WO2006078132A1 - Escherichia strain capable of converting xmp to gmp and maintaining the inactivated state of gene(s) associated with gmp degradation and methods of using the same - Google Patents

Escherichia strain capable of converting xmp to gmp and maintaining the inactivated state of gene(s) associated with gmp degradation and methods of using the same Download PDF

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WO2006078132A1
WO2006078132A1 PCT/KR2006/000221 KR2006000221W WO2006078132A1 WO 2006078132 A1 WO2006078132 A1 WO 2006078132A1 KR 2006000221 W KR2006000221 W KR 2006000221W WO 2006078132 A1 WO2006078132 A1 WO 2006078132A1
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gene
gmp
strain
escherichia
accession
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PCT/KR2006/000221
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French (fr)
Inventor
Young-Hoon Park
Hyoung-Suk Kim
Jin-Nam Lee
Ki-Hoon Oh
Jeong-Hwan Kim
Yoon-Suk Oh
Jae-Ick Sim
Kyung-Oh Choi
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Cj Corp.
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Priority to EP06702908.2A priority Critical patent/EP1844135B1/en
Priority to ES06702908.2T priority patent/ES2443099T3/en
Priority to CN2006800027775A priority patent/CN101137743B/en
Priority to DK06702908.2T priority patent/DK1844135T3/en
Priority to US11/814,417 priority patent/US7741101B2/en
Priority to JP2007552064A priority patent/JP4769255B2/en
Publication of WO2006078132A1 publication Critical patent/WO2006078132A1/en

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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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to an Escherichia strain capable of converting
  • XMP 5'-xanthylic acid
  • GMP 5'-guanylic acid
  • [2] 5'-guanylic acid can be produced by microbial enzymatic or chemical breakdown of yeast RNA, direct fermentation for directly producing nucleotides using mutant strains cultured in media containing a sugar, a nitrogen source, and a phosphate source, or a combination method including the fermentative synthesis of nucleotide intermediates and the chemical or enzymatic conversion of the nucleotide intermediates to nucleotides.
  • a combination method that combines fermentative or chemical synthesis and enzymatic conversion is industrially widely used due to its economical advantages.
  • GMP production by the combination method consists of fermentative synthesis of
  • XMP 5'-xanthylic acid
  • GMP 5'-xanthylic acid
  • microbial strains two types are used. That is, an XMP-producing strain is used in the fermentative synthesis, and a microbial strain capable of inducing a continuous high-level expression of a gene encoding XMP aminase activity is used in the enzymatic conversion. In this case, the survival rate of the microbial strain used in the enzymatic conversion is remarkably reduced during a later culture period relative to during an initial culture period, thereby decreasing the production of GMP.
  • guaA gene encoding XMP aminase is fatal to host cells, and thus, the continuous high-level expression of XMP aminase hinders the growth of the microbial strain used in the enzymatic conversion during the later culture period.
  • an internal gene responsible for GMP degradation in the microbial strain used for the enzymatic conversion is expressed, thereby facilitating the degradation of GMP.
  • GPDl 114 (Accession No. KCCM- 10543) including an XMP aminase-encoding gene capable of expressing XMP aminase according to cellular growth state and an inactivated internal glnh gene. Furthermore, the present inventors developed Escherichia sp. GPUl 114 (Accession No. KCCM- 10536) derived from the Escherichia sp. GPDl 114, in which the ushA gene encoding 5'-nucleotidase is inactivated to remove GMP degradability.
  • the present invention provides a microbial strain capable of converting
  • XMP 5'-xanthylic acid
  • GMP 5'-guanylic acid
  • the present invention also provides a method of accumulating GMP synthetase in a high concentration in a culture medium using the microbial strain.
  • the present invention also provides a method of producing GMP, guanosine
  • GDP 5'-diphosphate
  • GTP guanosine 5'-triphosphate
  • the present invention provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes an inactivated deoD gene.
  • the parent strain, Escherichia sp. GPUl 114 (Accession No. KCCM-10536) is an
  • Escherichia coli Escherichia coli (E. coli) strain in which glnL gene is inactivated, guaA gene encoding 5'-xanthylic acid (XMP) aminase is inserted by transformation, and an internal ushA gene encoding 5'-nucleotidase is inactivated.
  • XMP can be converted to 5'-guanylic acid (GMP) at a high level by the expression of the guaA gene, and GMP degradation activity is low due to no expression of the ushA gene, thereby increasing GMP productivity.
  • GMP 5'-guanylic acid
  • the deoD gene is not limited provided that it is a gene encoding purine-nucleoside phosphorylase activity.
  • the deoD gene may be NCBI accession no. NP_418801, but the present invention is not limited thereto.
  • the deoD gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for deoD.
  • the Escherichia strain of the present invention may be E. coli DKO-ud (Accession
  • the present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD and aphA genes.
  • the aphA gene is not limited provided that it is a gene encoding acid phosphatase activity.
  • the aphA gene may be NCBI accession no. NP_418479, but the present invention is not limited thereto.
  • the aphA gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for aphA.
  • the Escherichia strain of the present invention may be E. coli TKO-udh (A ccession No. KCCM- 10631).
  • the present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD, aphA, and appA genes.
  • the appA gene is not limited provided that it is a gene encoding acid
  • the appA gene may be NCBI accession no. NP_415500, but the present invention is not limited thereto.
  • the appA gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for appA.
  • the Escherichia strain of the present invention may be E. coli QKO-udhp
  • the present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD, aphA, appA, and hprt genes.
  • the hprt gene is not limited provided that it is a gene encoding hypoxanthine phos- phoribosyltransferase.
  • the hprt gene may be NCBI accession no. NP_414667, but the present invention is not limited thereto.
  • the hprt gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for hprt.
  • the Escherichia strain of the present invention may be E. coli QIKO (Accession
  • the term 'inactivation' indicates that a target gene is not expressed, or even if the expression is possible, the gene does not produce a functional gene product.
  • the 'inactivation' embraces the meaning that a target gene is completely inactivated or is not substantially expressed due to its significantly low expression level.
  • the inactivated genes can be obtained by any method known in the art, e.g., site-specific mutagenesis or homologous recombination, but the present invention is not limited thereto. Homologous recombination is preferable.
  • the inactivation of a gene can be induced by deletion, substitution, inversion, or a combination thereof, but the present invention is not limited thereto.
  • Gene inactivation by homologous recombination includes constructing inactivated gene cassettes by inserting foreign DNAs into target genomic DNA fragments, inserting the cassettes into microbial strains to induce homologous recombination between internal genes in the microbial cells and the cassettes, and selecting recombinant strains containing inactivated genes.
  • the foreign DNAs cloned into the target DNAs may contain a selectable marker, e.g., an antibiotic resistance gene.
  • Target genes inactivated with antibiotic resistance gene-containing cassettes can be easily selected on an antibiotic-containing agar plate. Occurrence of recombination can be confirmed by southern blotting, PCR (polymerase chain reaction), etc.
  • the removal of antibiotic resistance genes from selected strains includes transforming the selected strains with plasmids expressing genes capable of recognizing specific nucleotide sequences of inactivated cassettes and removing antibiotic resistance genes; and removing antibiotic resistance genes from the transformants.
  • the resultant antibiotic resistance gene-free transformants can be selected on an ampicillin-containing agar plate.
  • the removal of antibiotic resistance genes from target DNAs can be confirmed by colony transfer onto an agar plate containing an appropriate antibiotic with a toothpick and growth of colonies on the agar plate.
  • the Escherichia strains of the present invention can be prepared using the following illustrative methods.
  • Genomic DNAs are extracted from Escherichia sp. GPUl 114 (KCCM- 10536), and deoD, aphA, appA, and hprt genes are amplified by PCR using the genomic DNAs as templates.
  • the deoD, aphA, appA, and hprt genes are cloned into plasmids or other vectors to construct recombinant vectors (e.g., recombinant plasmids pDEO, pAPH, pAPP, pHPRT).
  • Host cells such as E. coli cells are transformed with the recombinant vectors.
  • recombinant vectors containing the deoD, aphA, appA, and hprt genes are extracted from the transformed cells.
  • a loxp- containing antibiotic resistance gene fragment is inserted into the deoD, aphA, appA, and hprt genes of the recombinant vectors to construct recombinant vectors containing inactivated deoD, aphA, appA, and hprt genes (e.g., pTdeoD:loxpKAT, pTaphA:loxpKAT, pTappA:loxpKAT, pThprt:loxpCAT).
  • the recombinant vectors are inserted in host cells and cultured. After the culture, recombinant vectors are extracted from the transformants and digested with appropriate restriction enzymes to obtain deoD, aphA, appA, and hprt gene-inactivated cassette fragments (e.g., ⁇ deoD:loxpKAT, ⁇ aph:loxpKAT, ⁇ app:loxpKAT, ⁇ hprt:loxpCAT).
  • deoD e.g., ⁇ deoD:loxpKAT, ⁇ aph:loxpKAT, ⁇ app:loxpKAT, ⁇ hprt:loxpCAT.
  • ⁇ deoD:loxpKAT is inserted into the GMP-producing strain GPUl 114 by elec- troporation, etc., and the transformed GMP-producing strain GPUl 114 is maintained under the antibiotic selection pressure to allow recombination between an antibiotic marker-containing deoD gene fragment and a chromosomal wild-type deoD gene to occur, thereby obtaining recombinant strains maintaining the inactivated state of the deoD gene even when generation continues.
  • the antibiotic marker must be removed.
  • recombinant vectors expressing genes recognizing loxp sites in antibiotic resistance genes and removing the antibiotic resistance genes are inserted into the selected recombinant strains by electroporation, etc.
  • the selection of the resultant transformants is performed on an ampicillin-containing agar plate using acquired antibiotic resistance.
  • the removal of antibiotic resistance genes from target DNAs can be confirmed by colony transfer onto an agar plate containing an appropriate antibiotic with a toothpick and growth of colonies on the agar plate.
  • the resultant transformants are maintained under the antibiotic selection pressure to allowrecombination between a deoD gene fragment containing a part of the antibiotic marker and a chromosomal wild-type deoD gene to occur, thereby obtaining transformed strains (e.g., E.
  • the present invention also provides a method of accumulating GMP synthetase in cells, which includes culturing an Escherichia strain of the present invention.
  • the present invention also provides a method of producing GMP, guanosine
  • GDP 5'-diphosphate
  • GTP guanosine 5'-triphosphate
  • the culturing of the Escherichia strain of the present invention can be performed under commonly known culture conditions, e.g., in a culture medium containing a carbon source, a nitrogen source, an amino acid, an inorganic compound, etc., while adjusting the temperature and pH of the culture medium.
  • the carbon source may be a carbohydrate such as glucose, fructose, or sterilized molasses (i.e., molasses converted to reducing sugar), and the nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium chloride, and ammonium sulfate, and an organic nitrogen source such as peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydroxylate, fish meal or its digest, defatted soybean or its digest.
  • the inorganic compound may be potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc.
  • the culture medium When needed, the culture medium may be supplemented with vitamins, bases, etc.
  • the culture may be performed under an aerobic condition, e.g., by shaking or aerobic stirring, preferably at a temperature of 30 to 40 0 C .
  • the pH of the culture medium may be maintained near neutral.
  • the culture may be performed for 1 to 2 days so that products having XMP aminase activity are accumulated in the culture medium.
  • GMP can be produced by converting XMP to GMP using
  • GMP synthetase produced by the culture of an Escherichia strain according to the present invention can be achieved by adding a material (e.g., xylene) increasing the cell membrane permeability of XMP into a culture medium so that XMP is introduced into a GMP-producing strain.
  • a material e.g., xylene
  • the material increasing the cell membrane permeability of XMP is well known in the art, e.g., a hydrophobic material (e.g., xylene, toluene, benzene, ethyl acetate), a surfactant (e.g., polyoxyethylenestearylamine (e.g., Nymeen S-215, Nihon Yushi Co.) a cationic surfactant such as cetyltrimethylammonium bromide, Cation FB, or Cation F2-40E, or an anionic surfactant such as sodium oleylamide sulfate, Newrex TAB, and Rapizole 80), a metal ion (e.g., calcium ion, magnesium ion), but the present invention is not limited to the above-illustrated examples.
  • a hydrophobic material e.g., xylene, toluene, benzene, ethyl acetate
  • a surfactant e
  • the content of the material increasing the cell membrane permeability of XMP varies according to the type of the material, and can be appropriately adjusted by those of ordinary skill in the art.
  • xylene may be used in an amount of 0.5 to 1 wt% based on the total weight of a culture medium.
  • GDP or GTP can be produced by commonly known enzymatic or chemical phosphorylation of GMP.
  • FIG. IA is a scheme of the construction of a recombinant plasmid pDEO of
  • FIG. IB is a scheme of the construction of a recombinant plasmid pTdeoD:loxpKAT of Example 1 of the present invention.
  • FIG. 2A is a scheme of the construction of a recombinant plasmid pAPH of
  • FIG. 2B is a scheme of the construction of a recombinant plasmid pTaphA:loxpKAT of Example 2 of the present invention.
  • FIG. 3A is a scheme of the construction of a recombinant plasmid pAPP of
  • FIG. 3B is a scheme of the construction of a recombinant plasmid pTappA:loxpKAT of Example 3 of the present invention.
  • FIG. 4A is a scheme of the construction of a recombinant plasmid pHPRT of Example 4 of the present invention.
  • FIG. 4B is a scheme of the construction of a recombinant plasmid pThprt:loxpCAT of Example 4 of the present invention.
  • Example 1 Construction of recombinant plasmid and inactivation of deoD gene using the recombinant plasmid
  • Nucleic Acid Research 24 (13), 1996, pp2519-2524) were digested with restriction enzymes EcoR V and Hinc II to obtain loxp site- containing kanamycin resistance gene fragments (about 1.7 kb).
  • the DNA fragments contained in the bands were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTdeoD::loxpKAT (about 4.5kb) (see FIG. IB).
  • DH5 ⁇ the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0 C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 niL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.3 kb DNA fragments containing the ORFs of deoD genes and the loxpKAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 1 and 2 as primers. The PCR was performed at 94 0 C for 1 minute for denaturation, 55 0 C for 1 minute for annealing, and 68 0 C for 1 minute for extension for 35 cycles.
  • GPUl 114 containing a foreign guaA gene and the obtained transformants were smeared onto solid media containing kanamycin to select colonies.
  • Example 2 Construction of recombinant plasmid and inactivation of aphA gene using the recombinant plasmid
  • plasmids pUG6 were digested with restriction enzymes EcoRY and Hindi to obtain loxp site-containing kanamycin resistance gene fragments (about 1.7 kb).
  • the blunt-ended DNA fragments were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTaphA::loxpKAT (about 5.4kb) (see FIG. 2B).
  • DH5 ⁇ the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0 C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.4 kb DNA fragments containing the ORFs of aphA genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 3 and 4 as primers. The PCR was performed at 94 0 C for 1 minute for denaturation, 55 0 C for 1 minute for annealing, and 68 0 C for 1 minute for extension for 35 cycles.
  • the DNA fragments were ligated into pGEM-T-easy cloning vectors at 16 0 C overnight (see FIG. 3A).
  • the resultant recombinant plasmids pAPP were transformed into E. coli DH5 ⁇ , the obtained transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 37 0 C overnight.
  • plasmids pUG6 were digested with restriction enzymes EcoRY and Hindi to obtain loxp site-containing kanamycin resistance gene fragments (about 1.7 kb).
  • the blunt-ended DNA fragments were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTappA::loxpKAT (about 6.3kb) (see FIG. 3B).
  • DH5 ⁇ the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0 C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 3.1 kb DNA fragments containing the ORFs of appA genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 5 and 6 as primers. The PCR was performed at 94 0 C for 1 minute for denaturation, 55 0 C for 1 minute for annealing, and 68 0 C for 1 minute for extension for 35 cycles.
  • Example 4 Construction of recombinant plasmid and inactivation of hprt gene using the recombinant plasmid
  • PCR using genomic DNAs extracted from the E. coli QKO-udhp obtained in Example 3 as templates and oligonucleotides as set forth in SEQ ID NOS: 7 and 8 as primers.
  • the PCR was performed at 94 0 C for 30 seconds for denaturation, 69 0 C for 30 seconds for annealing, and 72 0 C for 1 minute and 30 seconds for extension for 30 cycles.
  • the DNA fragments contained in the bands were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pThprt::loxpCAT (about 5.3kb) (see FIG. 4B).
  • DH5 ⁇ the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0 C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.1 kb DNA fragments containing the ORFs of hprt genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 7 and 8 as primers.
  • the PCR was performed at 94 0 C for 1 minute for denaturation, 60 0 C for 1 minute for annealing, and 72 0 C for 1 minute for extension for 30 cycles.
  • the obtained DNA fragments ⁇ hprt::loxpCAT were elec- troporated into the E. coli QKO-udhp obtained in Example 3, and the resultant transformants were smeared onto solid media containing chloramphenicol to select colonies.
  • the colonies were designated QIKO and deposited in the KCCM on November 30, 2004 (Accession No. KCCM- 10630).
  • Example 5 GMP productivity of E. coli QIKO of Example 4 [71] Among the E. coli QIKO colonies selected after smearing on the solid media containing chloramphenicol in Example 4, 30 colonies were cultured in Erlenmeyer flasks containing GMP-producing media with the medium composition of Table 1 below, and GMP production was evaluated. First, the 30 single colonies were cultured overnight in a 32 0 C incubator containing GMP-producing solid media. Then, one loopful of each of the single colonies was inoculated on 50 ml of the GMP-producing media and cultured for 7 hours at 37 0 C with shaking at 250 rpm.
  • the cell cultures were supplemented with 2% xylene to facilitate the cell membrane permeation of enzymes, substrates, and products in the cell cultures.
  • the resultant cultures were added to enzymatic reaction solutions and incubated at 42 0 C for 15 minutes with shaking at 250 rpm.
  • the concentration of GMP was determined by HPLC (high performance liquid chromatography) and the results are presented in Table 2 below.
  • Table 1 Medium composition for GMP production.
  • the parent strains GPUl 114 produced 46 U/mL of GMP, whereas the recombinant strains QIKO, in which the genes with nucleotide degradability were inactivated, produced 49 U/mL of GMP.
  • the recombinant strains QIKO of the present invention exhibited 6% higher GMP production than the parent strains.
  • Example 6 Comparison of GMP byproduct (guanine and guanosine) production for recombinant strains in Erlenmeyer flasks [76] The E. coli QIKO colonies selected after smearing on the solid media containing chloramphenicol in Example 4 were cultured in Erlenmeyer flasks containing GMP- producing media with the medium composition of the above Table 1. The cultures were added to reaction solutions for GMP byproduct production with the composition of Table 3 below and byproduct production was evaluated.
  • the E. coli QIKO single colonies were cultured overnight in a 32 0 C incubator containing GMP-producing solid media. Then, one loopful of each of the single colonies was inoculated on 50 ml of the GMP-producing media and cultured for 7 hours at 37 0 C with shaking at 250 rpm. Then, the cell cultures were supplemented with 2% xylene to facilitate the cell membrane permeation of enzymes, substrates, and products in the cell cultures, and incubated at 37 0 C for 20 minutes with shaking at 250 rpm. The resultant cultures were mixed with the reaction solutions for GMP byproduct production with the composition of Table 3 and incubated at 42 0 C for 8 hours with shaking at 250 rpm. The concentration of two types of byproducts, guanine and guanosine, with respect to time was determined by HPLC, and the results are presented in Table 4 below.
  • GMP-producing strains of the present invention exhibit high conversion efficiency of XMP to GMP and low GMP degradation activity.
  • GMP synthetase According to a method of accumulating GMP synthetase in cells of the present invention, GMP synthetase can be accumulated in a high concentration in cells.
  • GMP synthetase According to a method of producing GMP, GDP, or GTP of the present invention,
  • GMP, GDP, or GTP can be produced in high efficiency.
  • KCCM Green Culture Center of Microorganisms
  • KCCM Green Culture Center of Microorganisms
  • KCCM Green Culture Center of Microorganisms
  • KCCM Green Culture Center of Microorganisms

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Abstract

Provided are mutant strains derived from Escherichia sp. GPU1114 (Accession No. KCCM-10536), having cumulative inactivation of deoD, aphA, appA, and hprt genes, and methods of using the same.

Description

Description
ESCHERICHIA STRAIN CAPABLE OF CONVERTING XMP TO GMP AND MAINTAINING THE INACTIVATED STATE OF GENE(S) ASSOCIATED WITH GMP DEGRADATION AND
METHODS OF USING THE SAME
Technical Field
[1] The present invention relates to an Escherichia strain capable of converting
5'-xanthylic acid (XMP) to 5'-guanylic acid (GMP) and maintaining the inactivated state of a gene(s) associated with GMP degradation, and methods of using the same.
Background Art
[2] 5'-guanylic acid (GMP) can be produced by microbial enzymatic or chemical breakdown of yeast RNA, direct fermentation for directly producing nucleotides using mutant strains cultured in media containing a sugar, a nitrogen source, and a phosphate source, or a combination method including the fermentative synthesis of nucleotide intermediates and the chemical or enzymatic conversion of the nucleotide intermediates to nucleotides. In recent years, a combination method that combines fermentative or chemical synthesis and enzymatic conversion is industrially widely used due to its economical advantages.
[3] GMP production by the combination method consists of fermentative synthesis of
5'-xanthylic acid (XMP) and enzymatic conversion of XMP to GMP. At this time, two types of microbial strains are used. That is, an XMP-producing strain is used in the fermentative synthesis, and a microbial strain capable of inducing a continuous high-level expression of a gene encoding XMP aminase activity is used in the enzymatic conversion. In this case, the survival rate of the microbial strain used in the enzymatic conversion is remarkably reduced during a later culture period relative to during an initial culture period, thereby decreasing the production of GMP. Furthermore, it is known that the guaA gene encoding XMP aminase is fatal to host cells, and thus, the continuous high-level expression of XMP aminase hinders the growth of the microbial strain used in the enzymatic conversion during the later culture period. In addition, it is known that an internal gene responsible for GMP degradation in the microbial strain used for the enzymatic conversion is expressed, thereby facilitating the degradation of GMP.
[4] In view of the above problems, the present inventors developed Escherichia sp.
GPDl 114 (Accession No. KCCM- 10543) including an XMP aminase-encoding gene capable of expressing XMP aminase according to cellular growth state and an inactivated internal glnh gene. Furthermore, the present inventors developed Escherichia sp. GPUl 114 (Accession No. KCCM- 10536) derived from the Escherichia sp. GPDl 114, in which the ushA gene encoding 5'-nucleotidase is inactivated to remove GMP degradability.
[5] However, since the Escherichia sp. GPUl 114 still contains internal genes associated with GMP degradation except the ushA gene encoding 5'-nucleotidase, GMP can be degraded into guanosine or guanine. Therefore, a microbial strain with simultaneous inactivation of all genes associated with GMP degradation is required in the art. Disclosure of Invention
Technical Problem
[6] The present invention provides a microbial strain capable of converting
5'-xanthylic acid (XMP) to 5'-guanylic acid (GMP) and maintaining the inactivated state of an internal gene(s) associated with GMP degradation.
[7] The present invention also provides a method of accumulating GMP synthetase in a high concentration in a culture medium using the microbial strain.
[8] The present invention also provides a method of producing GMP, guanosine
5'-diphosphate (GDP), or guanosine 5'-triphosphate (GTP) using the microbial strain.
Technical Solution
[9] The present invention provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes an inactivated deoD gene.
[10] The parent strain, Escherichia sp. GPUl 114 (Accession No. KCCM-10536) is an
Escherichia coli (E. coli) strain in which glnL gene is inactivated, guaA gene encoding 5'-xanthylic acid (XMP) aminase is inserted by transformation, and an internal ushA gene encoding 5'-nucleotidase is inactivated. In the Escherichia sp. GPUl 114 (Accession No. KCCM-10536), XMP can be converted to 5'-guanylic acid (GMP) at a high level by the expression of the guaA gene, and GMP degradation activity is low due to no expression of the ushA gene, thereby increasing GMP productivity.
[11] As used herein, the deoD gene is not limited provided that it is a gene encoding purine-nucleoside phosphorylase activity. For example, the deoD gene may be NCBI accession no. NP_418801, but the present invention is not limited thereto. Thus, the deoD gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for deoD.
[12] The Escherichia strain of the present invention may be E. coli DKO-ud (Accession
No. KCCM- 10633).
[13] The present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD and aphA genes.
[14] The aphA gene is not limited provided that it is a gene encoding acid phosphatase activity. For example, the aphA gene may be NCBI accession no. NP_418479, but the present invention is not limited thereto. Thus, the aphA gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for aphA.
[15] The Escherichia strain of the present invention may be E. coli TKO-udh (A ccession No. KCCM- 10631).
[16] The present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD, aphA, and appA genes.
[17] The appA gene is not limited provided that it is a gene encoding acid
(poly)phosphatase activity. For example, the appA gene may be NCBI accession no. NP_415500, but the present invention is not limited thereto. Thus, the appA gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for appA.
[18] The Escherichia strain of the present invention may be E. coli QKO-udhp
(Accession No. KCCM- 10632).
[19] The present invention also provides a strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM-10536) and includes inactivated deoD, aphA, appA, and hprt genes.
[20] The hprt gene is not limited provided that it is a gene encoding hypoxanthine phos- phoribosyltransferase. For example, the hprt gene may be NCBI accession no. NP_414667, but the present invention is not limited thereto. Thus, the hprt gene is meant to embrace a wild-type gene and a natural or artificial mutant gene for hprt.
[21] The Escherichia strain of the present invention may be E. coli QIKO (Accession
No. KCCM- 10630).
[22] As used herein, the term 'inactivation' indicates that a target gene is not expressed, or even if the expression is possible, the gene does not produce a functional gene product. The 'inactivation' embraces the meaning that a target gene is completely inactivated or is not substantially expressed due to its significantly low expression level.
[23] In the Escherichia strains of the present invention, the inactivated genes can be obtained by any method known in the art, e.g., site-specific mutagenesis or homologous recombination, but the present invention is not limited thereto. Homologous recombination is preferable. The inactivation of a gene can be induced by deletion, substitution, inversion, or a combination thereof, but the present invention is not limited thereto.
[24] Gene inactivation by homologous recombination includes constructing inactivated gene cassettes by inserting foreign DNAs into target genomic DNA fragments, inserting the cassettes into microbial strains to induce homologous recombination between internal genes in the microbial cells and the cassettes, and selecting recombinant strains containing inactivated genes. For convenience, the foreign DNAs cloned into the target DNAs may contain a selectable marker, e.g., an antibiotic resistance gene. Target genes inactivated with antibiotic resistance gene-containing cassettes can be easily selected on an antibiotic-containing agar plate. Occurrence of recombination can be confirmed by southern blotting, PCR (polymerase chain reaction), etc.
[25] In the case of using inactivated cassettes containing antibiotic resistance genes during the preparation of the Escherichia strains of the present invention, it is preferable to remove the antibiotic resistance genes after selection is completed. For example, the removal of antibiotic resistance genes from selected strains includes transforming the selected strains with plasmids expressing genes capable of recognizing specific nucleotide sequences of inactivated cassettes and removing antibiotic resistance genes; and removing antibiotic resistance genes from the transformants. The resultant antibiotic resistance gene-free transformants can be selected on an ampicillin-containing agar plate. The removal of antibiotic resistance genes from target DNAs can be confirmed by colony transfer onto an agar plate containing an appropriate antibiotic with a toothpick and growth of colonies on the agar plate.
[26] The Escherichia strains of the present invention can be prepared using the following illustrative methods.
[27] Genomic DNAs are extracted from Escherichia sp. GPUl 114 (KCCM- 10536), and deoD, aphA, appA, and hprt genes are amplified by PCR using the genomic DNAs as templates. The deoD, aphA, appA, and hprt genes are cloned into plasmids or other vectors to construct recombinant vectors (e.g., recombinant plasmids pDEO, pAPH, pAPP, pHPRT). Host cells such as E. coli cells are transformed with the recombinant vectors. After the transformants are cultured, recombinant vectors containing the deoD, aphA, appA, and hprt genes are extracted from the transformed cells. A loxp- containing antibiotic resistance gene fragment is inserted into the deoD, aphA, appA, and hprt genes of the recombinant vectors to construct recombinant vectors containing inactivated deoD, aphA, appA, and hprt genes (e.g., pTdeoD:loxpKAT, pTaphA:loxpKAT, pTappA:loxpKAT, pThprt:loxpCAT). The recombinant vectors are inserted in host cells and cultured. After the culture, recombinant vectors are extracted from the transformants and digested with appropriate restriction enzymes to obtain deoD, aphA, appA, and hprt gene-inactivated cassette fragments (e.g., Δ deoD:loxpKAT, Δ aph:loxpKAT, Δ app:loxpKAT, Δ hprt:loxpCAT). First, Δ deoD:loxpKAT is inserted into the GMP-producing strain GPUl 114 by elec- troporation, etc., and the transformed GMP-producing strain GPUl 114 is maintained under the antibiotic selection pressure to allow recombination between an antibiotic marker-containing deoD gene fragment and a chromosomal wild-type deoD gene to occur, thereby obtaining recombinant strains maintaining the inactivated state of the deoD gene even when generation continues. To further inactivate the aphA gene in the selected recombinant strains, the antibiotic marker must be removed. For this, recombinant vectors expressing genes recognizing loxp sites in antibiotic resistance genes and removing the antibiotic resistance genes are inserted into the selected recombinant strains by electroporation, etc. The selection of the resultant transformants is performed on an ampicillin-containing agar plate using acquired antibiotic resistance. The removal of antibiotic resistance genes from target DNAs can be confirmed by colony transfer onto an agar plate containing an appropriate antibiotic with a toothpick and growth of colonies on the agar plate. The resultant transformants are maintained under the antibiotic selection pressure to allowrecombination between a deoD gene fragment containing a part of the antibiotic marker and a chromosomal wild-type deoD gene to occur, thereby obtaining transformed strains (e.g., E. coli DKO-ud) maintaining the inactivated state of the ushA and deoD genes even when generations are continued. . When the above-described procedure is sequentially applied to the aphA, appA, and hprt genes, transformed strains with cumulative in- activation of the deoD, aphA, appA, and hprt genes can be obtained.
[28] The present invention also provides a method of accumulating GMP synthetase in cells, which includes culturing an Escherichia strain of the present invention.
[29] The present invention also provides a method of producing GMP, guanosine
5'-diphosphate (GDP), or guanosine 5'-triphosphate (GTP), which includes culturing an Escherichia strain of the present invention.
[30] In the methods of the present invention, the culturing of the Escherichia strain of the present invention can be performed under commonly known culture conditions, e.g., in a culture medium containing a carbon source, a nitrogen source, an amino acid, an inorganic compound, etc., while adjusting the temperature and pH of the culture medium.
[31] The carbon source may be a carbohydrate such as glucose, fructose, or sterilized molasses (i.e., molasses converted to reducing sugar), and the nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium chloride, and ammonium sulfate, and an organic nitrogen source such as peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydroxylate, fish meal or its digest, defatted soybean or its digest. The inorganic compound may be potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. When needed, the culture medium may be supplemented with vitamins, bases, etc. The culture may be performed under an aerobic condition, e.g., by shaking or aerobic stirring, preferably at a temperature of 30 to 40 0C . The pH of the culture medium may be maintained near neutral. The culture may be performed for 1 to 2 days so that products having XMP aminase activity are accumulated in the culture medium.
[32] In the present invention, GMP can be produced by converting XMP to GMP using
GMP synthetase produced by the culture of an Escherichia strain according to the present invention. The conversion of XMP to GMP can be achieved by adding a material (e.g., xylene) increasing the cell membrane permeability of XMP into a culture medium so that XMP is introduced into a GMP-producing strain. The material increasing the cell membrane permeability of XMP is well known in the art, e.g., a hydrophobic material (e.g., xylene, toluene, benzene, ethyl acetate), a surfactant (e.g., polyoxyethylenestearylamine (e.g., Nymeen S-215, Nihon Yushi Co.) a cationic surfactant such as cetyltrimethylammonium bromide, Cation FB, or Cation F2-40E, or an anionic surfactant such as sodium oleylamide sulfate, Newrex TAB, and Rapizole 80), a metal ion (e.g., calcium ion, magnesium ion), but the present invention is not limited to the above-illustrated examples. The content of the material increasing the cell membrane permeability of XMP varies according to the type of the material, and can be appropriately adjusted by those of ordinary skill in the art. For example, xylene may be used in an amount of 0.5 to 1 wt% based on the total weight of a culture medium. In addition, GDP or GTP can be produced by commonly known enzymatic or chemical phosphorylation of GMP.
Description of Drawings
[33] The above and other features and advantages of the present invention will become m ore apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:
[34] FIG. IA is a scheme of the construction of a recombinant plasmid pDEO of
Example 1 of the present invention;
[35] FIG. IB is a scheme of the construction of a recombinant plasmid pTdeoD:loxpKAT of Example 1 of the present invention;
[36] FIG. 2A is a scheme of the construction of a recombinant plasmid pAPH of
Example 2 of the present invention;
[37] FIG. 2B is a scheme of the construction of a recombinant plasmid pTaphA:loxpKAT of Example 2 of the present invention;
[38] FIG. 3A is a scheme of the construction of a recombinant plasmid pAPP of
Example 3 of the present invention;
[39] FIG. 3B is a scheme of the construction of a recombinant plasmid pTappA:loxpKAT of Example 3 of the present invention;
[40] FIG. 4A is a scheme of the construction of a recombinant plasmid pHPRT of Example 4 of the present invention; and
[41] FIG. 4B is a scheme of the construction of a recombinant plasmid pThprt:loxpCAT of Example 4 of the present invention.
Best Mode
[42] Hereinafter, the present invention will be described more specifically with reference to the following Examples. The following Examples are for illustrative purposes and are not intended to limit the scope of the invention.
[43] Examples
[44] Example 1: Construction of recombinant plasmid and inactivation of deoD gene using the recombinant plasmid
[45] About 720 bp DNA fragments containing the open reading frames (ORFs) of deoD genes were amplified by PCR using genomic DNAs of E. coli GPUl 114 (Accession No. KCCM-10536) as templates and oligonucleotides as set forth in SEQ ID NOS: 1 and 2 as primers. The PCR was performed at 94 0C for 30 seconds for denaturation, 50 0C for 30 seconds for annealing, and 68 0C for 1 minute and 30 seconds for extension for 35 cycles.
[46] After the PCR products were size-fractionated by 0.1% agarose gel electrophoresis, bands containing 720 bp DNA fragments were purified. The DNA fragments were ligated into the Hindll site of pUC18 cloning vectors (Promega Co.) at 16 0C overnight (see FIG. IA). The resultant recombinant plasmids pDEO were transformed into E. coli DH5 α , the obtained transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 37 0C overnight.
[47] Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of ampicillin-containing liquid media and cultured overnight. Then, plasmid DNAs were isolated using a QIAGEN mini prep kit (QIAGEN) and digested with restriction enzymes EcoRl and HindlII to identify the presence of cloned deoD gene fragments. The identified pDEO plasmids were digested with restriction enzymes EcoRl and Hind III and bands of about 720 bp were resolved on 0.8% agarose gel. Meanwhile, plasmids pUG6 (U. Guldenre et al, A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Research 24 (13), 1996, pp2519-2524) were digested with restriction enzymes EcoR V and Hinc II to obtain loxp site- containing kanamycin resistance gene fragments (about 1.7 kb). The DNA fragments contained in the bands were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTdeoD::loxpKAT (about 4.5kb) (see FIG. IB).
[48] After the pTdeoD::loxpKAT recombinant plasmids were transformed into E. coli
DH5 α , the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 niL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.3 kb DNA fragments containing the ORFs of deoD genes and the loxpKAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 1 and 2 as primers. The PCR was performed at 94 0C for 1 minute for denaturation, 55 0C for 1 minute for annealing, and 68 0C for 1 minute for extension for 35 cycles.
[49] The obtained DNA fragments Δ deoD::loxpKAT were electroporated into E. coli
GPUl 114 containing a foreign guaA gene, and the obtained transformants were smeared onto solid media containing kanamycin to select colonies.
[50] To remove the antibiotic marker from recombinant strains of the selected colonies, pCP20 plasmids were transformed into the recombinant strains of the selected colonies, and the resultant transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 30 0C overnight. Colonies grown on the solid media were toothpicked to ampicillin-containing solid media, kanamycin-containing solid media, and antibiotic-free solid media and cultured at 43 0C overnight. Colonies grown only on the antibiotic-free solid media were harvested. The colonies were designated E. coli DKO-ud and deposited in the Korean Culture Center of Microorganisms (KCCM) on November 30, 2004 (Accession No. KCCM- 10633).
[51] Example 2: Construction of recombinant plasmid and inactivation of aphA gene using the recombinant plasmid
[52] About 714 bp DNA fragments containing the ORFs of αphA genes were amplified by PCR using genomic DNAs extracted from the E. coli DKO-ud obtained in Example 1 as templates and oligonucleotides as set forth in SEQ ID NOS: 3 and 4 as primers. The PCR was performed at 94 0C for 30 seconds for denaturation, 55 0C for 30 seconds for annealing, and 68 0C for 1 minute and 30 seconds for extension for 35 cycles.
[53] After the PCR products were size-fractionated by 0.1% agarose gel electrophoresis, bands containing 714 bp DNA fragments were purified. The DNA fragments were ligated into pGEM-T-easy cloning vectors (Promega Co.) at 16 0C overnight (see FIG. 2A). The resultant recombinant plasmids pAPH were transformed into E. coli DH5 α , the obtained transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 37 0C overnight.
[54] Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of ampicillin-containing liquid media and cultured overnight. Then, plasmid DNAs were isolated using a QIAGEN mini prep kit (QIAGEN) and digested with restriction enzyme Clαl to identify the presence of cloned αphA gene fragments. The identified pAPH plasmids were digested with restriction enzyme Clαl and bands of about 3.7 kb were resolved on 0.8% agarose gel. The DNA fragments contained in the bands were blunt-ended with T4 polymerase. Meanwhile, plasmids pUG6 were digested with restriction enzymes EcoRY and Hindi to obtain loxp site-containing kanamycin resistance gene fragments (about 1.7 kb). The blunt-ended DNA fragments were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTaphA::loxpKAT (about 5.4kb) (see FIG. 2B).
[55] After the pTaphA::loxpKAT recombinant plasmids were transformed into E. coli
DH5 α , the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.4 kb DNA fragments containing the ORFs of aphA genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 3 and 4 as primers. The PCR was performed at 94 0C for 1 minute for denaturation, 55 0C for 1 minute for annealing, and 68 0C for 1 minute for extension for 35 cycles.
[56] The obtained DNA fragments Δ aphA: :loxp KAT were electroporated into the E. coli DKO-ud obtained in Example 1, and the resultant transformants were smeared onto solid media containing kanamycin to select colonies.
[57] To remove the antibiotic marker from recombinant strains of the selected colonies, pCP20 plasmids were transformed into the recombinant strains of the selected colonies, and the resultant transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 30 0C overnight. Colonies grown on the solid media were toothpicked to ampicillin-containing solid media, kanamycin-containing solid media, and antibiotic-free solid media and cultured at 43 0C overnight. Colonies grown only on the antibiotic-free solid media were harvested. The colonies were designated E. coli TKO-udh and deposited in the KCCM on November 30, 2004 (Accession No. KCCM-10631).
[58] Example 3: Construction of recombinant plasmid and inactivation of appA gene using the recombinant plasmid
[59] About 1.3 kb DNA fragments containing the ORFs of αppA genes were amplified by PCR using the genomic DNAs extracted from the E. coli DKO-ud obtained in Example 1 as templates and oligonucleotides as set forth in SEQ ID NOS: 5 and 6 as primers. The PCR was performed at 94 0C for 30 seconds for denaturation, 55 0C for 30 seconds for annealing, and 68 0C for 1 minute and 30 seconds for extension for 35 cycles. [60] After the PCR products were size-fractionated by 0.1% agarose gel electrophoresis, bands containing 1.3 kb DNA fragments were purified. The DNA fragments were ligated into pGEM-T-easy cloning vectors at 16 0C overnight (see FIG. 3A). The resultant recombinant plasmids pAPP were transformed into E. coli DH5 α , the obtained transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 37 0C overnight.
[61] Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of ampicillin-containing liquid media and cultured overnight. Then, plasmid DNAs were isolated using a QIAGEN mini prep kit (QIAGEN) and digested with restriction enzyme CIaI to identify the presence of cloned appA gene fragments. The identified p APP plasmids were digested with restriction enzyme BcII and bands of about 4.3 kb were resolved on 0.8% agarose gel. The DNA fragments contained in the bands were blunt-ended with T4 polymerase. Meanwhile, plasmids pUG6 were digested with restriction enzymes EcoRY and Hindi to obtain loxp site-containing kanamycin resistance gene fragments (about 1.7 kb). The blunt-ended DNA fragments were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pTappA::loxpKAT (about 6.3kb) (see FIG. 3B).
[62] After the pTappA::loxpKAT recombinant plasmids were transformed into E. coli
DH5 α , the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 3.1 kb DNA fragments containing the ORFs of appA genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 5 and 6 as primers. The PCR was performed at 94 0C for 1 minute for denaturation, 55 0C for 1 minute for annealing, and 68 0C for 1 minute for extension for 35 cycles.
[63] The obtained DNA fragments Δ appA::loxpKAT were electroporated into the E. coli DKO-udh obtained in Example 2, and the resultant transformants were smeared onto solid media containing kanamycin to select colonies.
[64] To remove the antibiotic marker from recombinant strains of the selected colonies, pCP20 plasmids were transformed into the recombinant strains of the selected colonies, and the resultant transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 30 0C overnight. Colonies grown on the solid media were toothpicked to ampicillin-containing solid media, kanamycin-containing solid media, and antibiotic-free solid media and cultured at 43 0C overnight. Colonies grown only on the antibiotic-free solid media were harvested. The colonies were designated QKO-udhp and deposited in the KCCM on November 30, 2004 (Accession No. KCCM- 10632).
[65] Example 4: Construction of recombinant plasmid and inactivation of hprt gene using the recombinant plasmid
[66] About 1.7 kb DNA fragments containing the ORFs of hprt genes were amplified by
PCR using genomic DNAs extracted from the E. coli QKO-udhp obtained in Example 3 as templates and oligonucleotides as set forth in SEQ ID NOS: 7 and 8 as primers. The PCR was performed at 94 0C for 30 seconds for denaturation, 69 0C for 30 seconds for annealing, and 72 0C for 1 minute and 30 seconds for extension for 30 cycles.
[67] After the PCR products were size-fractionated by 0.1% agarose gel electrophoresis, bands containing 500 bp DNA fragments were purified. The DNA fragments were ligated into pETBlue-1 cloning vectors (Novagen Co.) at 16 0C overnight (see FIG. 4A). The resultant recombinant plasmids pHPRT were transformed into E. coli DH5 α , the obtained transformants were smeared onto solid media containing ampicillin (50 mg/L) and cultured at 37 0C overnight.
[68] Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of ampicillin-containing liquid media and cultured overnight. Then, plasmid DNAs were isolated using a QIAGEN mini prep kit (QIAGEN) and digested with restriction enzyme Hindll to identify the presence of cloned hprt gene fragments. The identified pHPRT plasmids were digested with restriction enzyme Hindll and bands of about 4.0 kb were resolved on 0.7% agarose gel. Meanwhile, plasmids pUG6 were digested with restriction enzymes EcoRY and Hindi to obtain loxp site-containing kanamycin resistance gene fragments (about 1.7 kb). The DNA fragments contained in the bands were ligated into the loxp site-containing kanamycin resistance gene fragments by blunt-end ligation to obtain recombinant plasmids, pThprt::loxpCAT (about 5.3kb) (see FIG. 4B).
[69] After the pThprt::loxpCAT recombinant plasmids were transformed into E. coli
DH5 α , the obtained transformants were smeared onto solid media containing ampicillin and kanamycin (50 mg/L for each) and cultured at 37 0C overnight. Colonies grown on the solid media were inoculated by loop transfer needles onto 3 mL of liquid media containing ampicillin and kanamycin and cultured overnight. Then, plasmid DNAs were extracted using a QIAGEN mini prep kit (QIAGEN). About 2.1 kb DNA fragments containing the ORFs of hprt genes and the loxp KAT sites were amplified by PCR using the plasmid DNAs as templates and the oligonucleotides as set forth in SEQ ID NOS: 7 and 8 as primers. The PCR was performed at 94 0C for 1 minute for denaturation, 60 0C for 1 minute for annealing, and 72 0C for 1 minute for extension for 30 cycles. The obtained DNA fragments Δ hprt::loxpCAT were elec- troporated into the E. coli QKO-udhp obtained in Example 3, and the resultant transformants were smeared onto solid media containing chloramphenicol to select colonies. The colonies were designated QIKO and deposited in the KCCM on November 30, 2004 (Accession No. KCCM- 10630).
[70] Example 5: GMP productivity of E. coli QIKO of Example 4 [71] Among the E. coli QIKO colonies selected after smearing on the solid media containing chloramphenicol in Example 4, 30 colonies were cultured in Erlenmeyer flasks containing GMP-producing media with the medium composition of Table 1 below, and GMP production was evaluated. First, the 30 single colonies were cultured overnight in a 32 0C incubator containing GMP-producing solid media. Then, one loopful of each of the single colonies was inoculated on 50 ml of the GMP-producing media and cultured for 7 hours at 37 0C with shaking at 250 rpm. Then, the cell cultures were supplemented with 2% xylene to facilitate the cell membrane permeation of enzymes, substrates, and products in the cell cultures. The resultant cultures were added to enzymatic reaction solutions and incubated at 42 0C for 15 minutes with shaking at 250 rpm. The concentration of GMP was determined by HPLC (high performance liquid chromatography) and the results are presented in Table 2 below.
[72] Table 1 : Medium composition for GMP production.
Figure imgf000014_0001
[73] Table 2: Flask test results for recombinant strains
Figure imgf000014_0002
[74] As shown in Table 2, the parent strains GPUl 114 produced 46 U/mL of GMP, whereas the recombinant strains QIKO, in which the genes with nucleotide degradability were inactivated, produced 49 U/mL of GMP. As seen from the above results, the recombinant strains QIKO of the present invention exhibited 6% higher GMP production than the parent strains.
[75] Example 6: Comparison of GMP byproduct (guanine and guanosine) production for recombinant strains in Erlenmeyer flasks [76] The E. coli QIKO colonies selected after smearing on the solid media containing chloramphenicol in Example 4 were cultured in Erlenmeyer flasks containing GMP- producing media with the medium composition of the above Table 1. The cultures were added to reaction solutions for GMP byproduct production with the composition of Table 3 below and byproduct production was evaluated.
[77] First, the E. coli QIKO single colonies were cultured overnight in a 32 0C incubator containing GMP-producing solid media. Then, one loopful of each of the single colonies was inoculated on 50 ml of the GMP-producing media and cultured for 7 hours at 37 0C with shaking at 250 rpm. Then, the cell cultures were supplemented with 2% xylene to facilitate the cell membrane permeation of enzymes, substrates, and products in the cell cultures, and incubated at 37 0C for 20 minutes with shaking at 250 rpm. The resultant cultures were mixed with the reaction solutions for GMP byproduct production with the composition of Table 3 and incubated at 42 0C for 8 hours with shaking at 250 rpm. The concentration of two types of byproducts, guanine and guanosine, with respect to time was determined by HPLC, and the results are presented in Table 4 below.
[78] Table 3: Reaction solution composition for GMP byproduct production .
Figure imgf000015_0001
[79] Table 4: Results of byproduct production test for recombinant strains.
Figure imgf000015_0002
[80] As shown in Table 4, the concentration of degraded GMP for the parent strains GPUl 114 was 2.54 mmol, whereas the concentration of degraded GMP for the recombinant strains QIKO, in which the genes with nucleotide degradability were in- activated, was 1.37 mmol. That is, the GMP degradation of the recombinant strains
QIKO was reduced by about 47% relative to that of the parent strains. [81] As described above, GMP-producing strains of the present invention exhibit high conversion efficiency of XMP to GMP and low GMP degradation activity. [82] According to a method of accumulating GMP synthetase in cells of the present invention, GMP synthetase can be accumulated in a high concentration in cells. [83] According to a method of producing GMP, GDP, or GTP of the present invention,
GMP, GDP, or GTP can be produced in high efficiency.
Figure imgf000017_0003
INDICATIONS RELATING TO DEPOSITED MICROORGANISM OROTHERBIOLOGICAL MATERIAL
Figure imgf000017_0001
A. The indications made below relate to the deposited microorganism or other biological material referred to in the description
In paragraph number 21 .
B. IDENTTFI CATT ON OF DEPOSIT Further deposits are identified on an additional sheet
Name of depositary institution
KCCM (Korean Culture Center of Microorganisms)
Address of depositary institution (including postal code and country)
361-221 , Yurim B/D, Honje 1 , Sudaemun, Seoul, 120-091 , Republic of Korea
Date of deposit Accession Number
30 November 2004 KCCM-10630
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Figure imgf000017_0002
Figure imgf000018_0003
INDICATIONS RELATING TO DEPOSITED MICROORGANISM OROTHERBIOLOGICAL MATERIAL
Figure imgf000018_0001
A. The indications made below relate to the deposited microorganism or other biological material referred to in the description
In paragraph number 1 5.
B. IDENTTFI CATT ON OF DEPOSIT Further deposits are identified on an additional sheet
Name of depositary institution
KCCM (Korean Culture Center of Microorganisms)
Address of depositary institution (including postal code and country)
361-221 , Yurim B/D, Honje 1 , Sudaemun, Seoul, 120-091 , Republic of Korea
Date of deposit Accession Number
30 November 2004 KCCM-10631
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
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Figure imgf000018_0002
Figure imgf000019_0003
INDICATIONS RELATING TO DEPOSITED MICROORGANISM OROTHERBIOLOGICAL MATERIAL
Figure imgf000019_0001
A. The indications made below relate to the deposited microorganism or other biological material referred to in the description in paragraph number : 18.
B. IDENTTFI CATT ON OF DEPOSIT Further deposits are identified on an additional sheet
Name of depositary institution
KCCM (Korean Culture Center of Microorganisms)
Address of depositary institution (including postal code and country)
361-221 , Yurim B/D, Honje 1 , Sudaemun, Seoul, 120-091 , Republic of Korea
Date of deposit Accession Number
30 November 2004 KCCM-10632
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g, "Accession ϊfumber of Deposit")
Figure imgf000019_0002
Figure imgf000020_0003
INDICATIONS RELATING TO DEPOSITED MICROORGANISM OROTHERBIOLOGICAL MATERIAL
Figure imgf000020_0001
A. The indications made below relate to the deposited microorganism or other biological material referred to in the description in paragraph number 1 2.
B. IDENTTFI CATT ON OF DEPOSIT Further deposits are identified on an additional sheet
Name of depositary institution
KCCM (Korean Culture Center of Microorganisms)
Address of depositary institution (including postal code and country)
361-221 , Yurim B/D, Honje 1 , Sudaemun, Seoul, 120-091 , Republic of Korea
Date of deposit Accession Number
30 November 2004 KCCM-10633
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g, "Accession ϊfumber of Deposit")
Figure imgf000020_0002

Claims

Claims
[I] A strain of the genus Escherichia which is derived from Escherichia sp. GPUl 114 (Accession No. KCCM- 10536) and comprises an inactivated deoD gene.
[2] The strain of claim 1, wherein the deoD gene is NCBI accession no. NP_418801.
[3] The strain of claim 1, which is Escherichia sp. DKO-ud (Accession No. KCCM-
10633).
[4] The strain of claim 1, wherein aphA gene is further inactivated.
[5] The strain of claim 4, wherein the aphA gene is NCBI accession no. NP_418479.
[6] The strain of claim 4, which is Escherichia sp. TKO-udh (Accession No.
KCCM-10631).
[7] The strain of claim 4, wherein appA gene is further inactivated.
[8] The strain of claim 7, wherein the appA gene is NCBI accession no. NP_415500.
[9] The strain of claim 7, which is Escherichia sp. QKO-udhp (Accession No.
KCCM- 10632). [10] The strain of claim 7, wherein hprt gene is further inactivated.
[I I] The strain of claim 10, wherein the hprt gene is NCBI accession no. NP_414667. [12] The strain of claim 10, which is Escherichia sp. QIKO (Accession No. KCCM-
10630). [13] The strain of claim 1, 4, 7, or 10, wherein the deoD, aphA, appA, and hprt genes are each inactivated by homologous recombination-based substitution. [14] A method of accumulating 5'-guanylic acid (GMP) synthetase in cells, the method comprising culturing the strain of any one of claims 1 through 13. [15] A method of producing GMP, guanosine 5'-diphosphate (GDP), or guanosine
5'-triphosphate (GTP), the method comprising culturing the strain of any one of claims 1 through 13.
PCT/KR2006/000221 2005-01-21 2006-01-20 Escherichia strain capable of converting xmp to gmp and maintaining the inactivated state of gene(s) associated with gmp degradation and methods of using the same WO2006078132A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP06702908.2A EP1844135B1 (en) 2005-01-21 2006-01-20 Escherichia strain capable of converting xmp to gmp and maintaining the inactivated state of gene(s) associated with gmp degradation and methods of using the same
ES06702908.2T ES2443099T3 (en) 2005-01-21 2006-01-20 Escherichia strain capable of converting XMP into GMP and maintaining the inactivated state of the gene or genes associated with GMP degradation and procedures for using it
CN2006800027775A CN101137743B (en) 2005-01-21 2006-01-20 Escherichia strain capable of converting xmp to gmp and maintaining the inactivated state of gene(s) associated with gmp degradation and methods of using the same
DK06702908.2T DK1844135T3 (en) 2005-01-21 2006-01-20 ESCHERICHIA TRIALS WHICH CAN CONVERT XMP TO GMP AND MAINTAIN THE INACTIVATED STATE OF GEN (S) ASSOCIATED WITH GMP DEGRADATION AND PROCEDURES TO USE THEREOF
US11/814,417 US7741101B2 (en) 2005-01-21 2006-01-20 Escherichia strain capable of converting XMP to GMP and maintaining the inactivated state of gene(s) associated with GMP degradation and methods of using the same
JP2007552064A JP4769255B2 (en) 2005-01-21 2006-01-20 An Escherichia strain capable of converting XMP to GMP, wherein a gene associated with degradation of GMP is inactivated, and a method using the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2005-0005863 2005-01-21
KR1020050005863A KR100664653B1 (en) 2005-01-21 2005-01-21 Escherichia coli strain capable of converting XMP to GMP and having inactivated genes for GMP degradation and method for using the same

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EP (1) EP1844135B1 (en)
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CN (1) CN101137743B (en)
DK (1) DK1844135T3 (en)
ES (1) ES2443099T3 (en)
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EP2295546A2 (en) 2009-08-10 2011-03-16 Ajinomoto Co., Inc. Method for producing 5'-guanylic acid
EP2358865A2 (en) * 2008-12-17 2011-08-24 CJ CheilJedang Corporation A corynebacteria strain for enhancement of 5'-guanosine monophosphate productivity and a method of producing 5'-guanosine monophosphate using the same

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KR100686561B1 (en) * 2005-11-25 2007-02-26 씨제이 주식회사 Corynebacterium ammoniagenes cjxcv19 kccm-10692p having low activity of degradation of guanine 5'- monophosphate and high-throughput screening method for mutant
JP7111878B1 (en) 2021-10-27 2022-08-02 旭化成ファーマ株式会社 Method for producing nicotinamide mononucleotide

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EP1170370A2 (en) 2000-07-05 2002-01-09 Ajinomoto Co., Inc. Method for producing nucleotide by fermentation
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EP2358865A2 (en) * 2008-12-17 2011-08-24 CJ CheilJedang Corporation A corynebacteria strain for enhancement of 5'-guanosine monophosphate productivity and a method of producing 5'-guanosine monophosphate using the same
EP2358865A4 (en) * 2008-12-17 2013-08-21 Cj Cheiljedang Corp A corynebacteria strain for enhancement of 5'-guanosine monophosphate productivity and a method of producing 5'-guanosine monophosphate using the same
EP2295546A2 (en) 2009-08-10 2011-03-16 Ajinomoto Co., Inc. Method for producing 5'-guanylic acid
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KR100664653B1 (en) 2007-01-04
US20080299620A1 (en) 2008-12-04
TW200637909A (en) 2006-11-01
JP2008543272A (en) 2008-12-04
KR20060084994A (en) 2006-07-26
DK1844135T3 (en) 2014-01-20
ES2443099T3 (en) 2014-02-17
CN101137743B (en) 2010-12-08
JP4769255B2 (en) 2011-09-07
CN101137743A (en) 2008-03-05
EP1844135B1 (en) 2013-12-25
EP1844135A4 (en) 2009-04-15
US7741101B2 (en) 2010-06-22
EP1844135A1 (en) 2007-10-17

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