleotide sequence is derived from a Medicago sativa strain.
• the DNA fragment is complementary DNA
• cDNA genomic DNA or synthetic DNA.
• a DNA fragment of the present invention encoding PEP carboxylase can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
• Isolation of theppc gene can be conducted, for example, by the following method. Although the following example refers to Corynebacterium for simplicity, it is to be recognized that bacteria from the genus Brevibacterium can likewise be used. First, a chromosomal gene is extracted from a Corynebacterium strain carrying a. ppc gene (utilizing, for example, the method of H.
• restriction enzymes can be employed by controlling the degree of cleavage, for example, by controlling the time of the cleavage reaction, the temperature, etc. Cleavage of DNA by restriction enzymes is well understood by those skilled in the art and need not be set forth here in detail.
• a PEP carboxylase-deficient mutant of coryneform bacteria or E. coli is transformed with the resulting recombinant DNA.
• Transformants thus obtained can be selected and isolated by conventional methods based on characteristics possessed by the vector DNA and/or the recipient. For example, bacterial strains which come to possess PEP carboxylase activity are isolated, and appc gene can be isolated therefrom.
• an enzyme which does not require acetyl-CoA for activation and is substantially desensitized to aspartic acid inhibition may be obtained. It becomes apparent, by measuring PEP carboxylase activity in the absence and/or presence of acetyl-CoA, for example, whether or not the enzyme requires acetyl- CoA as an activator. It also becomes apparent, by measuring the PEP carboxylase activity in the presence and/or absence of aspartic acid in an enzyme reaction system, for example, whether or not the enzyme thus obtained is substantially inhibited by aspartic acid.
• the measurement of the enzyme activity can use a spectrometric method (Yoshinage, T., etal,J. Biochem. 55:747-750 (1970)) and the like.
• a spectrometric method Yamashinage, T., etal,J. Biochem. 55:747-750 (1970)
• the reaction can be measured spectrophotometrically by following the decrease in the absorbance (usually at 340 nanometers).
• a method of selecting a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid comprises extracting a chromosomal gene from a Corynebacterium strain carrying a ppc gene, cleaving the chromosomal gene with an appropriate restriction enzyme, ligating the ppc gene with a plasmid vector capable of propagating in
• Corynebacterium transforming a Corynebacterium strain in which the ppc and pyc genes are nonfunctional, isolating strains which show superior growth on minimal medium with glucose as the only carbon source, and isolating a DNA fragment from the strain.
• Pyruvate carboxylase (EC 6.4.1.1 ) is an important anaplerotic enzyme that replenishes OAA, which is consumed for biosynthesis during growth, from pyruvate and is used in lysine and glutamic acid production in industrial fermentations.
• the biotin-dependent pyruvate carboxylase encoded by the pyc gene has recently been found to be an anaplerotic enzyme in Corynebacterium glutamicum.
• inhibitors of PEP carboxylase activity are also added to the medium.
• an analog of aspartic acid may be added.
• the analog compound preferably exhibits a growth inhibitory action against a microorganism belonging to the genus Corynebacterium which produces a wild- type PEP carboxylase, the aforementioned growth inhibitory action is recovered by existence of L-glutamic or L-aspartic acid, and the analog compound inhibits wild-type PEP carboxylase activity. If a strain being resistant to the analog compound is selected from a microorganism belonging to the genus Corynebacterium, it is much more likely that a host microorganism which produces PEP carboxylase with desensitized feedback inhibition by aspartic acid will be obtained.
• strains are isolating which show an increased production of an amino acid derived from OAA.
• amino acids include aspartate, lysine, methionine, threonine and isoleucine.
• strains can be grown on minimal medium in the absence of acetyl-CoA, and the PEP carboxylase activity can be measured.
• a recombinant DNA molecule comprising a plasmid and a gene encoding a polypeptide having PEP carboxylase activity operationally inserted therein, wherein the recombinant DNA molecule is capable of propagating and the gene is capable of being expressed in a host microorganism comprising the genus Escherichia, Corynebacterium and Brevibacterium, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the plasmid vector used in the present invention can be any vector as long as it can be propagated in cells of bacteria from Escherichia, Corynebacterium or
• the vector DNA is cleaved by the same restriction enzyme used for cleavage of the chromosomal gene or is connected to an oligonucleotide having a complementary base sequence at the respective terminals of the chromosomal DNA cleavage fragment and the cleaved vector DNA.
• the plasmid vector and the chromosomal gene-containing fragment are then subjected to a ligation reaction.
• the gene encoding the polypeptide having PEP carboxylase activity is derived from an alfalfa plant. Most preferably, the gene is derived from a Medicago sativa strain. In another preferred embodiment, the gene is modified by one or more nucleotide substitutions, deletions and/or insertions. Most preferably, the modification comprises deleting the nucleotides encoding the amino acid sequence: Met - Ala - Ser - He - Asp - Ala - Gin - Leu -
• a host microorganism transformed with a DNA fragment of the present invention containing a gene encoding a polypeptide having PEP carboxylase activity.
• microorganisms utilized for the production of L-amino acids may be used, for example, those belonging to the genus Brevibacterium, the genus Corynebacterium, the genus Bacillus, the genus Escherichia, the genus Seratia, the genus Providencia, and the genus Arthrobacter.
• the DNA fragment containing the ppc gene is expressed in a host microorganism belonging to the genus Escherichia,
• Corynebacterium or Brevibacterium there may be exemplified microorganisms belonging to the genus Escherichia, for example, Escherichia coli, preferably L-lysine-producing Escherichia coli, coryneform bacteria, preferably L-lysine-producing strains, and the like.
• the coryneform bacteria referred to in the present invention is a group of microorganisms which are aerobic
• Gram-positive non-acid-fast rods having no spore-forming ability including bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium having been hitherto classified into the genus Brevibacterium but being united as bacteria belonging to the genus Corynebacterium at present, and bacteria belonging to the genus Brevibacterium closely related to bacteria belonging to the genus Corynebacterium.
• the host microorganism when the DNA fragment is derived from a plant from the class Monocotyledonae or Dicotyledonae, the host microorganism may be transformed with a recombinant DNA molecule comprising a plasmid and the DNA fragment operationally inserted therein.
• the host microorganism may be transformed by integrating the DNA fragment of the present invention into the host chromosomal DNA.
• the DNA fragment is derived from an alfalfa plant, and most preferably, it is derived from a Medicago sativa strain.
• the plant-derived DNA fragment is modified by one or more nucleotide substitutions, deletions and/or insertions. Most preferably, the modification comprises deleting the nucleotides encoding the amino acid sequence: Met - Ala - Ser - He - Asp - Ala - Gin - Leu - Arg.
• the DNA sequence of the present invention is inserted into vector DNA capable of self-replication and introduced into the host.
• vector DNA a plasmid vector is preferable, and those capable of self-replication in a host cell are most preferable.
• a vector of phage DNA can be also utilized.
• the DNA fragment containing a gene is derived from a plant of the class Monocotyledonae or Dicotyledonae or from a microorganism belonging to the genus Corynebacterium or Brevibacterium
• the DNA fragment is integrated into the chromosomal DNA of a host microorganism by means of a method using, for example, transposons (Berg, D.E. and Berg, CM., Bio/Technol 7:417 (1983)), Mu phage (Japanese Patent Laid-open No. 2- 109985) or homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)).
• transposons Berg, D.E. and Berg, CM., Bio/Technol 7:417 (1983)
• Mu phage Japanese Patent Laid-open No. 2- 109985
• homologous recombination Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)
• the DNA fragment is derived from a Corynebacterium glutamicum strain and is integrated into the chromosomal DNA of a host microorganism.
• Corynebacterium glutamicum chromosome has been sequenced (SEQ ID NO: 3). According to the gene replacement strategy of the present invention, the chromosomal copy of the ppc gene is removed and replaced with an antibiotic resistance gene marker ( Figure 1 ). The marker is in turn replaced with a modified ppc gene of the present invention.
• this gene replacement strategy facilitates complete removal of the chromosomal ppc DNA sequence of a host microorganism and substitution of a new ppc gene without altering the expression of the two neighboring genes, the tpi gene and the secG gene.
• the tpi gene encodes the glycolytic enzyme triosephosphate isomerase
• the secG gene encodes secG, an integral membrane protein involved in protein export.
• a method of producing an amino acid by fermentation comprises cultivating a host microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium in a suitable medium and isolating from the culture medium an amino acid, wherein the host microorganism is transformed with a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the host microorganism expresses the gene, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
• the method for cultivating the aforementioned hosts is not especially different from a cultivation method for amino acid-producing microorganisms in the prior art.
• an ordinary medium is used containing a carbon source, a nitrogen source, inorganic ions, substances satisfying nutrient auxotrophy, and optionally organic trace nutrients such as amino acids, vitamins and the like.
• carbon source carbohydrates such as glucose, sucrose, lactose, etc., as well as organic acids such as acetic acid may be used.
• nitrogen source ammonia gas, aqueous ammonium, ammonium salt and the like can be used.
• inorganic ions potassium ions, sodium ions, magnesium ions, phosphate ions, and the like are appropriately added to the media as required.
• the cultivation is performed until the generation and accumulation of the amino acid substantially stops while suitably controlling pH and temperature of the medium under an aerobic condition.
• an ordinary method can be applied. For example, after the removal of the cells by filtration, ultrafiltration, centrifugation or other known means, the amino acid is recovered, for example, by concentration of the cell-free solution and crystallization of the amino acid (or a salt thereof). Alternatively, the compound can be recovered by ion exchange chromatography.
• the amino acid is one which is derived from OAA, such as L-aspartate, L-lysine, L-methionine, L-threonine and L-isoleucine. Most preferably, the amino acid is L-lysine.
• a method of increasing the rate of conversion of PEP to OAA comprises transforming a host microorganism with a DNA fragment of the present invention.
• the host microorganism is selected from the genus Escherichia, Corynebacterium or Brevibacterium.
• PEP carboxylase catalyzes the condensation reaction between PEP and carbon dioxide resulting in the formation of OAA.
• a PEP carboxylase of the present invention that is not substantially regulated by acetyl-CoA or aspartic acid therefore increases the rate of conversion of PEP to OAA.
• transformation may be by integration or by utilization of a recombinant DNA molecule, for example.
• the host microorganism is transformed by the integration of the DNA fragment of the invention into the chromosomal DNA of the host microorganism.
• a method of recycling carbon in a fermentation process comprises transforming a host microorganism with a DNA fragment of the present invention.
• the host microorganism is selected from the genus Escherichia, Corynebacterium or Brevibacterium.
• the TCA cycle requires continuous replenishment of C 4 molecules in order to replace the intermediates withdrawn for amino acid biosynthesis.
• PEP carboxylase aids in fulfilling this function by playing an anaplerotic role in supplying the four carbon OAA to the TCA cycle.
• transformation may be by integration or by utilization of a recombinant DNA molecule, for example.
• the host microorganism is transformed by the integration of the DNA fragment of the invention into the chromosomal DNA of the host microorganism.
• L-lysine and L-glutamic acid have been hitherto industrially produced by fermentative methods by using coryneform bacteria belonging to the genus
• PEP carboxylase does not require biotin for biological activity.
• PEP carboxylase one of the major physiological roles of PEP carboxylase is to replenish the TCA cycle by the assimilation of carbon.
• the de-regulated PEP carboxylase of the present invention improves the assimilation of carbon dioxide.
• a method of assimilating carbon in a fermentation process which does not require biotin.
• the method comprises transforming a host microorganism with a DNA fragment of the present invention.
• the host microorganism is selected from the genus Escherichia, Corynebacterium or Brevibacterium.
• transformation may be by integration or by utilization of a recombinant DNA molecule, for example.
• the host microorganism is transformed by the integration of the DNA fragment of the invention into the chromosomal DNA of the host microorganism.
• the anaplerotic enzyme PEP carboxylase is critical to the maintenance of an optimal pool of OAA, and consequently determines the biosynthetic levels of organic acids deriving from it.
• DNA fragment of the present invention the rate of production of OAA is increased. As such, the production of organic acids derived from OAA is increased as well.
• the host microorganism is selected from the genus Escherichia, Corynebacterium or Brevibacterium.
• transformation may be by integration or by utilization of a recombinant DNA molecule, for example.
• the host microorganism is transformed by the integration of the DNA fragment of the invention into the chromosomal DNA of the host microorganism.
• OAA is an important substrate for the production of cell metabolites such as amino acids.
• the ppc genes of the invention thereby increase the production of amino acids. Therefore, in another aspect of the invention there is provided a method of increasing the production of amino acids in a fermentation process.
• the method comprises transforming a host microorganism with a DNA fragment of the present invention.
• the host microorganism is selected from the genus Escherichia, Corynebacterium or Brevibacterium.
• the amino acid comprises L-aspartate, L-lysine, L-methionine, L- threonine and L-isoleucine. Most preferably, the amino acid is L-lysine.
• transformation may be by integration or by utilization of a recombinant DNA molecule, for example.
• the host microorganism is transformed by the integration of the DNA fragment of the invention into the chromosomal DNA of the host microorganism.
• Example 1 A Plant ppc Gene Functions in Escherichia coli
• the cDNA clone (APPC) of theppc gene from alfalfa (Medicago sativa) was functional in the Escherichia coli mutant CGSC3594 which lacks a functional PEP carboxylase and cannot grow on M9 medium with glucose as the sole carbon source.
• APPC plasmid pMS22
• E. coli mutant CGSC3594 was able to grow on M9 medium with glucose as the sole carbon source.
• the DNA and amino acid sequences of the alfalfa PEP carboxylase are provided in SEQ ID NO:l and SEQ ID NO:2, respectively.
• Theppc Gene from Alfalfa Shows Growth Stimulation in Corynebacterium in Shake Flasks
• the effect of the ppc gene from alfalfa (Medicago sativa) on growth stimulation in the lysine-producing Corynebacterium strain BF100 was determined. Growth was measured as the optical density at 660nm, the titer was measured as g lysine/liter of medium, and the yield was measured as (g lysine/g glucose consumed) x 100. 30 mg/L of isopropyl-beta-D-galactoside (IPTG), an inducer, was present. The results are shown in Table 1 :
• Theppc Gene from a Wild-Type Corynebacterium Strain Improves Productivity of a Lysine-Producing Corynebacterium Strain
• the cDNA clone (CPPC) of the ppc gene from Corynebacterium glutamicum ATCC 13032 was inserted into the pCPPC plasmid.
• CPPC cDNA clone
• the region flanking the ppc gene in the Corynebacterium glutamicum chromosome has been sequenced (SEQ ID NO: 3).
• the chromosomal copy of the ppc gene is removed and replaced with an antibiotic resistance gene marker ( Figure 1 ).
• the marker is in turn replaced with a modified ppc gene of the present invention.
• This gene replacement strategy facilitates complete removal of the chromosomal ppc DNA sequence of a host microorganism and substitution of a new gene without altering the expression of the two neighboring genes.

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• 1999
  • 1999-06-29 JP JP2001506844A patent/JP2003503064A/ja not_active Withdrawn
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