WO2012147989A1 - A method for producing an l-amino acid belonging to the glutamate family, using a coryneform bacterium - Google Patents

A method for producing an l-amino acid belonging to the glutamate family, using a coryneform bacterium Download PDF

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WO2012147989A1
WO2012147989A1 PCT/JP2012/061625 JP2012061625W WO2012147989A1 WO 2012147989 A1 WO2012147989 A1 WO 2012147989A1 JP 2012061625 W JP2012061625 W JP 2012061625W WO 2012147989 A1 WO2012147989 A1 WO 2012147989A1
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ncgl
gene
bacterium
amino acid
expression
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Svetlana Alekseevna SAMSONOVA
Viktor Vasilievich Samsonov
Yulia Georgievna Rostova
Mikhail Markovich Gusyatiner
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Ajinomoto Co Inc
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Ajinomoto Co Inc
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Priority to JP2013548522A priority patent/JP6011545B2/en
Publication of WO2012147989A1 publication Critical patent/WO2012147989A1/en
Priority to US14/045,205 priority patent/US9029104B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid belonging to the glutamate family using a coryneform bacterium which has been modified so that expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon in the bacterium is/are attenuated.
  • L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
  • EP1229121 A2 discloses a method for producing L- glutamine by culturing a coryneform bacterium which has L-glutamine producing ability and has been modified so that its intracellular glutamine synthetase activity is enhanced, and preferably has been further modified so that its intracellular glutamate dehydrogenase activity is enhanced, in a medium to produce and cause accumulation of L-glutamine in the medium, and collecting the L-glutamine.
  • glnA glutamine synthetase
  • the glnA gene is located in an operon with the glnR gene.
  • the glnR gene codes for a negative regulator which directly controls expression of the glutamine synthetase gene (glnA).
  • Certain mutations in glnR gene such as a large, in-frame deletion and, a start codon mutation, lead to high-level constitutivity of the operon; whereas other mutations caused low-level constitutivity (Schreier HJ et al., J Mol Biol. 1989, 210(l):51-63).
  • aspects of the present invention include enhancing the productivity of strains which produce L-amino acids belonging to the glutamate family, and providing a method for producing an L-amino acid belonging to the glutamate family, using these strains.
  • ORF open reading frame
  • NCgl_2066 is located in an operon with two other ORFs - NCgl_2067 and NCgl_2065.
  • the correspoding ORFs were named NCgl_2067,
  • NCgl_2066, and NCgl_2065 genes were given the name NCgl_2067- NCgl_2065 operon.
  • the present invention provides a coryneform bacterium having an increased ability to produce amino acids belonging to the glutamate family, such as L-glutamic acid, L- glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
  • amino acids belonging to the glutamate family such as L-glutamic acid, L- glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
  • L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine and L-citrulline.
  • L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine.
  • L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
  • L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine.
  • Figure 1 shows alignment of the GlnR (denoted as spr0433) protein from
  • Figure 2 shows structure of the NCgl 2067 -NCgl 2065 operon from
  • Figure 3 shows structure of the pBS4S plasmid.
  • a coryneform bacterium which can produce an L-amino acid belonging to the glutamate family, wherein the bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon.
  • bacterium producing an L-amino acid can mean a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
  • bacterium producing an L-amino acid also can mean a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain ofcoryneform bacterium, for example, bacterial strains listed below, such as Corynebacterium glutamicum ATCC 13032, ATCC 31833, Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 and B-6642, and can mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5g/l, or l.Og/1, of the target L- amino acid.
  • L-amino acid belonging to the glutamate family can include L- glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, L-citrulline, and combinations thereof.
  • coryneform bacteria can include conventional coryneform bacterium according to the classification known to a person skilled in the art of microbiology, and also can include bacteria that had been classified into the genus Brevibacterium, but are currently classified into the genus Corynebacterium (Liebl W et al., Int J Syst Bacteriol. 1991, 41(2):255-260), as well as the Brevibacterium bacteria that are highly related to Corynebacterium bacteria. Examples of such coryneform bacterium include the following:
  • coryneform bacterium examples include, but are not limited to, strains such as C. acetoacidophilum ATCC 13870, C. acetoglutamicum ATCC 15806, C alkanolyticum ATCC 2151 1, C callunae ATCC 15991, C glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, C lilium ATCC 15990, C. melassecola ATCC 17965, C. thermoaminogenes AJ12340 (FERM BP-1539), C herculis ATCC 13868, B. divaricatum ATCC 14020, B.
  • each strain is given a unique registration number which is listed in the catalogue of the ATCC. Strains can be ordered using this registration number.
  • the AJ 12340 strain was deposited at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology at Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305- 5466, Japan) on October 27, 1989 under the provisions of the Budapest Treaty and given an accession number of FERM BP-1539.
  • the AJ12418 strain was deposited at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on January 5, 1989 under the provisions of the Budapest Treaty and given an accession number of FERM BP-2205.
  • Brevibacterium' ' ' can mean that the bacterium is classified into the genus
  • Corynebacterium or Brevibacterium according to the classification known to a person skilled in the art of microbiology.
  • Examples of a bacterium belonging to the genus Corynebacterium or Brevibacterium include, but are not limited to, Corynebacterium glutamicum and Brevibacterium flavum.
  • bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl 2067 -NCgl 2065 operon
  • the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of one or more of the proteins, NCgl_2067, NCgl_2066 and NCgl_2065 as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize the NCgl_2067, NCgl_2066 and/or NCgl_2065 protein(s).
  • bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon
  • the bacterium has been modified in such a way that the modified gene(s) encodes mutant NCgl_2067, NCgl_2066 and or NCgl_2065 protein(s) with decreased activities. Since it is thought that genes NCgl_2067, NCgl_2066 and NCgl_2065 are organized as an operon, attenuation of expression of one of the genes can lead to polar effects on expression other genes of the NCgl 2067 -NCgl 2065 operon.
  • the presence or absence of gene(s) of the NCgl _2067 -NCgl 2065 operon in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like.
  • the level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
  • the amounts of the proteins encoded by the genes of the NCgl 206/ '-NCgl ' 2065 operon can be measured by well-known methods, including SDS-PAGE followed by an
  • activation of one or more gene(s) of the NCgl_2067-NCgl_2065 operon can mean that the modified gene(s) encodes a completely inactive protein(s). It is also possible that the modified DNA region is unable to naturally express the gene due to deletion of a part of or the entire gene, shifting of the reading frame of the gene, introduction of missense/nonsense mutation(s), or modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
  • the NCgl 2067 gene (synonyms - CG2358, Cgl2147) codes for a hypothetical protein with unknown function.
  • the NCgl_2067 gene of C glutamicum (nucleotides complement to the nucleotides from 2270870 to 2270256 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl 066 and the ileS genes, on the chromosome of C. glutamicum ATCC 13032.
  • the nucleotide sequence of the NCgl_2067 gene and the amino acid sequence of predicted NCgl_2067 protein encoded by the NCgl_2067 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the NCgl 2066 gene (synonyms - CG2357, Cgl2146) codes for a hypothetical putative transcriptional regulator.
  • the NCgl_2066 gene of C. glutamicum (nucleotides complement to the nucleotides from 2,270,262 to 2,269,258 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl_2067 and NCgl 065 gene on the chromosome of C. glutamicum ATCC 13032, and both genes are oriented in the same direction as the NCgl ' 2066 gene, and these three genes are presumably organized as an operon.
  • the nucleotide sequence of the NCgl_2066 gene and the amino acid sequence of predicted NCgl_2066 protein encoded by the NCgl_2066 gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the NCgl 2065 gene (synonyms - CG2356, Cgl2145) is predicted to encode a DMT family permease.
  • the NCgl_2065 gene of C glutamicum (nucleotides complement to the nucleotides from 2,269,246 to 2,268,386 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl_2066 and NCgl_2064 genes on the chromosome of C glutamicum ATCC 13032.
  • NCgl 2066 gene and the amino acid sequence of predicted NCgl_2066 protein encoded by the NCgl _2066 gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • genes of the NCgl 2067 -NCgl _2065 operon to be inactivated on the chromosome are not limited to the genes shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, but can include genes homologous to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, which encode a variant proteins of the NCgl_2067, NCgl_2066 and NCgl_2065 proteins.
  • the phrase "variant protein” can mean a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the product as the
  • NCgl_2067, NCgl_2066 or NCgl_2065 protein The number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It can be 1 to 30, in another example 1 to 15, and in another example 1 to 5 in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:6. These changes in the variant can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change.
  • the protein variant encoded by each gene of the NCgl 2067 -NCgl 2065 operon can have a homology of not less than 80%, in another example not less than 90%, and in another example not less than 95%, in another example not less than 98%, in another example not less than 99%, with respect to the entire amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:6, as long as the activity of the NCgl_2067, NCgl_2066 or NCgl_2065 protein prior to inactivation of each gene of the NCgl 2067 -NCgl _2065 operon is maintained.
  • Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
  • each gene of the NCgl_2067-NCgl_2065 operon can be a variant which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional NCgl_2067, NCgl_2066, or NCgl_2065 protein prior to inactivation.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, in another example not less than 70%, in another example not less than 80%, in another example not less than 90%, and in another example not less than 95%, in another example not less than 98% , in another example not less than 99%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed.
  • stringent conditions can be exemplified by washing one time or more, or in another example, two or three times at a salt concentration of 1 X SSC, 0.1% SDS, or in another example, 0.1 X SSC, 0.1% SDS at 60 °C.
  • Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes.
  • the washing step can be performed 2 to 3 times.
  • the length of the probe can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • Expression of gene(s) of the NCgl 2067 -NCgl 2065 operon can be attenuated by introducing a mutation into the gene(s) on the chromosome so that intracellular activity of the protein(s) encoded by the gene(s) are/is decreased as compared with an unmodified strain.
  • a mutation on the gene can be replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem.
  • NCgl 2067 -NCgl 2065 operon can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc.
  • SD Shine-Dalgarno
  • the following methods can be employed to introduce a mutation by gene recombination.
  • a mutant gene encoding a mutant protein having a decreased activity is prepared, and the bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected.
  • Such gene replacement using homologous recombination can be conducted by a method employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci.
  • Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Patent No. 5,175,107), or by conventional methods, such as a mutagenesis treatment using a UV irradiation or a mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).
  • Inactivation of the gene can also be performed by conventional methods, such as a mutagenesis treatment using UV irradiation or a mutagen such as nitrosoguanidine (N- methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA 2000, 97(12):5978-83 and Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45), also called "Red-driven integration".
  • a mutagenesis treatment using UV irradiation or a mutagen such as nitrosoguanidine (N- methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recomb
  • L-glutamic acid is a precursor in biosynthesis of L-glutamine, L-proline, L- histidine L-arginine, L-ornithine, and L-citrulline, increasing production of L-glutamic acid results in increased production of a part or all of these amino acids.
  • Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory
  • the bacterium in accordance with the presently disclosed subject matter can be obtained by attenuating expression of one or more genes of the NCgl_2067-NCgl_2065 operon in a bacterium which inherently has the ability to produce L-amino acids.
  • the bacterium can be obtained by imparting the ability to produce L-amino acids to a bacterium already having the attenuated expression of one or more genes of the NCgl_2067-NCgl 065 operon.
  • parent strains which can be used to derive L-glutamic acid-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum ATCC 13032, ATCC 31833, C. acetoacidophilum ATCC 13870, C. herculis ATCC 13868, C. lilium ATCC 15990, B. divaricatum ATCC 14020, B.
  • Examples of parent strains which can be used to derive L-glutamic acid-producing bacteria also include strains in which expression of one or more genes encoding an L- glutamic acid enzyme are enhanced.
  • genes include genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3- phophate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase
  • Methods of enhancing gene expression can include increasing the gene copy number by introducing a gene into a vector that is able to function in a coryneform bacterium.
  • Gene expression can also be enhanced by introducing multiple copies of the gene into a bacterial chromosome by, for example, homologous recombination, Mu integration, or the like.
  • the copy number of a gene can also be increased by introducing multiple copies of the gene into the chromosomal DNA of the bacterium.
  • homologous recombination can be carried out using multiple copies of a sequence as targets in the chromosomal DNA.
  • Gene expression can also be enhanced by placing the objective DNA under the control of a potent promoter.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter.
  • RBS ribosome binding site
  • the substitution of several nucleotides in the spacer region between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon can profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol. 1981, 35:365-403; Hui et al., EMBO J. 1984, 3:623-629).
  • a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function.
  • the alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
  • microorganisms modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced can include those microorganisms disclosed in the following:
  • JP2001-333769A EP1078989A
  • JP2000-106869A EP955368A
  • JP2000- 189169A JP952221A
  • JP2001-333769A EP1078989A
  • Examples of a method of imparting L-glutamic acid-producing ability to coryneform bacterium can include increasing expression of the fasR gene and increasing of sensitivity to a surfactant (WO2007024010 Al).
  • Examples of an L-glutamic acid-producing coryneform bacterium can include a strain which has been modified to enhance expression of the yggB gene (JP2007-097573 A).
  • genes include genes encoding isocitrate lyase, a-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, and glutamate decarboxylase.
  • the methods described above may be used.
  • L-glutamic acid-producing coryneform bacterium can include a strain in which the a-ketoglutarate dehydrogenase activity is decreased (JP7-834672A and JP06-237779), strain with inactive gluX gene (US 2010279363 Al), a strain with attenuated expression of sucC and/or sucD genes (EP1 10361 1 Al), and so forth.
  • strains having an L-glutamic acid-producing ability can also include strains which are resistant to benzopirone or naphtoquinone (JP56-1889A), monofluoroacetic acid (JP 50-1 13209 A), to adenine and thymine (JP57-065198), to a- ketomalonic acid (JP57-2689A), to guanidine (JP56-35981A), to daunomicin (JP58- 158192A), and stains which are sensitive to penicillin (JP04-88994A).
  • L-glutamic acid-producing coryneform bacterium can also include the following strains: B. flavum AB949 (FERM BP-2632) (JP 50- 1 13209 A), AJ11355 (FERM P-5007) (JP56-1889A), AJ11217 (FERM P-4318) (JP57-2689A), AJ11564 (FERM P-5472) (JP56-140895A), AJ11439 (FERM P-5136) (JP56-35981A), B. lactofermentum AJ11426 (FERM P5123) (JP56-048890A), AJ1 1796 (FERM P6402) (JP58-158192A), C.
  • B. flavum AB949 (FERM BP-2632) (JP 50- 1 13209 A), AJ11355 (FERM P-5007) (JP56-1889A), AJ11217 (FERM P-4318) (JP57-2689A),
  • parent strains which can be used to derive L-glutamine-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. flavum AJ1 1573 (FERM P-5492) (JP56-161495A) AJ1 1576 (FERM BP- 10381) (JP56-161495A) AJ12212 (FERM P-8123) (JP61-202694A), AJ12418 (FERM BP-2205) (JP02- 186994 A), DH18 (FERM P-l 1116) (JP03-232497A), C. glutamicum AJ11 ⁇ 4 (FERM P-5493) (JP56-151495A), C.
  • strains belonging to coryneform bacteria such as strains B. flavum AJ1 1573 (FERM P-5492) (JP56-161495A) AJ1 1576 (FERM BP- 10381) (JP56-161495A) AJ12212 (FER
  • Examples of parent strains which can be used to derive L-glutamine-producing bacteria can also include strains in which expression of one or more genes encoding enzyme(s) of L-glutamine biosynthesis pathway are enhanced. Examples of such genes include genes encoding glutamine synthetase and glutamic acid dehydrogenase
  • parent strains which can be used to derive L-glutamine- producing bacteria can also include strains in which expression of one or more genes encoding enzyme(s) that catalyze synthesis of compound other than L-glutamine, but which are directed away from the L-glutamine biosynthesis pathway, are attenuated. For attenuation of expression levels of such genes, the methods described above may be used.
  • an L-glutamine-producing strain with decreased glutaminase activity EP 1424397 Bl
  • WO2008026698 Al a strain in which the gluABCD operon system for glutamine transport has been down-regulated or deleted
  • strains having an L-glutamic acid-producing ability can also include strains which are resistant to an L-amino acid analog.
  • parent strains can include strains resistant to 6-diazo-5-oxo-norleucine (JP-A-3-232497), to purine analogs and/or methionine sulfoxide (JP-A-61-202694), to a-ketomalonic acid (JP-A-56- 151495), and so forth.
  • parent strains which can be used to derive L-histidine-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum FERM BP-485 and FERM BP-486 (U.S. Patent No. 4,495,283), C. acetoacidophilum AJ 1562 and Microbacterium flavum (C. flavescens) ATCC 10340 (JP 47002549 B), C. glutamicum AJ 12092 (FERM P-7273) and A J 12426 (FERM BP-2213),
  • strains belonging to coryneform bacteria such as strains C. glutamicum FERM BP-485 and FERM BP-486 (U.S. Patent No. 4,495,283), C. acetoacidophilum AJ 1562 and Microbacterium flavum (C. flavescens) ATCC 10340 (JP 47002549 B), C. glutamicum AJ 12092 (FERM P-727
  • strains having an L-histidine-producing ability include strains belonging to coryneform bacteria, such as B. flavum FERM-P 2317, B. lactofermentum FERM-P 1565 (AJ 3386) C. glutamicum ATCC 14297 B. lactofermentum ATCC 21086
  • strains having an L-histidine-producing ability include strains belonging to genus Brevibacterium, such as a strain B. flavum AJ1 1846 which is resistant to thiamin antagonists (2-thiazolalanine, triazolecarboxamid, and cobalamine) (JP 02018838 B), and B. falvum AJ 3579 strain which requires threonine, proline, shikimic acid, xanthine, or guanine and is resistant to 2-thiazolalanine.(JP 51024594).
  • parent strains which can be used to derive L-proline-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. lactofermentum A J 11225 (FERM P-4370) (Japanese Patent Application Laid- Open No. 60-87788), B. flavum AJ 11512 (FERM P-5332), AJ 11513 (FERM P-5333), AJ 1 1514 (FERM P-5334), C. glutamicum AJ 11522 (FERM P-5342), AJ 11523 (FERM P-5343) (see Japanese Patent Publication No. 62-36679), and so forth.
  • strains belonging to coryneform bacteria such as strains B. lactofermentum A J 11225 (FERM P-4370) (Japanese Patent Application Laid- Open No. 60-87788), B. flavum AJ 11512 (FERM P-5332), AJ 11513 (FERM P-5333), AJ 1 15
  • Examples of parent strains which can be used to derive L-proline producing bacteria also include strains in which expression of one or more genes encoding an L-proline biosynthetic enzyme are enhanced.
  • Examples of an L-proline-biosynthetic enzyme include glutamate kinase, ⁇ -glutamyl phosphate reductase, and pyroline-5-carboxylate reductase (Ankri S et al., J Bacteriol. 1996, 178(15):4412-4419.).
  • L-proline producing bacteria can be used which have decreased activity of an enzyme that catalyzes a reaction which is directed away from the L-proline biosynthetic pathway and results in the production of other compounds.
  • L-proline-producing ability may be imparted by decreasing ornithine-aminotransferase activity (J Bacteriol. 1996 Aug;
  • parent strains which can be used to derive L-arginine-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum AJ12092 and B. flavum AJ11169 (EP 378223 Bl, JP 2817155 B2, US 5284757 A), B. flavum AJ 12144 (FERM P-7642) and C. glutamicum AJ 12145 (FERM P-7643) (Japanese Patent Publication No. 5-27388), B. flavum ATCC 21493 and C. glutamicum ATCC 21659; (Japanese Patent Application Laid-Open No. 5-3793), B. flavum FERM-P 4948, C. glutamicum FERM-P 7274 (AJ 12093), B. flavum NRRL 12235 (AJ 1 1337) C. acetoglutamicum NRRL 12239 (AJ 1 1342).
  • strains belonging to coryneform bacteria such as strain
  • strains having an L-arginine-producing ability include strains belonging to the genus Brevibacterium or Corynebacterium, and which are resistant to arginine hydroxamate, argi ine antagonists, argininol, sulfamides, cysteine or its analogs (EP 336387 Bl, JP 2578468 B2, US 5034319 A), aliphatic guanidine or aliphatic chain derivative guanidine (JP 2817155 B2, EP 0378223 Bl), and so forth.
  • the productivity of L-arginine strains of coryneform bacterium can be also improved by imparting resistance to sulfa drugs or argininol, or resistance to chemicals such as 8-azaguanine, alpha-amino- beta-hydroxyvaleric acid, etc.
  • parent strains which can be used to derive L-arginine producing bacteria also include strains auxotrophic for amino acids such as L-histidine, L-proline, L- threonine, L-tryptophan, L-lysine, etc.
  • parent strains which can be used to derive L-arginine producing bacteria also include strains that are known as citrulline or arginine auxotrophs (Japanese Patent Publication No. 43-8712), variants of arginine auxotroph (Japanese Patent Application Laid-Open No.
  • Examples of parent strains which can be used to derive L-arginine producing bacteria also include strains in which expression of one or more genes encoding an L- arginine biosynthetic enzyme are enhanced.
  • Examples of such genes include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argj), N-acetylglutamate kinase (argB), acetylornithine transaminase ( rgD), ornithine carbamoyl transferase ( rgF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase ( rgH), and carbamoyl phosphate synthetase (carAB).
  • argC N-acetylglutamyl phosphate reductase
  • argj ornithine acet
  • L-arginine producing bacterium can be improved by inactivation of the arginine repressor encoded by ctrgR gene. Methods for inactivation of argR gene are described above.
  • parent strains which can be used to derive L-ornithine-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. lactofermentum FERM BP-2344, FERM-P 5936 (AJ 1 1678) and C. glutamicum FERM BP-2345 (JP 2817185 (B2), EP 393708 Bl), C. glutamicum FERM-P 5644 (AJ 1 1589), ATCC 13232, and the like.
  • strains belonging to coryneform bacteria such as strains B. lactofermentum FERM BP-2344, FERM-P 5936 (AJ 1 1678) and C. glutamicum FERM BP-2345 (JP 2817185 (B2), EP 393708 Bl), C. glutamicum FERM-P 5644 (AJ 1 1589), ATCC 13232, and the like.
  • L-ornithine is an intermediate of L-arginine biosynthetic pathway
  • parent strains which can be used to derive L-ornithine-producing bacteria include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme is enhanced.
  • L-ornithine producing bacterium can be easily obtained from any arginine producing bacterium, by inactivation of ornithine carbamoyltransferase encoded by both argF and argl genes. Methods for inactivation of ornithine carbamoyltransferase are described above.
  • parent strains which can be used to derive L-citrulline-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, strains C. glutamicum FERM-P 5643 (AJ 1 1588), B. flavum FERM-P 1645 (AJ 3408), and so forth.
  • L-citrulline is an intermediate in the L-arginine biosynthetic pathway
  • examples of parent strains, which can be used to derive L-citrulline producing bacterium can be easily obtained from any L-arginine producing bacterium, for example, by inactivation of argininosuccinate synthase encoded by argG gene.
  • the bacterium as described herein is cultured in a culture medium to produce and excrete the L-amino acid into the medium, and the L-amino acid is collected from the medium.
  • the cultivation of the bacterium, collection, and purification of an L-amino acid from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
  • the medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • the carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen
  • alcohol including ethanol and glycerol
  • nitrogen source various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean- hydrolysate, and digested fermentative microorganism can be used.
  • minerals potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
  • vitamins, thiamine, yeast extract, and the like can be used.
  • the cultivation can be performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40°C, or 30 to 38°C.
  • the pH of the culture is usually between 5 and 9, or between 6.5 and 7.2.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
  • solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
  • Example 1 Construction of a strain with attenuated expression of NCgl_2067 gene 1 - 1. Deletion of the NCsl 2067 gene
  • a strain in which the NCgl_2067 gene is deleted was constructed by using the non- replicative plasmid pSB4S (see Fig.3; the construction of the plasmid is described in US patent No. 7,794,989).
  • the in-frame deletion of the NCgl_2067 gene was constructed.
  • overlap extension PCR was conducted. First, a DNA fragment was obtained by PCR with primers NC2067F (SEQ ID NO: 7) and NC2067R1 (SEQ ID NO: 8) and chromosomal DNA of B. flavum B-6642 (described in patent of Russian Federation RU 2084520 C2) as the template.
  • the strain B. flavum B-6642 was deposited as an
  • VKPM National Collection of Industrial Microorganisms
  • a DNA fragment was obtained in PCR with oligonucleotides NC2067F1 (SEQ ID NO: 9) and NC2067R (SEQ ID NO: 10) and chromosomal DNA oiB. flavum B- 6642 as the template.
  • the two DNA fragments described above were used as the template in the first round of PCR with primers NC2067-SmF (SEQ ID NO: 1 1) and NC2067-SmR (SEQ ID NO: 12).
  • Conditions for all PCRs were as follows: denaturation step for 30 sec at 94°C; profile for 25 cycles: 30 sec at 94°C, 30 sec at 58°C, 60 sec at 72°C; final step: 2 min at 72°C.
  • the obtained PCR product was purified and treated with endonuclease Smal and then ligated into a S/wal-cleaved plasmid pBS4S to obtain pBS4SA2067.
  • Electrocompetent cells were prepared as follows: B. flavum B-6642 was grown overnight at 30 °C in CM2GxYE medium (peptone - lOg/1, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0), and then the culture was diluted about 20 times with fresh CM2GxYE medium. The cells were grown with aeration at 30°C to an OD 600 of «0.6 and ampicillin was added (10 ⁇ g/ml) and cultivated for 1 hour. The obtained cells were washed three times with ice-cold deionized H 2 0.
  • CM2GxYE medium peptone - lOg/1, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0
  • the cells were grown with aeration at 30°C to an OD 600 of «0.6 and ampicillin was added (10 ⁇ g/m
  • Electroporation was performed using 50 ⁇ of cells and * ⁇ g of the plasmid pBS4SA2067 with the following parameters: 2.0 kV, 0,1 cm electrode gap.
  • Cells after electroporation were incubated with 1 ml CM2GxYE medium at 30°C for 2 hours with shaking and then were plated onto 2LA- agar (peptone - lOg/1, yeast extract - lOg/1, NaCl - 5g/l, agar - 20g/l, pH is adjusted to 7.0) containing kanamycin (25 ⁇ g/ml) and grown at 30°C to select Km R recombinants for 48 h. The obtained integrants were also unable to grow on 2LA medium containing 10% sucrose.
  • the mutants in which the NCgl 2067 gene was deleted were verified by PCR.
  • Locus-specific primers PI (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) were used in PCR for the verification.
  • the PCR product obtained in the reaction with the cells of the parental strain B. flavum B-6642 as the template was -1.35 kbp in length.
  • the PCR product obtained in the reaction with the cells of mutant strain as the template was -0.78 kbp in length.
  • Deletion of NCgl_2067 gene_ was also confirmed by the sequencing of the corresponding chromosomal DNA fragment.
  • the mutant strain was given the name B. flavum B-6642ANCgl_2067.
  • a strain in which the NCgl 2066 gene was deleted was constructed as the same manner as described in Example 1.
  • the in-frame deletion of the NCgl 2066 gene was constructed.
  • overlap extension PCR was conducted.
  • a DNA fragment was obtained in PCR with primers NC2066F (SEQ ID NO: 15) and NC2066R1 (SEQ ID NO: 16) and chromosomal DNA of B. flavum B-6642 as the template.
  • Second, a DNA fragment was obtained in PCR with oligonucleotides NC2066F1 (SEQ ID NO: 17) and NC2066R (SEQ ID NO: 18) and chromosomal DNA of B. flavum B-6642 as the template.
  • the two DNA fragments described above were used as the template in the first round of PCR with primers NC2066-SmF (SEQ ID NO: 19) and NC2066-SmR (SEQ ID NO: 20). After that, the obtained PCR product was purified and treated with endonuclease Smal, and then ligated into Smal-cleaved plasmid pBS4S to obtain pBS4SA2066.
  • Electrocompetent cells were prepared as follows: B. flavum B-6642 was grown overnight at 30 °C in CM2GxYE medium (peptone - lOg/l, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0), and then the culture was diluted about 20 times with fresh CM2GxYE medium. The cells were grown with aeration at 30°C to an OD 600 of «0.6 and ampicillin was added (10 ⁇ g/ml) and cultivated for 1 hour. The obtained cells were washed three times with ice-cold deionized H 2 0.
  • CM2GxYE medium peptone - lOg/l, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0
  • the cells were grown with aeration at 30°C to an OD 600 of «0.6 and ampicillin was added (10 ⁇ g
  • Electroporation was performed using 50 ⁇ of cells and «5 ⁇ g of the plasmid pBS4SA2066 with the following parameters: 2.0 kV, 0.1 cm electrode gap.
  • Cells after electroporation were incubated with 1 ml CM2GxYE medium at 30°C for 2 hours with shaking and then were plated onto 2LA-agar (peptone - lOg/1, yeast extract - lOg/1, NaCl - 5g/l, agar - 20g/l, pH is adjusted to 7.0) containing kanamycin (25 ⁇ g/ml) and grown at 30°C to select Km R recombinants for 48 h. The obtained integrants were also unable to grow on 2LA medium containing 10% sucrose.
  • the mutants in which the NCgl_2066 gene was deleted were verified by PCR.
  • Locus-specific primers NC2066F (SEQ ID NO: 15) and NC2066R (SEQ ID NO: 18) were used in PCR for the verification.
  • the PCR product obtained in the reaction with the cells of parental strain B. flavum B-6642 as the template was -2.12 kbp in length.
  • the PCR product obtained in the reaction with the cells of mutant strain as the template was -1.15 kbp in length.
  • Deletion of the NCgl_2066 gene_ was also confirmed by the sequencing of the corresponding chromosomal DNA fragment.
  • the mutant strain was given the name B. flavum B-6642ANCgl_2066.
  • B. flavum strains B-6642ANCgl_2067, and B-6642, were separately grown for 18- 24 hours at 30°C on 2LA-agar plates. Then, one loop of the cells was transferred into test tubes containing 2ml of fermentation medium.
  • the fermentation medium contains (NH 4 ) 2 S0 4 (70g/l), KH 2 P0 4 (2,5g/l), thiamine (350mg/l), biotin (lOOmg/l), MgS0 4 7H 2 0 (0.4g/l), Mameno (soybean protein hydrolysate, 5.7mg/l), sucrose (120g/l), and CaC0 3 (50g/l).
  • the pH is adjusted to 7.
  • B. flavum strains B-6642ANCgl_2066 and B-6642, were separately grown for 18- 24 hours at 30°C on 2LA-agar plates. Then, one loop of the cells was transferred into test tubes containing 2ml of fermentation medium.
  • the fermentation medium contains (NH 4 ) 2 S0 4 (70g/l), KH 2 P0 4 (2,5g/l), thiamine (350mg/l), biotin (lOOmg/l), MgS0 4 7H 2 O(0.4g/l), Mameno (5.7mg/l), sucrose (120g/l), and CaC0 3 (50g/l).
  • the pH is adjusted to 7.
  • NCgl_2066 gene produced higher amounts of L-glutamic acid and L-glutamine, as compared with the parent strain B-6642.
  • an L-amino acid belonging to the glutamate family is efficently produced by a coryneform bacterium.

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Abstract

The present invention provides a method for producing an L-amino acid belonging to the glutamate family, using a coryneform bacterium which has been modified so that expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon in said bacterium is/are attenuated.

Description

DESCRIPTION
A METHOD FOR PRODUCING AN L-AMINO ACID BELONGING TO THE GLUTAMATE FAMILY, USING A CORYNEFORM BACTERIUM
Tecnical Field
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid belonging to the glutamate family using a coryneform bacterium which has been modified so that expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon in the bacterium is/are attenuated.
Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including by transforming microorganisms with recombinant DNA (see, for example, US patent No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 or US patent Nos. 4,346,170; 5,661,012 and 6,040,160).
Many methods for producing L-glutamine by culturing a coryneform bacterium have been disclosed. For example, EP1229121 A2 discloses a method for producing L- glutamine by culturing a coryneform bacterium which has L-glutamine producing ability and has been modified so that its intracellular glutamine synthetase activity is enhanced, and preferably has been further modified so that its intracellular glutamate dehydrogenase activity is enhanced, in a medium to produce and cause accumulation of L-glutamine in the medium, and collecting the L-glutamine.
It is known that transcription of the Bacillus subtilis gene coding of glutamine synthetase (glnA) is regulated by the nitrogen source. The glnA gene is located in an operon with the glnR gene. The glnR gene codes for a negative regulator which directly controls expression of the glutamine synthetase gene (glnA). Certain mutations in glnR gene, such as a large, in-frame deletion and, a start codon mutation, lead to high-level constitutivity of the operon; whereas other mutations caused low-level constitutivity (Schreier HJ et al., J Mol Biol. 1989, 210(l):51-63). Recently, regulation of glutamine and glutamate metabolism by the nitrogen regulatory protein GlnR in Streptococcus pneumoniae was analyzed. Using DNA microarray analyses of S. pneumoniae D39 wild-type and its isogenic glnR mutant which had been grown in nitrogen-rich medium supplemented with glutamine, a list of operons and genes that were up-regulated the most in the glnR mutant was determined, and includes the following genes: glnA (encoding glutamine synthetase GlnA), glnPQ
(encoding ABC transporter amino acid-binding protein/permease), and gdhA (encoding glutamate dehydrogenase glutamate dehydrogenase). All of these genes have a GlnR operator in their promoter regions. Also the zwf gene, encoding the glucose-6-phosphate dehydrogenase and located downstream of, and in the same orientation as glnPQ, was up- regulated (Kloosterman et al., J Biol Chem. 2006, 281(35):25097-25109). Currently, there have been no reports about the presence of the putative glnR gene in the genomes of coryneform bacteria.
There have been no reports of attenuating expression of one or more genes of the NCgl_2067-NCgl_2065 operon for the purpose of improving productivity of L-amino acid belonging to the glutamate family.
Disclosure of the Invention
Aspects of the present invention include enhancing the productivity of strains which produce L-amino acids belonging to the glutamate family, and providing a method for producing an L-amino acid belonging to the glutamate family, using these strains.
An open reading frame, (ORF) NCgl_2066, was fourid in the genome of
Corynebacterium glutamicum which is homologous to the glnR gene from Streptococcus pneumoniae. The ORF NCgl_2066 is located in an operon with two other ORFs - NCgl_2067 and NCgl_2065. The correspoding ORFs were named NCgl_2067,
NCgl_2066, and NCgl_2065 genes, and the operon was given the name NCgl_2067- NCgl_2065 operon.
The above aspects were achieved by finding that attenuation of the expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon can enhance production of L-amino acids belonging to the glutamate family, such as L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
The present invention provides a coryneform bacterium having an increased ability to produce amino acids belonging to the glutamate family, such as L-glutamic acid, L- glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
It is an aspect of the present invention to provide a coryneform bacterium which is able to produce an L-amino acid belonging to the glutamate family, wherein said bacterium has been modified so that the expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon in said bacterium is attenuated.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been modified to attenuate the expression of the NCgl 067 gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression of the NCgl 2067 gene is attenuated by inactivation of the NCgl_2067 gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been modified to attenuate the expression of the NCgl 066 gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression of the NCgl_2066 gene is attenuated by inactivation of the NCgl ' 2066 gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium belongs to genus Corynebacterium or Brevibacterium.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium is Brevibacterium flavum.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine and L-citrulline.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine.
It is a further aspect of the present invention to provide a method for producing an L-amino acid belonging to the glutamate family comprising:
- cultivating the bacterium as described above in a medium, and
- collecting said L-amino acid from the medium.
It is a further aspect of the present invention to provide the method as described above, wherein said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
It is a further aspect of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine. The present invention is described in detail below.
Brief Description of the Drawings
Figure 1 shows alignment of the GlnR (denoted as spr0433) protein from
Streptococcus pneumoniae R6 and putative NCgl_2066 protein from Corynebacterium glutamicum ATCC13032.
Figure 2 shows structure of the NCgl 2067 -NCgl 2065 operon from
Corynebacterium glutamicum ATCC 13032
Figure 3 shows structure of the pBS4S plasmid.
Detailed Description of the Preferred Embodiments
The present invention is described in detail below.
1. Bacterium of the present invention
A coryneform bacterium is described which can produce an L-amino acid belonging to the glutamate family, wherein the bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon.
The phrase "bacterium producing an L-amino acid" can mean a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
The phrase "bacterium producing an L-amino acid" as used herein also can mean a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain ofcoryneform bacterium, for example, bacterial strains listed below, such as Corynebacterium glutamicum ATCC 13032, ATCC 31833, Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 and B-6642, and can mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5g/l, or l.Og/1, of the target L- amino acid.
The phrase "L-amino acid belonging to the glutamate family" can include L- glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, L-citrulline, and combinations thereof.
The term "coryneform bacteria" can include conventional coryneform bacterium according to the classification known to a person skilled in the art of microbiology, and also can include bacteria that had been classified into the genus Brevibacterium, but are currently classified into the genus Corynebacterium (Liebl W et al., Int J Syst Bacteriol. 1991, 41(2):255-260), as well as the Brevibacterium bacteria that are highly related to Corynebacterium bacteria. Examples of such coryneform bacterium include the following:
Corynebacterium acetoacidophil um
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium glutamicum {Micrococcus glutamicus)
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum
Brevibacterium flavum
Brevibacterium immariophilum
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Brevibacterium ammoniagenes
Brevibacterium album
Brevibacterium cerinum
Microbacterium ammoniaphilum,
Microbacterium flavum {Corynebacterium flavescens) etc.
Specific examples of the coryneform bacterium include, but are not limited to, strains such as C. acetoacidophilum ATCC 13870, C. acetoglutamicum ATCC 15806, C alkanolyticum ATCC 2151 1, C callunae ATCC 15991, C glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, C lilium ATCC 15990, C. melassecola ATCC 17965, C. thermoaminogenes AJ12340 (FERM BP-1539), C herculis ATCC 13868, B. divaricatum ATCC 14020, B. flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205), B. immariophilum ATCC 14068, B. lactofermentum (C. glutamicum) ATCC 13869, B. roseum ATCC 13825 B. saccharolyticum ATCC 14066, B. thiogenitalis ATCC 19240, B. ammoniagenes ATCC 6871, ATCC 6872, B. album ATCC 15111, 5. cerinum ATCC 15112, Microbacterium ammoniaphilum ATCC 15354, and so forth. These strains are available from the American Type Culture Collection (ATCC, Address: P.O. Box 1549, Manassas, VA 20108, United States of America). That is, each strain is given a unique registration number which is listed in the catalogue of the ATCC. Strains can be ordered using this registration number. The AJ 12340 strain was deposited at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology at Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305- 5466, Japan) on October 27, 1989 under the provisions of the Budapest Treaty and given an accession number of FERM BP-1539. The AJ12418 strain was deposited at National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on January 5, 1989 under the provisions of the Budapest Treaty and given an accession number of FERM BP-2205.
The phrase "a bacterium belonging to the genus Corynebacterium or
Brevibacterium''' can mean that the bacterium is classified into the genus
Corynebacterium or Brevibacterium according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Corynebacterium or Brevibacterium include, but are not limited to, Corynebacterium glutamicum and Brevibacterium flavum.
The phrase "bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl 2067 -NCgl 2065 operon" can mean that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of one or more of the proteins, NCgl_2067, NCgl_2066 and NCgl_2065 as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize the NCgl_2067, NCgl_2066 and/or NCgl_2065 protein(s). The phrase "bacterium has been modified to attenuate expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon" also can mean that the bacterium has been modified in such a way that the modified gene(s) encodes mutant NCgl_2067, NCgl_2066 and or NCgl_2065 protein(s) with decreased activities. Since it is thought that genes NCgl_2067, NCgl_2066 and NCgl_2065 are organized as an operon, attenuation of expression of one of the genes can lead to polar effects on expression other genes of the NCgl 2067 -NCgl 2065 operon.
The presence or absence of gene(s) of the NCgl _2067 -NCgl 2065 operon in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amounts of the proteins encoded by the genes of the NCgl 206/ '-NCgl ' 2065 operon can be measured by well-known methods, including SDS-PAGE followed by an
immunoblotting assay (Western blotting analysis), and the like.
The phrase "inactivation of one or more gene(s) of the NCgl_2067-NCgl_2065 operon" can mean that the modified gene(s) encodes a completely inactive protein(s). It is also possible that the modified DNA region is unable to naturally express the gene due to deletion of a part of or the entire gene, shifting of the reading frame of the gene, introduction of missense/nonsense mutation(s), or modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
The NCgl 2067 gene (synonyms - CG2358, Cgl2147) codes for a hypothetical protein with unknown function. The NCgl_2067 gene of C glutamicum (nucleotides complement to the nucleotides from 2270870 to 2270256 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl 066 and the ileS genes, on the chromosome of C. glutamicum ATCC 13032. The nucleotide sequence of the NCgl_2067 gene and the amino acid sequence of predicted NCgl_2067 protein encoded by the NCgl_2067 gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The NCgl 2066 gene (synonyms - CG2357, Cgl2146) codes for a hypothetical putative transcriptional regulator. The NCgl_2066 gene of C. glutamicum (nucleotides complement to the nucleotides from 2,270,262 to 2,269,258 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl_2067 and NCgl 065 gene on the chromosome of C. glutamicum ATCC 13032, and both genes are oriented in the same direction as the NCgl ' 2066 gene, and these three genes are presumably organized as an operon. The nucleotide sequence of the NCgl_2066 gene and the amino acid sequence of predicted NCgl_2066 protein encoded by the NCgl_2066 gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The NCgl 2065 gene (synonyms - CG2356, Cgl2145) is predicted to encode a DMT family permease. The NCgl_2065 gene of C glutamicum (nucleotides complement to the nucleotides from 2,269,246 to 2,268,386 in the GenBank accession number NC 003450; gi: 58036263) is located between the NCgl_2066 and NCgl_2064 genes on the chromosome of C glutamicum ATCC 13032. The nucleotide sequence of the
NCgl 2066 gene and the amino acid sequence of predicted NCgl_2066 protein encoded by the NCgl _2066 gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
Since there may be some differences in DNA sequences between the genera or strains of coryneform bacteria, genes of the NCgl 2067 -NCgl _2065 operon to be inactivated on the chromosome are not limited to the genes shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, but can include genes homologous to SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, which encode a variant proteins of the NCgl_2067, NCgl_2066 and NCgl_2065 proteins. The phrase "variant protein" can mean a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the activity of the product as the
NCgl_2067, NCgl_2066 or NCgl_2065 protein. The number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It can be 1 to 30, in another example 1 to 15, and in another example 1 to 5 in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:6. These changes in the variant can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change. Therefore, the protein variant encoded by each gene of the NCgl 2067 -NCgl 2065 operon can have a homology of not less than 80%, in another example not less than 90%, and in another example not less than 95%, in another example not less than 98%, in another example not less than 99%, with respect to the entire amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:6, as long as the activity of the NCgl_2067, NCgl_2066 or NCgl_2065 protein prior to inactivation of each gene of the NCgl 2067 -NCgl _2065 operon is maintained.
Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
Moreover, each gene of the NCgl_2067-NCgl_2065 operon can be a variant which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional NCgl_2067, NCgl_2066, or NCgl_2065 protein prior to inactivation. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, in another example not less than 70%, in another example not less than 80%, in another example not less than 90%, and in another example not less than 95%, in another example not less than 98% , in another example not less than 99%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times at a salt concentration of 1 X SSC, 0.1% SDS, or in another example, 0.1 X SSC, 0.1% SDS at 60 °C. Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. The length of the probe can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
Expression of gene(s) of the NCgl 2067 -NCgl 2065 operon can be attenuated by introducing a mutation into the gene(s) on the chromosome so that intracellular activity of the protein(s) encoded by the gene(s) are/is decreased as compared with an unmodified strain. Such a mutation on the gene can be replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem. 1997, 272:8611-8617; Kwon, D. H. et al., J. Antimicrob. Chemother. 2000, 46:793-796). Expression of genes of the NCgl 2067 -NCgl 2065 operon can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc.
(W095/34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 1999, 15:58-64).
For example, the following methods can be employed to introduce a mutation by gene recombination. A mutant gene encoding a mutant protein having a decreased activity is prepared, and the bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement using homologous recombination can be conducted by a method employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-6645), or by methods employing a plasmid containing a temperature-sensitive replication origin (U.S. Patent 6,303,383 or JP 05-007491 A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid lacking the ability to replicate in the host.
Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Patent No. 5,175,107), or by conventional methods, such as a mutagenesis treatment using a UV irradiation or a mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).
Inactivation of the gene can also be performed by conventional methods, such as a mutagenesis treatment using UV irradiation or a mutagen such as nitrosoguanidine (N- methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA 2000, 97(12):5978-83 and Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45), also called "Red-driven integration".
As L-glutamic acid is a precursor in biosynthesis of L-glutamine, L-proline, L- histidine L-arginine, L-ornithine, and L-citrulline, increasing production of L-glutamic acid results in increased production of a part or all of these amino acids.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory
Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).
L-amino acid-producing bacteria
As a bacterium in accordance with the presently disclosed subject matter which is modified to attenuate expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon, bacteria which are able to produce L-amino acids belonging to the glutamate family are described.
The bacterium in accordance with the presently disclosed subject matter can be obtained by attenuating expression of one or more genes of the NCgl_2067-NCgl_2065 operon in a bacterium which inherently has the ability to produce L-amino acids.
Alternatively, the bacterium can be obtained by imparting the ability to produce L-amino acids to a bacterium already having the attenuated expression of one or more genes of the NCgl_2067-NCgl 065 operon.
L-glutamic acid-producing bacteria
Examples of parent strains which can be used to derive L-glutamic acid-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum ATCC 13032, ATCC 31833, C. acetoacidophilum ATCC 13870, C. herculis ATCC 13868, C. lilium ATCC 15990, B. divaricatum ATCC 14020, B.
flavum ATCC 14067, B. imaliophilum ATCC 14068, B. lactofermentum ATCC 13869, B. thiogenitalis ATCC 19240, and so forth.
Examples of parent strains which can be used to derive L-glutamic acid-producing bacteria also include strains in which expression of one or more genes encoding an L- glutamic acid enzyme are enhanced. Examples of such genes include genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3- phophate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, and glucose phosphate isomerase.
Methods of enhancing gene expression can include increasing the gene copy number by introducing a gene into a vector that is able to function in a coryneform bacterium. Gene expression can also be enhanced by introducing multiple copies of the gene into a bacterial chromosome by, for example, homologous recombination, Mu integration, or the like. The copy number of a gene can also be increased by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. To introduce multiple copies of the gene into a bacterial chromosome, homologous recombination can be carried out using multiple copies of a sequence as targets in the chromosomal DNA. Gene expression can also be enhanced by placing the objective DNA under the control of a potent promoter.
Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter. Furthermore, it is known that the substitution of several nucleotides in the spacer region between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, can profoundly affect the mRNA translatability. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol. 1981, 35:365-403; Hui et al., EMBO J. 1984, 3:623-629). Moreover, it is also possible to introduce a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function. The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
Examples of microorganisms modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced can include those microorganisms disclosed in the following:
JP2001-333769A (EP1078989A), JP2000-106869A (EP955368A), JP2000- 189169A(EP952221A), and JP2001-333769A (EP1078989A). Examples of a method of imparting L-glutamic acid-producing ability to coryneform bacterium can include increasing expression of the fasR gene and increasing of sensitivity to a surfactant (WO2007024010 Al). Examples of an L-glutamic acid-producing coryneform bacterium can include a strain which has been modified to enhance expression of the yggB gene (JP2007-097573 A).
Other examples of parent strains which can be used to derive L-glutamic acid- producing bacteria include strains in which expression of one or more genes encoding enzymes that catalyze synthesis of a compound other than L-glutamic acid, but which direct away from the L-glutamic acid biosynthesis pathway, are attenuated. Examples of such genes include genes encoding isocitrate lyase, a-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, and glutamate decarboxylase. For attenuation of expression levels of such genes, the methods described above may be used.
Examples of L-glutamic acid-producing coryneform bacterium can include a strain in which the a-ketoglutarate dehydrogenase activity is decreased (JP7-834672A and JP06-237779), strain with inactive gluX gene (US 2010279363 Al), a strain with attenuated expression of sucC and/or sucD genes (EP1 10361 1 Al), and so forth.
Specific examples of strains having an L-glutamic acid-producing ability can also include strains which are resistant to benzopirone or naphtoquinone (JP56-1889A), monofluoroacetic acid (JP 50-1 13209 A), to adenine and thymine (JP57-065198), to a- ketomalonic acid (JP57-2689A), to guanidine (JP56-35981A), to daunomicin (JP58- 158192A), and stains which are sensitive to penicillin (JP04-88994A).
Specific examples of L-glutamic acid-producing coryneform bacterium can also include the following strains: B. flavum AB949 (FERM BP-2632) (JP 50- 1 13209 A), AJ11355 (FERM P-5007) (JP56-1889A), AJ11217 (FERM P-4318) (JP57-2689A), AJ11564 (FERM P-5472) (JP56-140895A), AJ11439 (FERM P-5136) (JP56-35981A), B. lactofermentum AJ11426 (FERM P5123) (JP56-048890A), AJ1 1796 (FERM P6402) (JP58-158192A), C. glutamicum AJ11628 (FERM P-5736) (JP 57-065198A), AJ11440 (FERM P5137) (JP56-048890A), H7684 (FERM BP-3004; JP04-88994A), AJ11355 (FERM P-5020) (JP56-1889A), AJ1 1218 (FERM P-4319) (JP57-2689A).
L-glutamine-producing bacteria
Examples of parent strains which can be used to derive L-glutamine-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. flavum AJ1 1573 (FERM P-5492) (JP56-161495A) AJ1 1576 (FERM BP- 10381) (JP56-161495A) AJ12212 (FERM P-8123) (JP61-202694A), AJ12418 (FERM BP-2205) (JP02- 186994 A), DH18 (FERM P-l 1116) (JP03-232497A), C. glutamicum AJ11^ 4 (FERM P-5493) (JP56-151495A), C. melassecola DH344 (FERM P-l 1117) (JP3-232497A). Examples of parent strains which can be used to derive L-glutamine-producing bacteria can also include strains in which expression of one or more genes encoding enzyme(s) of L-glutamine biosynthesis pathway are enhanced. Examples of such genes include genes encoding glutamine synthetase and glutamic acid dehydrogenase
(US7262035 B2, EP1229121 Bl), and the like.
Other examples of parent strains which can be used to derive L-glutamine- producing bacteria can also include strains in which expression of one or more genes encoding enzyme(s) that catalyze synthesis of compound other than L-glutamine, but which are directed away from the L-glutamine biosynthesis pathway, are attenuated. For attenuation of expression levels of such genes, the methods described above may be used. As a parent strain, an L-glutamine-producing strain with decreased glutaminase activity (EP 1424397 Bl), or a strain in which the gluABCD operon system for glutamine transport has been down-regulated or deleted (WO2008026698 Al) can be used, and the like.
Specific examples of strains having an L-glutamic acid-producing ability can also include strains which are resistant to an L-amino acid analog. Examples of parent strains can include strains resistant to 6-diazo-5-oxo-norleucine (JP-A-3-232497), to purine analogs and/or methionine sulfoxide (JP-A-61-202694), to a-ketomalonic acid (JP-A-56- 151495), and so forth.
L-histidine-producing bacteria
Examples of parent strains which can be used to derive L-histidine-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum FERM BP-485 and FERM BP-486 (U.S. Patent No. 4,495,283), C. acetoacidophilum AJ 1562 and Microbacterium flavum (C. flavescens) ATCC 10340 (JP 47002549 B), C. glutamicum AJ 12092 (FERM P-7273) and A J 12426 (FERM BP-2213),
B. flavum AJ 3420 (FERM P-2316) and B. flavum AJ 12425 (FERM BP-2212) (U.S. Patent. No. 5,294,547), C. glutamicum (B. flavum) ATCC 21604 (VKPM B-1080) (U.S. Patent No. 3,713,977), C. glutamicum ("Micrococcus glutamicus"); ATCC 13761; (NCIB 10334, VKPM B-4382) (U.S. Patent No. 3,220,929), C. glutamicum ATCC 21339 (VKPM B-1003) (U.S. Patent No. 3,676,301).
Specific examples of strains having an L-histidine-producing ability include strains belonging to coryneform bacteria, such as B. flavum FERM-P 2317, B. lactofermentum FERM-P 1565 (AJ 3386) C. glutamicum ATCC 14297 B. lactofermentum ATCC 21086
C. acetoacidophilum ATCC 21407, B. flavum ATCC 21406 (AJ 3225), and so forth.
Specific examples of strains having an L-histidine-producing ability include strains belonging to genus Brevibacterium, such as a strain B. flavum AJ1 1846 which is resistant to thiamin antagonists (2-thiazolalanine, triazolecarboxamid, and cobalamine) (JP 02018838 B), and B. falvum AJ 3579 strain which requires threonine, proline, shikimic acid, xanthine, or guanine and is resistant to 2-thiazolalanine.(JP 51024594).
L-proline producing bacteria
Examples of parent strains which can be used to derive L-proline-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. lactofermentum A J 11225 (FERM P-4370) (Japanese Patent Application Laid- Open No. 60-87788), B. flavum AJ 11512 (FERM P-5332), AJ 11513 (FERM P-5333), AJ 1 1514 (FERM P-5334), C. glutamicum AJ 11522 (FERM P-5342), AJ 11523 (FERM P-5343) (see Japanese Patent Publication No. 62-36679), and so forth.
Examples of parent strains which can be used to derive L-proline producing bacteria also include strains in which expression of one or more genes encoding an L-proline biosynthetic enzyme are enhanced. Examples of an L-proline-biosynthetic enzyme include glutamate kinase, γ-glutamyl phosphate reductase, and pyroline-5-carboxylate reductase (Ankri S et al., J Bacteriol. 1996, 178(15):4412-4419.). Also, L-proline producing bacteria can be used which have decreased activity of an enzyme that catalyzes a reaction which is directed away from the L-proline biosynthetic pathway and results in the production of other compounds. For example, L-proline-producing ability may be imparted by decreasing ornithine-aminotransferase activity (J Bacteriol. 1996 Aug;
178(15):4412-9).
L-arginine-producing bacteria
Examples of parent strains which can be used to derive L-arginine-producing bacteria can include, but are not limited to, strains belonging to coryneform bacteria, such as strains C. glutamicum AJ12092 and B. flavum AJ11169 (EP 378223 Bl, JP 2817155 B2, US 5284757 A), B. flavum AJ 12144 (FERM P-7642) and C. glutamicum AJ 12145 (FERM P-7643) (Japanese Patent Publication No. 5-27388), B. flavum ATCC 21493 and C. glutamicum ATCC 21659; (Japanese Patent Application Laid-Open No. 5-3793), B. flavum FERM-P 4948, C. glutamicum FERM-P 7274 (AJ 12093), B. flavum NRRL 12235 (AJ 1 1337) C. acetoglutamicum NRRL 12239 (AJ 1 1342).
It is known that an L-arginine-producing ability can also be efficiently enhanced by using a mutant N-acetylglutamate kinase which is resistant to feedback inhibition by L- arginine (WO 2006035831 Al).
Specific examples of strains having an L-arginine-producing ability include strains belonging to the genus Brevibacterium or Corynebacterium, and which are resistant to arginine hydroxamate, argi ine antagonists, argininol, sulfamides, cysteine or its analogs (EP 336387 Bl, JP 2578468 B2, US 5034319 A), aliphatic guanidine or aliphatic chain derivative guanidine (JP 2817155 B2, EP 0378223 Bl), and so forth. The productivity of L-arginine strains of coryneform bacterium can be also improved by imparting resistance to sulfa drugs or argininol, or resistance to chemicals such as 8-azaguanine, alpha-amino- beta-hydroxyvaleric acid, etc.
Examples of parent strains which can be used to derive L-arginine producing bacteria also include strains auxotrophic for amino acids such as L-histidine, L-proline, L- threonine, L-tryptophan, L-lysine, etc. Examples of parent strains which can be used to derive L-arginine producing bacteria also include strains that are known as citrulline or arginine auxotrophs (Japanese Patent Publication No. 43-8712), variants of arginine auxotroph (Japanese Patent Application Laid-Open No. 53-24096), variants belonging to the genus Corynebacterium having a resistance to 2-thiazole-alanine, sulfaguanidine or 2- fluoropyruvic acid (Japanese Patent Application Laid-Open No. 61-119194) and so forth.
Examples of parent strains which can be used to derive L-arginine producing bacteria also include strains in which expression of one or more genes encoding an L- arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argj), N-acetylglutamate kinase (argB), acetylornithine transaminase ( rgD), ornithine carbamoyl transferase ( rgF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase ( rgH), and carbamoyl phosphate synthetase (carAB).
L-arginine producing bacterium can be improved by inactivation of the arginine repressor encoded by ctrgR gene. Methods for inactivation of argR gene are described above.
L-ornithine producing bacteria
Examples of parent strains which can be used to derive L-ornithine-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, such as strains B. lactofermentum FERM BP-2344, FERM-P 5936 (AJ 1 1678) and C. glutamicum FERM BP-2345 (JP 2817185 (B2), EP 393708 Bl), C. glutamicum FERM-P 5644 (AJ 1 1589), ATCC 13232, and the like.
As L-ornithine is an intermediate of L-arginine biosynthetic pathway, examples of parent strains, which can be used to derive L-ornithine-producing bacteria include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme is enhanced. L-ornithine producing bacterium can be easily obtained from any arginine producing bacterium, by inactivation of ornithine carbamoyltransferase encoded by both argF and argl genes. Methods for inactivation of ornithine carbamoyltransferase are described above.
L-citrulline producing bacteria
Examples of parent strains which can be used to derive L-citrulline-producing bacteria include, but are not limited to, strains belonging to coryneform bacteria, strains C. glutamicum FERM-P 5643 (AJ 1 1588), B. flavum FERM-P 1645 (AJ 3408), and so forth.
As L-citrulline is an intermediate in the L-arginine biosynthetic pathway, examples of parent strains, which can be used to derive L-citrulline producing bacterium can be easily obtained from any L-arginine producing bacterium, for example, by inactivation of argininosuccinate synthase encoded by argG gene.
2. Method of the present invention
In a method for producing an L-amino acid of the presently disclosed subject matter, the bacterium as described herein is cultured in a culture medium to produce and excrete the L-amino acid into the medium, and the L-amino acid is collected from the medium.
The cultivation of the bacterium, collection, and purification of an L-amino acid from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
The medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen
microorganism, alcohol, including ethanol and glycerol, may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean- hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.
The cultivation can be performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40°C, or 30 to 38°C. The pH of the culture is usually between 5 and 9, or between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
Examples
The present invention will be more concretely explained below with reference to the following non-limiting Examples.
Example 1. Construction of a strain with attenuated expression of NCgl_2067 gene 1 - 1. Deletion of the NCsl 2067 gene
A strain in which the NCgl_2067 gene is deleted was constructed by using the non- replicative plasmid pSB4S (see Fig.3; the construction of the plasmid is described in US patent No. 7,794,989). The in-frame deletion of the NCgl_2067 gene was constructed. For this purpose, overlap extension PCR was conducted. First, a DNA fragment was obtained by PCR with primers NC2067F (SEQ ID NO: 7) and NC2067R1 (SEQ ID NO: 8) and chromosomal DNA of B. flavum B-6642 (described in patent of Russian Federation RU 2084520 C2) as the template. The strain B. flavum B-6642 was deposited as an
international deposit at the Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on June 28, 1993 under the accession number of VKPM B-6642 and then converted to a deposit under the Budapest Treaty on March 5, 2012.
Second, a DNA fragment was obtained in PCR with oligonucleotides NC2067F1 (SEQ ID NO: 9) and NC2067R (SEQ ID NO: 10) and chromosomal DNA oiB. flavum B- 6642 as the template. After that, the two DNA fragments described above were used as the template in the first round of PCR with primers NC2067-SmF (SEQ ID NO: 1 1) and NC2067-SmR (SEQ ID NO: 12). Conditions for all PCRs were as follows: denaturation step for 30 sec at 94°C; profile for 25 cycles: 30 sec at 94°C, 30 sec at 58°C, 60 sec at 72°C; final step: 2 min at 72°C.
After that, the obtained PCR product was purified and treated with endonuclease Smal and then ligated into a S/wal-cleaved plasmid pBS4S to obtain pBS4SA2067.
Electrocompetent cells were prepared as follows: B. flavum B-6642 was grown overnight at 30 °C in CM2GxYE medium (peptone - lOg/1, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0), and then the culture was diluted about 20 times with fresh CM2GxYE medium. The cells were grown with aeration at 30°C to an OD600 of «0.6 and ampicillin was added (10 μg/ml) and cultivated for 1 hour. The obtained cells were washed three times with ice-cold deionized H20. Electroporation was performed using 50 μΐ of cells and *^g of the plasmid pBS4SA2067 with the following parameters: 2.0 kV, 0,1 cm electrode gap. Cells after electroporation were incubated with 1 ml CM2GxYE medium at 30°C for 2 hours with shaking and then were plated onto 2LA- agar (peptone - lOg/1, yeast extract - lOg/1, NaCl - 5g/l, agar - 20g/l, pH is adjusted to 7.0) containing kanamycin (25 μg/ml) and grown at 30°C to select KmR recombinants for 48 h. The obtained integrants were also unable to grow on 2LA medium containing 10% sucrose.
1- 2. Verification of the NCel 2067 gene deletion by PCR
The mutants in which the NCgl 2067 gene was deleted were verified by PCR. Locus-specific primers PI (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) were used in PCR for the verification. The PCR product obtained in the reaction with the cells of the parental strain B. flavum B-6642 as the template, was -1.35 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template was -0.78 kbp in length. Deletion of NCgl_2067 gene_was also confirmed by the sequencing of the corresponding chromosomal DNA fragment. The mutant strain was given the name B. flavum B-6642ANCgl_2067.
Example 2. Construction of a strain with attenuated expression of NCgl 2066 gene
2- 1. Deletion of the NCsl 2066 gene
A strain in which the NCgl 2066 gene was deleted was constructed as the same manner as described in Example 1. The in-frame deletion of the NCgl 2066 gene was constructed. For this purpose, overlap extension PCR was conducted. First, a DNA fragment was obtained in PCR with primers NC2066F (SEQ ID NO: 15) and NC2066R1 (SEQ ID NO: 16) and chromosomal DNA of B. flavum B-6642 as the template. Second, a DNA fragment was obtained in PCR with oligonucleotides NC2066F1 (SEQ ID NO: 17) and NC2066R (SEQ ID NO: 18) and chromosomal DNA of B. flavum B-6642 as the template. After that, the two DNA fragments described above were used as the template in the first round of PCR with primers NC2066-SmF (SEQ ID NO: 19) and NC2066-SmR (SEQ ID NO: 20). After that, the obtained PCR product was purified and treated with endonuclease Smal, and then ligated into Smal-cleaved plasmid pBS4S to obtain pBS4SA2066.
Electrocompetent cells were prepared as follows: B. flavum B-6642 was grown overnight at 30 °C in CM2GxYE medium (peptone - lOg/l, yeast extract - 20g/l, NaCl - 5g/l, glucose - 5g/l, pH is adjusted to 7.0), and then the culture was diluted about 20 times with fresh CM2GxYE medium. The cells were grown with aeration at 30°C to an OD600 of «0.6 and ampicillin was added (10 μg/ml) and cultivated for 1 hour. The obtained cells were washed three times with ice-cold deionized H20. Electroporation was performed using 50 μΐ of cells and «5 μg of the plasmid pBS4SA2066 with the following parameters: 2.0 kV, 0.1 cm electrode gap. Cells after electroporation were incubated with 1 ml CM2GxYE medium at 30°C for 2 hours with shaking and then were plated onto 2LA-agar (peptone - lOg/1, yeast extract - lOg/1, NaCl - 5g/l, agar - 20g/l, pH is adjusted to 7.0) containing kanamycin (25 μg/ml) and grown at 30°C to select KmR recombinants for 48 h. The obtained integrants were also unable to grow on 2LA medium containing 10% sucrose.
2-2. Verification of the NCsl 2066 gene deletion by PCR
The mutants in which the NCgl_2066 gene was deleted were verified by PCR. Locus-specific primers NC2066F (SEQ ID NO: 15) and NC2066R (SEQ ID NO: 18) were used in PCR for the verification. The PCR product obtained in the reaction with the cells of parental strain B. flavum B-6642 as the template, was -2.12 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template was -1.15 kbp in length. Deletion of the NCgl_2066 gene_was also confirmed by the sequencing of the corresponding chromosomal DNA fragment. The mutant strain was given the name B. flavum B-6642ANCgl_2066.
Example 3. Production of L-glutamic acid and L-glutamine by B. flavum strain B- 6642ANCgl 2067
B. flavum strains, B-6642ANCgl_2067, and B-6642, were separately grown for 18- 24 hours at 30°C on 2LA-agar plates. Then, one loop of the cells was transferred into test tubes containing 2ml of fermentation medium. The fermentation medium contains (NH4)2S04 (70g/l), KH2P04 (2,5g/l), thiamine (350mg/l), biotin (lOOmg/l), MgS047H20 (0.4g/l), Mameno (soybean protein hydrolysate, 5.7mg/l), sucrose (120g/l), and CaC03 (50g/l). The pH is adjusted to 7.
Cultivation was carried out at 30°C for 48 hours with shaking. After the cultivation, the amount of L-glutamic acid (Glu) and L-glutamine (Gln)which are produced was determined by TLC (thin-layer chromatography) using the following mobile phase:
butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (1%) in acetone was used as a visualizing reagent. The spots containing L-glutamic acid and L-glutamine were cut out, and L-glutamic acid and L-glutamine were eluted with 0.5% water solution of CdCl2. The amounts were estimated spectrophotometrically at 540 nm. The results of two independent test tube fermentations are shown in Table 1. As follows from Table 1 , strain B-6642ANCgl_2067 with attenuated expression of NCgl_2067 gene produced higher amounts of L- glutamic acid and L-glutamine, as compared with the parent strain B-6642.
Table 1
Figure imgf000021_0001
Example 4. Production of L-glutamic acid and L-glutamine by B. flavum strain B- 6642A NCgl 2066
B. flavum strains, B-6642ANCgl_2066 and B-6642, were separately grown for 18- 24 hours at 30°C on 2LA-agar plates. Then, one loop of the cells was transferred into test tubes containing 2ml of fermentation medium. The fermentation medium contains (NH4)2S04 (70g/l), KH2P04 (2,5g/l), thiamine (350mg/l), biotin (lOOmg/l), MgS04 7H2O(0.4g/l), Mameno (5.7mg/l), sucrose (120g/l), and CaC03 (50g/l). The pH is adjusted to 7.
Cultivation was carried out at 30°C for 48 hours with shaking. After the cultivation, the amount of L-glutamic acid and L-glutamine which is produced was determined by TLC (thin-layer chromatography) using the following mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (1%) in acetone was used as a visualizing reagent. The spots containing L-glutamic acid and L-glutamine were cut out, L-glutamic acid and L-glutamine were eluted with 0.5% water solution of CdCl2, and the amounts were estimated spectrophotometrically at 540 ran.
The results of two independent test tube fermentations are shown in Table 2. As follows from Table 2, strain B-6642ANCgl_2066 with attenuated expression of
NCgl_2066 gene produced higher amounts of L-glutamic acid and L-glutamine, as compared with the parent strain B-6642.
Table 2
Figure imgf000021_0002
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.
Industrial Applicability
According to the present invention, an L-amino acid belonging to the glutamate family is efficently produced by a coryneform bacterium.

Claims

1. A coryneform bacterium which is able to produce an L-amino acid belonging to the glutamate family, wherein said bacterium has been modified to attenuate the expression of one or more gene(s) of the NCgl_2067-NCgl_2065 operon.
2. The bacterium according to claim 1, wherein said bacterium has been modified to attenuate the expression of the NCgl_2067 gene.
3. The bacterium according to claim 2, wherein said expression of the NCgl_2067 gene is attenuated by inactivation of the NCgl_2067 gene.
4. The bacterium according to claim 1 , wherein said bacterium has been modified to attenuate the expression of the NCgl_2066 gene.
5. The bacterium according to claim 4, wherein said expression of the NCgl_2066 gene is attenuated by inactivation of the NCgl_2066 gene.
6. The bacterium according to claim 1, wherein said bacterium belongs to the genus Corynebacterium or Brevibacterium.
7. The bacterium according to claim 6, wherein said bacterium is Brevibacterium flavum.
8. The bacterium according to any of claims 1 to 7, wherein said L-amino acid is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L- histidine, L-arginine, L-ornithine, and L-citrulline.
9. The bacterium according to claim 8, wherein said L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine.
10. A method for producing an L-amino acid belonging to the glutamate family comprising:
- cultivating the bacterium according to any of claims 1 to 9 in a medium, and
- collecting said L-amino acid from the medium.
11. The method according to claim 10, wherein said L-amino acid is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-histidine, L-arginine, L-ornithine, and L-citrulline.
12. The method according to claim 1 1, wherein said L-amino acid is selected from the group consisting of L-glutamic acid and L-glutamine.
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