WO2007069782A1 - L-amino acid producing bacterium and method of producing l-amino acid - Google Patents

L-amino acid producing bacterium and method of producing l-amino acid Download PDF

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WO2007069782A1
WO2007069782A1 PCT/JP2006/325452 JP2006325452W WO2007069782A1 WO 2007069782 A1 WO2007069782 A1 WO 2007069782A1 JP 2006325452 W JP2006325452 W JP 2006325452W WO 2007069782 A1 WO2007069782 A1 WO 2007069782A1
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gene
amino acid
dna
seq
protein
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PCT/JP2006/325452
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French (fr)
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Mayu Iyo
Ryo Takeshita
Shinichi Sugimoto
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Ajinomoto Co., Inc.
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Priority to EP06842964.6A priority Critical patent/EP1963486B1/en
Publication of WO2007069782A1 publication Critical patent/WO2007069782A1/en
Priority to US12/137,666 priority patent/US8008047B2/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
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to a method of producing an L-amino acid using a bacterium, and more specifically, to a method of producing L-lysine, L-threonine and L-glutamic acid.
  • L-lysine and L-threonine are useful as additives for animal feeds, components of health foods, amino acid infusion, and the like.
  • L-glutamic acid is useful as a food seasoning.
  • L-amino acids are industrially produced by fermentation methods using bacteria belonging to the genus Brevibacterium, Corynebacterium, Escherichia, and the like.
  • Examples of a method of producing L-lysine include the methods described in EP 0643135 B, EP 0733712 B, EP 1477565 A, EP 0796912 A, EP 0837134 A, WO 01/53459, EP 1170376 A, and WO 2005/010175.
  • bacterial strains isolated from nature or artificially mutated strains thereof can be used.
  • bacteria which have been modified by a recombinant DNA technique so that the activity of an L-amino acid biosynthetic enzyme is enhanced have been used.
  • L-lysine WO 97/23597
  • L-arginine U.S. Patent Publication No. 2003-0113899
  • enhancing expression of a gene involved in the uptake of a sugar which is a substrate in fermentation improves the L-amino acid-producing ability.
  • Examples of such a method include producing an L-amino acid using an Escherichia bacterium which has been modified to enhance expression of the ptsG gene (WO 03/04670), and producing an L-amino acid using an Escherichia bacterium modified to enhance expression of the ptsH gene, ptsl gene, or err gene (WO 03/04674).
  • the nhaA gene and nhaB gene each encode a membrane protein known as the Na + /H + antiporter (Biochim Biophys Acta. 2005 Sep 30; 1709 (3): 240-50.).
  • the nhaR gene is known to encode a regulator that positively regulates expression of the nhaA gene (EMBO J. 1997 Oct 1; 16(19): 5922-9.).
  • enhancing the activity of the Na + /H + antiporter has an effect on L-amino acid production has not been previously reported.
  • the chaA gene is a membrane protein which is known as a sodium-calcium/proton antiporter, but it has never been reported to have an Na + /H + antiporter function (Biochim Biophys Acta. 2002 Dec 2; 1556 (2-3): 142-8.).
  • the mdfA gene is known to function as a multidrug/chloramphenicol efflux transporter (J Biol Chem. 2004 Mar 5; 279(10): 8957-65), but it has never been reported to have an NaVH + antiporter function, and the involvement of these genes in the production and accumulation of L-amino acids has not been reported.
  • An object of the present invention is to provide a bacterium which is capable of effectively producing an L-amino acid and to provide a method for effectively producing an L-amino acid using the bacterium.
  • the inventors of the present invention have made extensive studies to solve the above-mentioned object. As a result, they have found that L-amino acid productivity is improved by amplifying genes that encode a Na /H antiporter or a gene that positively regulates Na + /H + antiporter activity in an L-amino acid-producing bacterium. Moreover, they have found that enhancing the Na + /H + antiporter activity in a bacterium results in efficient production of an L-amino acid, and thereby the present invention has been accomplished. [0009]
  • nhaA gene encodes a protein selected from the group consisting of:
  • nhaB gene encodes a protein selected from the group consisting of:
  • (D) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 4, and has Na + /H + antiporter activity.
  • nhaR gene encodes a protein selected from the group consisting of:
  • (G) a protein comprising the amino acid sequence of SEQ ID NO: 24; and (H) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 24, and has Na + /H + antiporter activity.
  • (J) a protein comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 26, and has Na + /H + antiporter activity.
  • nhaA gene is a DNA selected from the group consisting of:
  • nhaB gene is a DNA selected from the group consisting of:
  • nhaR gene is a DNA selected from the group consisting of:
  • chaA gene is a DNA selected from the group consisting of:
  • mdfA gene is a DNA selected from the group consisting of:
  • L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
  • It is a further object of the present invention to provide a method of producing an L-amino acid comprising culturing the bacterium as described above in a medium to produce and accumulate an L-amino acid in the medium or cells of the bacterium and collecting the L-amino acid from the medium or the cells.
  • L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
  • the bacterium of the present invention belongs to the Enterobacte ⁇ aceae family and has an L-amino acid-producing ability. This bacterium also is modified so that the NaVH + antiporter activity is enhanced.
  • the term "L-amino acid-producing ability" means an ability to produce and accumulate an L-amino acid in a medium or bacterial cells when the bacterium of the present invention is cultured in the medium.
  • the bacterium of the present invention may produce a plurality of L-amino acids. The ability may be native to the bacterium, or the ability may be obtained by modifying the bacterium by mutation and or with recombinant DNA techniques. [0013]
  • L-amino acid is not particularly limited, but examples thereof include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline, aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and L-glycine, hydroxy monoaminocarboxylic acid such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine, and acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine, and L-asparagine.
  • the bacterium of the present invention may produce two or more kinds of amino acids. [0014] ⁇ 1-1>
  • the bacterium is not limited thereto, as long as it has an L-amino acid-producing ability.
  • Bacteria belonging to the Enterobacteriaceae family can be used as the parent strain of the bacterium of the present invention.
  • Other examples of bacteria belonging to the Enterobacteriaceae family include y-Proteobacteria such as Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, and Morganella.
  • Escherichia bacteria reported in Neidhardt et al. ((Backmann, B.J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K- 12, p. 2460-2488. Table 1.
  • Escherichia coli can be utilized.
  • Examples of a wild-type strain of Escherichia coli include the K- 12 strain or derivatives thereof, Escherichia coli MG 1655 strain (ATCC No. 47076), and W3110 strain (ATCC No. 27325). These strains are available from the American Type Culture Collection (ATCC) (Address: P.O. Box 1549, Manassas, VA 20108, 1, United States of America). [0016]
  • Enterobacter bacteria examples include Enterobacter agglomerans and Enterobacter aerogenes
  • Pantoea bacteria is Pantoea ananatis
  • Enterobacter agglomerans has been reclassif ⁇ ed in some cases as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like based on an analysis of the nucleotide sequence of 16S rRNA. Therefore, bacteria to be used in the present invention may belong to either the genus Enterobacter or the genus Pantoea as long as they are classified in the Enterobacteriaceae family.
  • Pantoea ananatis When Pantoea ananatis is bred using genetic engineering techniques, Pantoea ananatis AJ 13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ 13601 strain (FERM BP-7207), derivatives thereof, and the like, may be used. These strains were identified and deposited as Enterobacter agglomerans when they were isolated, but as described above, these strains have been reclassified as Pantoea ananatis based on an analysis of the nucleotide sequence of 16S rRNA. [0017]
  • the L-amino acid-producing ability can be imparted to a parent strain as described above, as follows.
  • L-amino acid-producing ability In order to impart the L-amino acid-producing ability, methods which have been conventionally adopted in the breeding of Escherichia bacteria or the like, may be used, such as by acquiring nutrient-auxotrophic mutant strains, analogue resistant strains, or metabolic regulation mutant strains, or by creating a recombinant strain in which expression of an L-amino acid biosynthetic enzyme is enhanced (Amino Acid Fermentation, Japan Scientific Societies Press, first edition publication: May 30, 1986, p.77 to 100).
  • properties such as nutrient- ' auxotrophy, analogue-resistance, and metabolic regulation may be imparted alone or in combination with the imparting of L-amino acid-producing ability.
  • L-amino acid biosynthetic enzymes may be enhanced. Furthermore, imparting of such properties as nutrient-auxotrophy, analogue-resistance and metabolic regulation mutation may be combined with enhancing the expression of L-amino acid biosynthetic enzymes.
  • Nutrient-auxotrophic mutant strains, L-amino acid-analogue resistant strains, and metabolic regulation mutant strains that have an L-amino acid-producing ability can be obtained as follows.
  • a parent strain or a wild-type strain is mutated by a typical mutation treatment, such as irradiation with X-ray or ultraviolet ray or by treating with a mutagen, including N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS), followed by selection of strains that exhibit nutrient-auxotrophy, analogue-resistance or a metabolic regulation mutation and have an L-amino acid-producing ability.
  • NTG N-methyl-N'-nitro-N-nitrosoguanidine
  • EMS ethylmethanesulfonate
  • L-lysine analogue examples include oxalysine, lysinehydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), ⁇ -methyllysine, ⁇ -chlorocaprolactam, and norleucine.
  • L-arginine analogue examples include arginine hydroxamate, homoarginine, D-arginine, and canavanine.
  • an L-lysine analogue resistant strain or metabolic regulation mutant strain having an L-lysine-producing ability examples include Escherichia coli AJl 1442 strain (FERM BP-1543, NRRL B-12185; JP 56-18596 A and U.S. Patent No. 4346170) and Escherichia coli VL611 strain (JP 2000- 189180 A).
  • WC 196 strain (WO 96/17930) may be used as an L-lysine producing strain of Escherichia coli.
  • WC 196 strain has been obtained by imparting AEC (S-(2-aminoethyl)-cysteine)-resistance to W3110 strain which was derived from Escherichia coli K- 12 strain.
  • the WC 196 strain was named Escherichia coli AJ 13069 strain and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan) on December 6, 1994 and given an accession number of FERM P- 14690, and the deposit was then converted to an international deposit under the provisions of Budapest Treaty on September 29, 1995 and given an accession number of
  • An L-amino acid-producing ability can also be imparted by enhancing the expression of a gene encoding an L-amino acid biosynthetic enzyme.
  • an L-lysine-producing ability may be imparted by enhancing the activities of dihydrodipicolinate synthase and aspartokinase. That is, a gene fragment encoding dihydrodipicolinate synthase and a gene fragment encoding aspartokinase are ligated to a vector which functions in a host bacterium to be used for L-lysine production.
  • the vector is preferably a multi-copy vector, and the obtained vector DNA is used to transform a host bacterium.
  • dihydrodipicolinate synthase aspartokinase, and aspartokinase III are abbreviated as DDPS, AK, and AKIIl, respectively.
  • the gene encoding DDPS and the gene encoding AK are not particularly limited as long as the DDPS and AK activities are expressed in the host bacterium, and examples thereof include the genes of Escherichia coli, Methylophilus methylotrophus, Corynebacterium glutamicum, and the like. Nucleotide sequences of the DDPS gene derived from an Escherichia bacterium (dapA, Richaud, F. et al. J. Bacterid., 297 (1986)) and the AKIII gene derived from an Escherichia bacterium (lysC, Cassan, M., Parsot, C, Cohen, GN. and Patte, J.C., J.
  • mutant DDPS which is not subject to feedback inhibition by L-lysine
  • DNA encoding the mutant DDPS is referred to as "mutant dapA or dapA*”.
  • mutant AKIII derived from Escherichia coli which is not subject to feedback inhibition by L-lysine is referred to as "mutant AKIN”
  • mutant lysC the DNA encoding the mutant AKIII
  • DDPS and AK which can be used in the present invention are not necessarily these type of mutants since DDPS derived from Corynebacterium bacterium is not subject to feedback inhibition.
  • An example of a DNA encoding mutant DDPS not subject to feedback inhibition by L-lysine includes a DNA encoding a DDPS which has an amino acid sequence in which the histidine at position 118 is substituted with tyrosine.
  • an example of a DNA encoding mutant AKIII not subject to feedback inhibition by L-lysine includes a DNA encoding an AKIII having an amino acid sequence in which the threonine at position 352, the glycine at position 323, and the methionine at position 318 are substituted with isoleucine, asparagine and isoleucine, respectively (U.S. Patent No. 5661012 and U.S. Patent No. 6040160).
  • Such mutant DNAs can be obtained by a site-specific mutation using PCR or the like.
  • Enhancing expression of the L-lysine biosynthetic genes as described above can be attained by transformation or homologous recombination using a plasmid or the like, in the same way as the nhaA gene, nhaB gene, nhaR gene, chaA gene, and mdfA gene described below.
  • Wide host-range plasmids RSFD80, pCABl, and pCABD2 are known to contain a mutant dapA gene encoding a mutant DDPS and a mutant lysC gene encoding a mutant AKIII (U.S. Patent No. 6040160).
  • Escherichia coli JM109 strain transformed with RSFD80 was named AJ12396 (U.S. Patent No.
  • RSFD80 can be obtained from AJl 2396 strain by a conventional method.
  • An L-lysine-producing ability can also be imparted by enhancing expression of genes encoding enzymes, other than DDPS and AK, which are involved in biosynthesis of L-lysine.
  • enzymes include enzymes in the diaminopimelate pathway such as dihydrodipicolinate reductase (dapB: hereinafter, the words in parentheses represent the gene names), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (WO96/40934), phosphoenolpyruvate carboxylase (pepC) (JP 60-87788 A), aspartate aminotransferase (aspC) (JP 06-102028 B), diaminopimelate epimerase gene (dapF) (JP 2003-135066), aspartate semialdehyde dehydrogenase (asd) (WO 00/61723), tetrahydrodipicolinate succin
  • genes other than L-lysine biosynthetic genes may also be enhanced to impart L-lysine-producing ability, and examples of such genes include those encoding enzymes involved in sugar uptake, sugar metabolism (glycolytic pathway), the TCA cycle, the pentose phosphate cycle, complementary pathway, and energy metabolism.
  • expression of genes that impart an amino acid-resistance to a host bacterium, genes encoding amino acid-export enzymes, and genes encoding enzymes involved in uptake of by-products may be enhanced.
  • Genes involved in sugar metabolism include genes encoding enzymes in the glycolytic pathway or enzymes involved in sugar uptake. Examples thereof include the glucose-6-phosphate isomerase gene (pgi; WO 01/02542), phosphoenolpyruvate synthase gene (pps; EP 877090 A), phosphoglucomutase gene (pgm; WO 03/04598), fructose bisphosphate aldolase gene (fbp; WO 03/04664), pyruvate kinase gene (pykF; WO 03/008609), transaldolase gene (talB; WO 03/008611), fumarase gene (fum; WO 01/02545), phosphoenolpyruvate synthase gene (pps; EP 877090 A), non-PTS sucrose uptake gene (esc; EP 149911 A), sucrose-assimilating gene (scrAB operon; WO 90/04636), PTS glucose uptake gene (pt
  • genes encoding TCA cycle enzymes include citrate synthase gene (git A; WO 03/008607), isocitrate dehydrogenase gene (icd; WO 03/008607), 2-ketoglutarate dehydrogenase gene (sucAB; WO 03/008614), and succinate dehydrogenase gene (sdh; WO 01/02544), glutamate dehydrogenase gene (gdh; U.S.Patent No 6,004,773). [0030]
  • genes encoding pentose phosphate cycle enzymes include glucose-6-phosphate dehydrogenase gene (zwf; WO 03/008607) and ribose-5-phosphate isomerase gene (rpiB; WO 03/008607).
  • genes encoding anaplerotic pathway include the phosphoenolpyruvate carboxylase gene (pepC; U.S. Patent No. 5,876,983), pyruvate carboxylase gene (pyc; EP 1092776), malate dehydrogenase gene (mdh; WO 01/02546), and pyruvate decarboxylase gene (pckA; WO 04/090125).
  • pepC phosphoenolpyruvate carboxylase gene
  • pyc pyruvate carboxylase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh malate dehydrogenase gene
  • mdh
  • genes encoding enzymes involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and cytochromoe bo type oxidase gene (cyoB; EP 1070376).
  • genes that impart L-amino acid-resistance include the rhtB gene (U.S. Patent No. 6,887,691), rhtC gene (EP 1013765), yedA gene (EP 1449917), yddG gene (EP 1449918), ygaZH gene (EP 1239041), yahN, yfiK, and yeaS genes (EP 1016710), rhtA gene (Res Microbiol. 2003 Mar; 154(2): 123-35.), and ybjE gene (WO 2005/073390). [0033]
  • an activity of an enzyme that catalyzes a reaction which branches off from L-lysine biosynthetic pathway and produces a compound other than L-lysine may be decreased or may be made deficient.
  • an enzyme include homoserine dehydrogenase, lysine decarboxylase, and malic enzyme, and strains in which activities of such enzymes are decreased or deficient are described in WO 95/23864, WO 96/17930, WO 2005/010175, and the like.
  • lysine decarboxylases are encoded by a cadA gene (Genbank Accession No.
  • NP_418555, SEQ ID NO: 19) and ldcC gene (Genbank Accession No. NP_414728, SEQ ID NO: 21) (WO 96/17930), so these genes may be disrupted to enhance L-lysine-producing ability.
  • DNA molecules homologous to the cadA gene and ldcC gene may be used as long as they can cause homologous recombination with the cad A gene and ldcC gene on the chromosome of a host bacterium.
  • a DNA molecule homologous to the cadA gene may hybridize to a complementary strand of SEQ ID NO: 19 under stringent conditions
  • a DNA molecule homologous to the ldcC gene may hybridize to a complementary strand of SEQ ID NO: 21 under stringent conditions.
  • Activities of these enzymes can be decreased or eliminated by introducing a mutation to the genes encoding the enzymes on the chromosome using a known mutation treatment, to thereby decrease or eliminate the activities of the enzymes in a cell.
  • decreasing or eliminating the activities of the enzymes can be attained by disrupting the genes encoding the enzymes on the chromosome by gene recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence.
  • SD Shine-Dalgarno
  • this can also be attained by introducing an amino acid substitution (missense mutation) to the region encoding the enzymes on the chromosome, introducing a stop codon (nonsense mutation), introducing a frameshift mutation that adds or deletes one or two nucleotides, or deleting part of the gene (Journal of biological Chemistry 272: 8611-8617 (1997).
  • the activities of the enzymes can also be decreased or eliminated by constructing a mutant gene encoding a mutant enzyme which has a deletion in the coding region, and then replacing the normal gene on the chromosome with the mutant gene by homologous recombination, or introducing the mutant gene using a transposon or an IS factor.
  • the following gene recombination method can be used to introduce a mutation that decreases or eliminates the activities of the above-mentioned enzymes.
  • a mutant gene is prepared by modifying a partial sequence of a target gene so that it does not encode an enzyme that can function normally. Then, a bacterium belonging to the Enterobacteriaceae family is transformed with a DNA containing the mutant gene to cause recombination of a gene on the bacterial chromosome with the mutant gene, thereby substituting the target gene on the chromosome with the mutant gene.
  • Examples of this type of gene substitution using homologous recombination include the method using a linear DNA called "Red-driven integration" (Datsenko, K.A, and Wanner, B. L.
  • Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E.
  • E. coli TDH-6/pVIC40 VKPM B-3996
  • E.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371 E. coli 472T23/pYN7 (ATCC 98081)
  • the strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine.
  • the strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSFlOlO-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine.
  • the strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Nagatinskaya Street 3 -A, 117105 Moscow, Russian Federation) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on April 7, 1987 under the accession number B-3996. [0040]
  • E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain to derive L-threonine-producing bacteria of the present invention.
  • the strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage Cl repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40.
  • the strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on May 3, 1990 under accession number of VKPM B-5318. [0041]
  • the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes:
  • mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
  • the sequence of the thrA gene of Escherichia coli which encodes aspartokinase homoserine dehydrogenase I has been elucidated (nucleotide positions 337 to 2799, GenBank accession NC 000913.2, gi: 49175990).
  • the thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12.
  • the nucleotide sequence of the thrB gene of Escherichia coli which encodes homoserine kinase has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990).
  • the thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K- 12.
  • the nucleotide . sequence of the thrC gene of Escherichia coli which encodes threonine synthase has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990).
  • the thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function together as a single threonine operon.
  • the attenuator region which affects the transcription can be removed from the operon (WO2005/049808, WO2003/097839). [0043]
  • a thrA gene which has been mutated so that it encodes aspartokinase homoserine dehydrogenase I which is resistant to feedback inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40, which is present in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371. [0044]
  • the rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system.
  • the rhtA gene is identical to ORFl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and located between the pexB and ompX genes.
  • the sequence expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine).
  • the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).
  • the nucleotide sequence of the asd gene of E.coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) by utilizing primers based on the nucleotide sequence of the gene.
  • the asd genes from other microorganisms can be obtained in a similar marine (US 2005-0124048) .
  • nucleotide sequence of the aspC gene of E. col '/ has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC_000913.1, gi:16128895), and can be obtained by PCR.
  • the aspC genes from other microorganisms can be obtained in a similar manner (WO03/072786).
  • Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM 15 which has been transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601), E. coli W3110 which over-expresses genes which encode proteins suitable for secreting toxic substances (U.S. Patent No. 5,972,663), E. coli strains with decreased cysteine desulfohydrase activity (JPl 1155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for the cysteine regulon encoded by the cysB gene (WO0127307A1), and the like. [0048] L-leucine-producing bacteria
  • parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including ⁇ -2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the genetic engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like. [0049]
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis.
  • these genes include those of the leu ABCD operon, which preferably include a leuA gene which has been mutated so that it encodes isopropylmalate synthase which is resistant to feedback inhibition by L-leucine (US Patent 6,403,342).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acids from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2). [0050] L-histidine-producing bacteria
  • Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U,S. Patent No. 6,258,554) and the like. [0051]
  • Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced.
  • Examples of these L-histidine-biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisl), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-arninoirnidazole carboxamide ribotide isomerase (his A), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth. [0052]
  • strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E.coli strains transformed with rht, a gene for an amino acid-export (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL- 1 ,2,4-triazole-3 -alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth. [0053]
  • Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC + (EP 1172433).
  • E. coli VL334 (VKPM B- 1641) is auxotrophic for L-isoleucine and L-threonine and is mutated in thrC and ilvA genes (U.S. Patent No. 4,278,765).
  • a wild-type allele of the thrC gene was transferred by general transduction using a bacteriophage Pl grown on the wild-type E. coli strain Kl 2 (VKPM B-7).
  • an L-isoleucine auxotrophic strain VL334thrC + (VKPM B-8961) was obtained. This strain is able to produce L-glutamic acid.
  • parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced.
  • the enzymes involved in L-glutamic acid biosynthesis include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), en
  • strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A, EP955368A, and EP952221A.
  • Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid, and branches off from an L-glutamic acid biosynthesis pathway.
  • Examples of such enzymes include isocitrate lyase, ⁇ -ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, and glutamate decarboxylase.
  • strains include the following:
  • E. coli W3110sucA::Kmr is obtained by disrupting the ⁇ -ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in ⁇ -ketoglutarate dehydrogenase.
  • L-glutamic acid-producing bacterium examples include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the ⁇ -ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (F ⁇ RM BP-5807) (U.S. Patent No. 5.908,768), F ⁇ RM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (F ⁇ RM BP-5565) (U.S. Patent No. 6,110,714), and the like. [0060]
  • L-glutamic acid-producing bacteria examples include mutant strains belonging to the genus Pantoea which are deficient in ⁇ -ketoglutarate dehydrogenase activity or have a decreased ⁇ -ketoglutarate dehydrogenase activity, and can be obtained as described above.
  • Such strains include Pantoea ananatis AJl 3356 (U.S. Patent No. 6,331 ,419).
  • Pantoea ananatis AJ 13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615.
  • Pantoea ananatis AJ13356 is deficient in ⁇ -ketoglutarate dehydrogenase activity as a result of disruption of the ⁇ KGDH-El subunit gene (sucA).
  • the above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomercms AJl 3356.
  • Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth.
  • AJ 13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.
  • Examples of parent strains for deriving L-phenylalanine-prod ⁇ cing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E.coli AJ12739 (tyrA::Tnl0, tyrR) (VKPM B-8197); £.c ⁇ /z HW1089 (ATCC 55371) harboring the pheA34 gene (U.S. Patent No. 5,354,672); E.coli MWEC 101 -b (KR8903681); E.coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No. 4,407,952).
  • E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl).
  • L-phenylalanine producing bacteria belonging to the genus Escherichia which have an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al). [0062] L-tryptophan-producing bacteria
  • parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E.
  • coli SV 164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase resistant to feedback inhibition by serine and a trpE allele encoding anthranilate synthase resistant to feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGXl 7 (pGX44) (NRRL B-12263) and AGX6( ⁇ GX50)aroP (NRRL B- 12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E.
  • parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and tryptophan synthase (trpAB) are enhanced.
  • the anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so a mutation which results in desensitizes the feedback inhibition may be introduced into these enzymes.
  • Specific examples of strains having such a mutation include a E.
  • E. coli SV 164 which harbors desensitized anthranilate synthase and a strain obtained by transforming the plasmid pGH5 into E. coli SV 164 (WO 94/08031), which contains a serA gene which has been mutated so that it encodes feedback-desensitized phosphoglycerate dehydrogenase.
  • Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains transformed with the tryptophan operon which contains a gene encoding desensitized anthranilate synthase (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614).
  • L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA).
  • the tryptophan synthase consists of ⁇ and ⁇ subunits which are encoded by trpA and trpB, respectively.
  • L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).
  • parent strains for deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline (EP 1172433).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis.
  • examples of such preferred genes for L-proline producing bacteria include the proB gene coding for glutamate kinase which is desensitized to feedback inhibition by L-proline (DE Patent 3127361).
  • the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from the bacterial cell.
  • genes are exemplified by the b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).
  • Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B- 12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15th Miami winter symposium, 1983, p.34), and the like. [0068] L-arginine-producing bacteria
  • parent strains for deriving L-arginine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N-acetylglutamate synthase ( Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 170361 Al), and the like. [0069]
  • Examples of parent strains for deriving L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced.
  • Examples of the L-arginine biosynthetic enzymes include N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argj), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase (carAB).
  • argC N-acetylglutamyl phosphate reductase
  • argj ornithine ace
  • Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased. Examples of parent strains for deriving L-valine-producing bacteria of the present invention also include mutants of amino-acyl t-RNA synthetase (U.S. Patent No.
  • E. coli VL1970 which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used.
  • E coli VL 1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) ( Russian, 113545 Moscow, 1 Dorozhny Proezd.) on June 24, 1988 under accession number VKPM B-4411. [0071]
  • mutants requiring lipoic acid for growth and/or lacking H + -ATPase can also be used as parent strains (WO96/06926).
  • parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A).
  • the bacterium of the present invention can be obtained by modifying a bacterium having an L-amino acid-producing ability as described above so that the Na 4 VH + antiporter activity is enhanced.
  • L-amino acid-producing ability may be imparted after the bacterium is modified so that the Na + /H + antiporter activity is enhanced.
  • the Na + /H + antiporter activity may be enhanced by increasing the expression of a gene encoding a Na + /H + antiporter or a gene that positively regulates the NaVH + antiporter activity.
  • Gene expression can be enhanced by modifying an expression regulatory sequence such as a promoter of an endogenous gene, or enhancing expression of an exogenous gene by introducing a plasmid containing the gene, or the like. These methods may be combined. [0074]
  • NaVH + antiporter refers to a membrane protein involved in uptake of H + into a cytoplasm and export of Na +
  • the phrase "modifying so that the NaVH + antiporter activity is enhanced” includes when the number of NaVH + antiporter molecules per cell increases and when the Na + /H + antiporter activity per molecule is increased as compared to a wild-type strain or an unmodified strain.
  • the bacterium is modified so that NaVH + antiporter activity is preferably not less than 150%, more preferably not less than 200%, further more preferably not less than 300% per bacterial cell as compared to a wild-type strain or an unmodified strain.
  • examples of a wild-type bacterium belonging to the Enterobacteriaceae family to be used as a control include Escherichia coli MGl 655 strain (ATCC No. 47076), W3110 strain (ATCC No. 27325), and Pantoea ananatis AJ13335 strain (FERM BP-6615). [0075]
  • the NaVH + antiporter activity can be enhanced increasing expression of a gene encoding a NaVH + antiporter or a gene encoding a protein that positively regulates Na + /H + antiporter activity.
  • the increased NaVH + antiporter activity can be confirmed by measuring the increase in expression of the gene encoding the NaVH + antiporter.
  • the increased expression can be confirmed by comparing the amount of NaVH + antiporter mRNA in the bacterium of the present invention with a wild-type or unmodified strain. Examples of a method of measuring the amount of expression include Northern hybridization and RT-PCR (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).
  • the expression may be at any level as long as the expression is increased as compared to a wild-type or unmodified strain, and for example, the amount of expression is preferably increased not less than 1.5 -fold, more preferably not less than 2-fold, and more preferably not less than 3-fold as compared to a wild-type or unmodified strain.
  • enhancement of the NaVH + antiporter activity can also be confirmed by an increase in the amount of Na + /H + antiporter protein as compared to a wild-type or unmodified strain, and the amount of the protein can be evaluated, for example, by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).
  • Examples of a gene that encodes the Na + /H + antiporter include the nhaA gene, nhaB gene, chaA gene, and mdfA gene, and homologues thereof.
  • examples of these genes from Escherichia coli include the nhaA gene of SEQ ID NO: 1 (nucleotide numbers 17489-18655 of GenBank Accession No. NC 000913), nhaB gene of SEQ ID NO: 3 (a complementary strand of nucleotide numbers 1232399-1233940 of GenBank Accession No. NC 000913), chaA gene of SEQ ID NO: 23 (a complementary strand of nucleotide numbers 1269972..1271072 of GenBank Accession No.
  • nhaA gene and nha B gene derived from other bacteria include a gene that is derived from Yersinia pestis strain CO92 and encodes a NhaA protein having the amino acid sequence of SEQ ID NO: 7, and a gene that is derived from Salmonella enterica CTl 8 strain and encodes a NhaB protein having the amino acid sequence of SEQ ID NO: 8.
  • Homologues of the above-mentioned genes can be obtained by cloning, based on homologies to the above-listed genes, from coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium lactofermentum, y-Proteobacteria such as those belonging to the genus Escherichia, Enterobacter, Klebsiella, Serratia, Erwinia, or Yersinia, Pseudomonas bacteria such as Pseudomonas aeruginosa, and Mycobacterium bacteria such as Mycobacterium tuberculosis.
  • coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium lactofermentum
  • y-Proteobacteria such as those belonging to the genus Escherichia, Enterobacter, Klebsiella, Serratia, Erwinia, or Yersinia
  • Pseudomonas bacteria
  • Homologue genes may be amplified by using, for example, synthetic oligonucleotides shown in SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, or SEQ ID NOS: 13 and 14 as primers.
  • the homologies of amino acid sequences and nucleotide sequences can be determined by using the algorithm BLAST created by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or the algorithm FASTA created by Pearson (Methods Enzymol., 183, 63 (1990)). Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (www.ncbi.nlm.nih.gov). [0078]
  • Examples of a gene that encodes a protein that positively regulates the Na'/tT antiporter activity include a nhaR gene or a homologue thereof, and examples of nhaR gene from Escherichia coli include the nhaR gene of SEQ ID NO: 5 (nucleotide numbers 18715-19620 of GenBank Accession No. NC_000913).
  • Homologues of a gene encoding a Na + /H + antiporter or of a gene encoding a protein that positively regulates Na + /H + antiporter activity include mutant genes derived from another bacteria or a natural or artificial mutant genes, which show high structural similarity to a gene such as nhaA, nhaB, chaA, mdfA, and nhaR of Escherichia coli and functions to enhance the Na 4 VH + antiporter activity when introduced or amplified in a host.
  • Each of the homologue genes encoding the Na + /H + antiporter include genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 98% to each of the entire amino acid sequences of SEQ ID NO: 2, 4, 24 or 26, and encode a protein which functions as a Na + /H + antiporter. Whether a protein functions as a Na + /H + antiporter can be confirmed by expressing the genes in a host cell and examining the transport of Na + and H + across a membrane (Biochim Biophys Acta. 2005 Sep 30; 1709(3): 240-50.).
  • Homologues of a gene encoding a protein that positively regulates the Na + /H + antiporter activity include genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 98% to the entire amino acid sequence of SEQ ID NO: 6, and encode a protein capable of increasing expression amount of Na + /H + antiporter gene, in particular, nhaA gene.
  • the increase in the expression of the Na + /H + antiporter gene can be confirmed by expressing the nhaR gene in a host cell and determining the transcription amount of the nhaA gene (EMBO J. 1997 Oct 1 ; 16(19): 5922-9.).
  • nhaA gene, nhaB gene, chaA gene, and mdfA gene to be used in the present invention are not limited to wild-type genes and may be mutant or artificially modified genes that encode a protein having an amino acid sequence of SEQ ID NO: 2, 4, 24, or 26 and which may include substitution, deletion, insertion, addition of one or several amino acids at one or a plurality of positions, as long as the function of the NhaA protein, NhaB protein, ChaA protein, or MdfA protein encoded by these genes, that is, a Na + /H + antiporter function, is maintained.
  • nhaR gene to be used in the present invention is not limited to a wild-type gene and may be a mutant or an artificially modified gene that encodes a protein having an amino acid sequence of SEQ ID NO: 6 including substitution, deletion, insertion, addition of one or several amino acids at one or a plurality of positions as long as the function of NhaR protein encoded by the gene, that is, the ability to increase expression of the Na + /H + antiporter is maintained.
  • the term "one or several" specifically means 1 to 20, preferably 1 to 10, and more preferably 1 to 5, although this determination depends on the position in the protein's tertiary structure or the types of amino acid residues in the protein.
  • the above-mentioned substitution is preferably a conservative substitution, which may include substitutions between aromatic amino acids such as substitution among Phe, Trp and Tyr, substitution between hydrophobic amino acids such as substitution among Leu, He and VaI, substitution between polar amino acids such as substitution between GIn and Asn, substitution between basic amino acids such as substitution among Lys, Arg and His, substitution between acidic amino acids such as substitution between Asp and GIu, substitution between hydroxyl group-containing amino acids such as substitution between Ser and Thr.
  • conservative substitutions include substitution of Ser or Thr with ala; substitution of GIn, His or Lys with Arg; substitution of GIu, GIn, Lys, His or Asp with Asn; substitution of Asn, GIu or GIn with Asp; substitution of Ser or Ala with Cys; substitution of Asn, GIu, Lys, His, Asp or Arg with GIn; substitution of GIy, Asn, GIn, Lys or Asp with GIu; substitution of Pro with GIy; substitution of Asn, Lys, GIn, Arg or Tyr with His; substitution of Leu, Met, VaI or Phe with He; substitution of He, Met, VaI or Phe with Leu; substitution of Asn, GIu, GIn, His or Arg with Lys; substitution of He, Leu, VaI or Phe with Met; substitution of Trp, Tyr, Met, He or Leu with Phe; substitution of Thr or Ala with Ser; substitution of Ser or Ala with Thr; substitution of Phe or Tyr with Tr
  • the above-mentioned amino acid substitution, deletion, insertion, addition or inversion may be a result of a naturally-occurring mutation (mutant or variant) due to an individual difference, a difference of species of a bacterium harboring the nhaA gene, nhaB gene, chaA gene, mdfA gene, or nhaR gene.
  • each of the nhaA gene, nhaB gene, chaA gene and mdfA gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 1, 3, 23, or 25 or a probe that can be prepared from each of the sequences under stringent conditions and encodes a protein having the Na + /H + antiporter activity.
  • the nhaR gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 5 or a probe that can be prepared from the sequence under stringent conditions and encodes a protein having a function to increase expression of Na + /H + antiporter.
  • stringent conditions refers to conditions where a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • conditions having high homology for example, at least 80%, preferably 90%, more preferably 95%, and further more preferably 98% homology hybridize with each other and DNAs having homology less than the value do not hybridize with each other; and specifically include washing typical of Southern hybridization, e.g., washing at 60°C, I xSSC, 0.1% SDS, preferably 60°C, 0.1 *SSC, 0.1% SDS, more preferably 68°C, 0.1 xSSC, 0.1% SDS, once or preferably twice or three times.
  • washing typical of Southern hybridization e.g., washing at 60°C, I xSSC, 0.1% SDS, preferably 60°C, 0.1 *SSC, 0.1% SDS, more preferably 68°C, 0.1 xSSC, 0.1% SDS, once or preferably twice or three times.
  • nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be increased by, for example, increasing the copy number of the genes in a cell using a gene recombination technique.
  • a DNA fragment containing the gene is ligated to a vector that functions in the host bacterium, preferably a multi-copy vector, to thereby prepare a recombinant DNA, and the recombinant DNA is used to transform the host bacterium.
  • the nhaA gene, nhaB gene, and nhaR gene can be obtained by PCR (polymerase chain reaction; White, T.J. et al., Trends Genet. 5, 185 (1989)) using primers based on the nucleotide sequence of SEQ ID NOS: 1, 3 or 5, for example, primers of SEQ ID NOS: 9 and 10, 11 and 12 or 13 and 14 and a chromosomal DNA of Escherichia coli as a template.
  • the nhaA gene, nhaB gene, and nhaR gene from another bacterium can also be obtained by PCR from the chromosomal DNA or genomic DNA library of the bacterium using, as primers, oligonucleotides prepared based on the known sequences of the nhaA gene, nhaB gene or nhaR gene of the bacterium or of the nhaA gene, nhaB gene or nhaR gene of another kind of bacterium, or the amino acid sequence of the NhaA protein, NhaB protein, or NhaR protein; or hybridization using an oligonucleotide prepared based on the sequence as a probe.
  • a chromosomal DNA can be prepared from a bacterium that serves as a DNA donor by the method of Saito and Miura (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual for Biotechnology, edited by The Society for Biotechnology, Japan, p97-98, Baifukan Co., Ltd., 1992) or the like.
  • the chaA gene and mdf gene can also be obtained in the same way.
  • a recombinant DNA is prepared by ligating the nhaA gene, nhaB gene, chaA gene, mdfA gene or nhaR gene which have been amplified by PCR to a vector DNA which is capable of functioning in the host bacterium.
  • the vector capable of functioning in the host bacterium include vectors autonomously replicable in the host bacterium.
  • Examples of a vector which is autonomously replicable in Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSFlOlO (Gene vol. 75(2), p271-288, 1989), pBR322, pMW219, pMW119 (pMW is available form Nippon Gene Co., Ltd.), pSTV28, and pSTV29 (Takara Bio Inc.).
  • a phage DNA vector can also be used.
  • the vector is digested with a restriction enzyme corresponding to a recognition site in the terminus of a DNA fragment containing the nhaA gene, nhaB gene, chaA gene, mdfA gene or nhaR gene. Ligation is generally performed using a ligase such as T4 DNA ligase.
  • a ligase such as T4 DNA ligase.
  • the thus-prepared recombinant DNA is introduced into a bacterium in accordance with a conventional transformation method.
  • the method include electroporation (Canadian Journal of Microbiology, 43. 197 (1997)).
  • Bacillus subtilis Uncan, C. H., Wilson, GA and Young, RE, Gene, 1, 153 (1977)
  • the copy number of the nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can also be increased by introducing multiple copies of the genes into the chromosomal DNA of a bacterium.
  • Introduction of multiple copies of the genes into the chromosomal DNA of a bacterium can be attained by homologous recombination using a target sequence present on the chromosomal DNA in multiple copies.
  • a sequence present on a chromosomal DNA in multiple copies may be a repetitive DNA or an inverted repeat present on the edge of a transposing element.
  • nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be introduced into the chromosomal DNA by inserting the genes into a transposon, and transferring it so that multiple copies of the gene are integrated into the chromosomal DNA. Integration of these genes into the chromosome can be confirmed by Southern hybridization using a portion of the genes as a probe. [0089]
  • nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene may be enhanced by, as described in WO 00/18935, substituting an expression regulatory sequence such as a promoter of the genes on a chromosomal DNA or of the genes on a plasmid with a stronger promoter, amplifying a regulator that increases expression of the genes, or deleting or attenuating a regulator that decreases expression of the genes.
  • strong promoters include the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and tet promoter.
  • the nhaR gene encodes a factor that positively regulates expression of the nhaA gene, so expression of the nhaA gene can be enhanced by amplifying expression of the nhaR gene.
  • a promoter of nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be made stronger so that expression of the genes are enhanced by introducing nucleotide substitution into it.
  • Examples of a method of evaluating the strength of a promoter and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the like.
  • a spacer sequence between the ribosome binding site (RBS) and translation initiation codon, especially, several nucleotides just upstream of the initiation codon has a great influence on translation efficiency. Therefore, this sequence may be modified.
  • a mutation that increases the activity of the Na 4 VH + antiporter may be introduced into the genes.
  • a mutation include a mutation in a promoter sequence to increase the transcription level of nhaA gene, nhaB gene, chaA gene, and/or mdfA gene, and a mutation in the coding region of these genes to increase the specific activities of the NhaA, NhaB, ChaA, or MdfA proteins.
  • a mutation to enhance the activity of the NhaR protein that positively regulates expression of these genes may be introduced into the nhaR gene.
  • the method of producing an L-amino acid of the present invention comprises culturing the bacterium of the present invention in a medium to produce and accumulate an L-amino acid in the medium or bacterial cells, and collecting the L-amino acid from the medium or the bacterial cells.
  • a conventional medium to be used for fermentative production of an L-amino acid using a bacterium can be used. That is, a general medium containing a carbon source, nitrogen source, inorganic ion, and if necessary, other organic components can be used.
  • the carbon source include sugars such as glucose, sucrose, lactose, galactose, fructose and a starch hydrolysate; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid and succinic acid.
  • the nitrogen source examples include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; an organic nitrogen such as a soybean hydrolysate; ammonia gas; and aqueous ammonia.
  • inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate
  • an organic nitrogen such as a soybean hydrolysate
  • ammonia gas examples of the nitrogen source
  • aqueous ammonia examples of the nitrogen source.
  • auxotrophic substances such as vitamin B 1 and L-homoserine, yeast extract, and the like are preferably contained in the medium in an appropriate amount.
  • potassium phosphate, magnesium sulfate, iron ion, manganese ion, or the like may be added in small amounts.
  • the medium to be used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, nitrogen source, inorganic ion,
  • the culture is preferably performed under aerobic conditions for 1 to 7 days at a temperature of 24°C to 37°C and a pH of 5 to 9.
  • the pH can be adjusted with an inorganic or organic acidic or alkaline substance, ammonia gas or the like.
  • the L-amino acid can be collected from the fermentation liquid by a conventional method such as ion-exchange resin, precipitation, and other known methods.
  • the L-amino acid When the L-amino acid accumulates in the bacterial cells, the L-amino acid can be collected, for example, by disrupting the bacterial cells by ultrasonication or the like to release L-amino acid into the supernatant fraction, and then the bacterial cells are removed by centrifugation, followed by subjecting the resulting supernatant to an ion-exchange resin or the like.
  • fermentation may be performed while controlling the pH of the medium during culture to 6.5-9.0 and controlling the pH of the medium after completion of the culture to 7.2-9.0, as well as controlling the pressure in the fermentation tank during fermentation so that it is positive.
  • carbon dioxide or a mixed gas containing carbon dioxide may be added to the medium so that a bicarbonate ion and/or carbonate ion are present in an amount of at least 2 g/L in the culture medium during the culture period.
  • These ions then function as counter ions against the cation of the basic amino acids, and the target basic amino acid may be collected(JP 2002-065287 A).
  • the medium was sterilized by steam at 120°C for 20 minutes.
  • the medium was sterilized by steam at 120°C for 20 minutes.
  • the medium was adjusted to pH 7.0 with potassium hydroxide and sterilized by steam at
  • Glucose and MgSO 4 -7H 2 O were separately sterilized.
  • Calcium carbonate (official grade) was separately sterilized by dry heat at 180°C.
  • Chloramphenicol (25 mg/L) was added as an antibiotic.
  • the medium was adjusted to pH 7.5 with potassium hydroxide and sterilized by steam at
  • Glucose and MgSO 4 -7H 2 O were separately sterilized.
  • Calcium carbonate (official grade) was separately sterilized by dry heat at 180°C.
  • Streptomycin 100 mg/L
  • chloramphenicol 25 mg/L
  • telomeres were used to perform PCR using a chromosomal DNA of Escherichia coli MG 1655 strain as a template.
  • the chromosomal DNA was obtained using Bacterial Genomic DNA purification kit (Edge Bio Systems). PCR was performed using Pyrobest DNA polymerase (Takara Bio Inc.) such that a cycle of 96 0 C for 20 seconds, 65°C for 20 seconds and 72°C for 2 minutes was repeated 25 times.
  • PCR products were ligated to a Smal-digested vector pSTV28 (Takara Bio Inc.), to construct a plasmid for amplifying nhaA (pSnhaA), a plasmid for amplifying nhaB (pSnhaB), and a plasmid for amplifying nhaR (pSnhaR).
  • pSnhaA amplifying nhaA
  • pSnhaB plasmid for amplifying nhaB
  • pSnhaR plasmids in which each of the above-mentioned genes was arranged downstream of a lac promoter in the forward direction were obtained.
  • Lysine decarboxylases are encoded by the cadA gene (Genbank Accession No. NP 418555, SEQ ID NO: 19) and the ldcC gene (Genbank Accession No. NP_414728, SEQ ID NO: 21) (WO 96/17930).
  • WC196 FERM BP-5252 strain was used as the parent strain.
  • Disruption of the cadA gene and the ldcC gene were performed by the method developed by Datsenko and Wanner, which is called “Red-driven integration" (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) and by an excision system derived from ⁇ phage (J. Bacteriol. 2002 Sep; 184(18): 5200-3. Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex. Cho EH, Gumport RI, Gardner JF.).
  • Red-driven integration makes it possible to construct a gene-disrupted strain in one step by employing a PCR product obtained by using as primers synthetic oligonucleotides designed to have a part of the targeted gene on the 5 '-ends and a part of an antibiotic-resistance gene on the 3'-ends. Combining the ⁇ phage-derived excision system permits the removal of the antibiotic-resistance gene that has been incorporated into the gene-disrupted strain ( WO2005/010175). [0103] (1) Disruption of the cadA gene
  • a plasmid pMWl 18-attL-Cm-attR (WO2005/010175) was used as a template for PCR.
  • the plasmid pMW 118-attL-Cm-attR was obtained by inserting attL gene and attR gene, which are attachment sites of ⁇ phage, and the cat gene, which is an antibiotic resistance gene, into pMWl 18 (Takara Bio Inc.)
  • the genes are arranged in the following order: attL-cat-attR.
  • PCR was performed using, as primers, the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16, which have sequences corresponding to attL and attR on the 3'-terminals and a sequence corresponding to a part of the targeted cadA gene on the 5 '-terminals.
  • the amplified PCR product was purified on an agarose gel and introduced into Escherichia coli WC 196 strain by electroporation.
  • This strain contains plasmid pKD46 which has temperature-sensitive replicability.
  • the plasmid pKD46 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) contains a DNA fragment of 2,154 nucleotides derived from ⁇ phage which contains the Red recombinase-encoding genes ( ⁇ , ⁇ , and exo genes) of the ⁇ Red homologous recombination system which is controlled by an arabinose-inducible ParaB promoter (GenBank/EMBL Accession No. J02459, nucleotide numbers 31088 to 33241).
  • the plasmid pKD46 is necessary to integrate the PCR product into the chromosome of the WC 196 strain. [0106]
  • Competent cells for electroporation were prepared as follows. That is, cells of the Escherichia coli WC 196 strain were cultured overnight at 30°C in LB medium containing 100 mg/L ampicillin, and then diluted 100-fold with 5 mL of SOB medium (Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) containing ampicillin (20 mg/L) and L-arabinose (1 mM). The diluted cells were grown with aeration at 30°C until OD600 reached about 0.6, and then concentrated 100-fold and washed three times with 10% glycerol so that the cells were available for electroporation.
  • the electroporation was performed with 70 ⁇ L of the competent cells and about 100 ng of the PCR product. After the electroporation, 1 mL of SOC medium (Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) was added to the cells, cultured at 37°C for 2.5 hours, and then subjected to plate culture onto L-agar medium containing Cm (chloramphenicol) (25 mg/L), to thereby select Cm-resistant recombinant strains.
  • SOC medium Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)
  • the cadA-disrupted strain was named WC196 ⁇ cadA::att-cat strain.
  • helper plasmid pMW-intxis-ts JP 2005-058827 A was used to remove the art-cat gene which had been introduced into the cadA gene.
  • the plasmid pMW-intxis-ts carries the gene encoding integrase (Int) of ⁇ phage, and the gene encoding excisionase (Xis), and has temperature-sensitive replicability.
  • Competent cells of the WC196 ⁇ cadA::att-cat strain were prepared by a conventional method, and were then transformed with the helper plasmid pMW-intxis-ts, and then subjected to plate culture at 30°C onto L-agar medium containing 50 mg/L ampicillin, to thereby select ampicillin-resistant strains.
  • the cells were subcultured twice at 42°C on L-agar medium, and ampicillin resistance and chloramphenicol resistance of the resulting colonies were examined, to thereby yield a chloramphenicol and ampicillin-sensitive strain, in which the cadA gene was disrupted, and art-cat and pMW-intxis-ts were removed.
  • the strain was named WC196 ⁇ cadA.
  • WC196 ⁇ cadA ⁇ ldcC strain was transformed with a plasmid for lysine production, pCABD2 (WO 01/53459), which carries the dapA gene, dapB gene, and lysC gene, to thereby yield WC 196 ⁇ cad A ⁇ ldcC/pC ABD2 strain (WC196LC/pCABD2).
  • WC 196LC/pCABD2 strain was transformed with each of the plasmids prepared in Example 1: i.e., pSnhaA for amplifying nhaA; pSnhaB for amplifying nhaB; pSnhaR for amplifying nhaR; and a control plasmid pSTV28 (Takara Bio Inc.), to thereby yield chloramphenicol-resistant strains.
  • pSnhaA-introduced strain pSnhaB-introduced strain
  • pSnhaR-introduced strain pSTV28-introduced strain
  • WC196LC/pCABD2/pSnhaA strain WC196LC/pCABD2/pSnhaB strain
  • WC 196LC/ ⁇ C ABD2/pSnhaR strain WC196LC/pCABD2/pSTV28 strain
  • WC 196LC/pCABD2/pSnhaA strain was cultured at 37°C in L-medium containing 50 mg/L chloramphenicol until the final OD600 reached about 0.6, and an equal volume of 40% glycerol solution was added to each culture, followed by stirring. Then, the resulting solution was dispensed in appropriate amounts and stored at -80°C as glycerol stocks.
  • the glycerol stocks of the strains were thawed, and 100 ⁇ L of each stock was uniformly applied to an L-plate containing 25 mg/L chloramphenicol and 20 mg/L streptomycin and cultured at 37°C for 24 hours.
  • the bacterial cells which grew on the plate were suspended in 3 mL of a fermentation medium, and 1 mL of the suspension whose OD620 was about 13.5 was inoculated into 20 mL of a fermentation medium (L-lysine production medium for Escherichia bacterium) containing 25 mg/L chloramphenicol and 20 mg/L streptomycin in a 500 mL-Sakaguchi flask and cultured at 37°C using a reciprocal shaker, followed by determination of the amount of L-lysine which had accumulated in the medium at 24 hours after beginning the culture.
  • the amounts of L-lysine which accumulated in the medium were determined using Biotech Analyzer AS210 (Sakura Seiki Co,. Ltd.).
  • Table 1 shows the amounts of L-lysine which had accumulated in the medium.
  • the amounts of L-lysine accumulated in the medium were higher as compared to the control WCl 96LC/pCABD2/pSTV28 strain, which revealed that the lysine-producing ability was improved by amplifying the nhaA gene, nhaB gene, and nhaR gene.
  • B-5318 strain was used as an L-threonine-producing strain.
  • B-5318 strain was deposited in the Russian National Collection of Industrial Bacteria (VKPM), GNII Genetika) on May 3, 1990, under accession No. VKPM B-5318.
  • the nhaA gene, nhaB gene, and nhaR gene were each amplified in the L-threonine-producing bacterium using the plasmids prepared in Example 1.
  • the B-5318 strain was transformed with the plasmids prepared in Example 1 : i.e., the pSnhaA for amplifying nhaA gene, pSnhaB for amplifying nhaB gene, and pSnhaR for amplifying nhaR, to thereby yield three different chloramphenicol-resistant strains.
  • the plasmids was confirmed, and the pSnhaA-introduced strain, pSnhaB-introduced strain, and pSnhaR-introduced strain were named B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain, respectively.
  • the B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain were cultured at 37°C in an L-medium containing 25 mg/L chloramphenicol and 100 mg/L streptomycin and the control B5318 strain was cultured in an L-medium containing 100 mg/L streptomycin until the final OD600 reached about 0.6. Then, an equal volume of a 40% glycerol solution was added to the culture, followed by stirring. Then, the solution was dispensed in appropriate amounts and stored at -80°C as glycerol stocks.
  • the glycerol stocks of the strains were thawed, and 100 ⁇ L of each of the plasmid-introduced strains was uniformly applied onto an L-plate containing 25 mg/L chloramphenicol and 100 mg/L streptomycin, while 100 ⁇ L of B5318 strain was uniformly applied on an L-plate containing 100 mg/L streptomycin, and they were cultured at 37°C for 24 hours.
  • the bacterial cells which grew on the plate were suspended in 6 mL of physiological saline, and 1 mL of each of the suspensions whose OD620 was 22.0 was inoculated into 20 mL of a fermentation medium (L-threonine production medium for Escherichia bacterium) containing 25 mg/L chloramphenicol and 100 mg/L streptomycin (in the case of B-5318 strain, the medium contains only 100 mg/L streptomycin) in a 500 mL-Sakaguchi flask and cultured at 37°C for 24 hours using a reciprocal shaker. After the culture, the amounts of L-threonine which had accumulated in the medium were determined using high performance liquid chromatography.
  • a fermentation medium L-threonine production medium for Escherichia bacterium
  • streptomycin in the case of B-5318 strain, the medium contains only 100 mg/L streptomycin
  • Table 2 shows the amounts of L-threonine accumulated in the medium 6 and 24 hours after the beginning of the culture.
  • B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain the amounts of L-threonine which had accumulated after 6 and 24 hours were higher as compared to the control B5318 strain, which revealed that the L-threonine-producing ability was improved by amplifying nhaA gene, nhaB gene, and nhaR gene.
  • bacterium of the present invention enables efficient fermentative production of basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine and L-citrulline, aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine and L-glycine, hydroxy monoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acid such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, or acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine.
  • basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine and L-

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Abstract

L-amino acid is produced by culturing a bacterium belonging to the Enterobacteriaceae family which has L-amino acid-producing ability and is modified so that expression of the nhaA gene, nhaB gene, nhaR gene, chaA gene, mdfA gene, or combinations thereof is enhanced.

Description

DESCRIPTION
L-AMINO ACID PRODUCING BACTERIUM AND METHOD OF PRODUCING
L-AMINO ACID
Technical Field [0001]
The present invention relates to a method of producing an L-amino acid using a bacterium, and more specifically, to a method of producing L-lysine, L-threonine and L-glutamic acid. L-lysine and L-threonine are useful as additives for animal feeds, components of health foods, amino acid infusion, and the like. L-glutamic acid is useful as a food seasoning.
Background Art [0002]
L-amino acids are industrially produced by fermentation methods using bacteria belonging to the genus Brevibacterium, Corynebacterium, Escherichia, and the like. Examples of a method of producing L-lysine include the methods described in EP 0643135 B, EP 0733712 B, EP 1477565 A, EP 0796912 A, EP 0837134 A, WO 01/53459, EP 1170376 A, and WO 2005/010175. In these production methods, bacterial strains isolated from nature or artificially mutated strains thereof can be used. Also, bacteria which have been modified by a recombinant DNA technique so that the activity of an L-amino acid biosynthetic enzyme is enhanced have been used. [0003]
Methods of modifying the uptake or export of L-amino acids have been known to improve L-amino acid-producing ability. An example of a method of enhancing export of an L-amino acid is a method of producing L-lysine (WO 97/23597) or L-arginine (U.S. Patent Publication No. 2003-0113899) using a bacterium belonging to the genus Corynebacterium which has been modified so that the expression of an L-lysine/L-arginine export gene (LysE) is enhanced. In addition, a method of producing an L-amino acid using a bacterium belonging to the Enter obacteriaceae family which has been modified so that expression of the rhtA gene, rhtB gene, and rhtC gene (JP 2000-189177 A), yfiK gene and yahN gene (EP 1016710 A), ybjE gene (WO 2005/073390), or yhfK gene (WO 2005/085419), have been reported, and it has been suggested that such methods are involved in enhanced export of an L-amino acid. [0004]
It is also known that enhancing expression of a gene involved in the uptake of a sugar which is a substrate in fermentation improves the L-amino acid-producing ability. Examples of such a method include producing an L-amino acid using an Escherichia bacterium which has been modified to enhance expression of the ptsG gene (WO 03/04670), and producing an L-amino acid using an Escherichia bacterium modified to enhance expression of the ptsH gene, ptsl gene, or err gene (WO 03/04674). [0005]
The nhaA gene and nhaB gene each encode a membrane protein known as the Na+/H+ antiporter (Biochim Biophys Acta. 2005 Sep 30; 1709 (3): 240-50.). On the other hand, the nhaR gene is known to encode a regulator that positively regulates expression of the nhaA gene (EMBO J. 1997 Oct 1; 16(19): 5922-9.). However, whether or not enhancing the activity of the Na+/H+ antiporter has an effect on L-amino acid production has not been previously reported. [0006]
The chaA gene is a membrane protein which is known as a sodium-calcium/proton antiporter, but it has never been reported to have an Na+/H+ antiporter function (Biochim Biophys Acta. 2002 Dec 2; 1556 (2-3): 142-8.). The mdfA gene is known to function as a multidrug/chloramphenicol efflux transporter (J Biol Chem. 2004 Mar 5; 279(10): 8957-65), but it has never been reported to have an NaVH+ antiporter function, and the involvement of these genes in the production and accumulation of L-amino acids has not been reported.
Disclosure of the Invention [0007]
An object of the present invention is to provide a bacterium which is capable of effectively producing an L-amino acid and to provide a method for effectively producing an L-amino acid using the bacterium. [0008]
The inventors of the present invention have made extensive studies to solve the above-mentioned object. As a result, they have found that L-amino acid productivity is improved by amplifying genes that encode a Na /H antiporter or a gene that positively regulates Na+/H+ antiporter activity in an L-amino acid-producing bacterium. Moreover, they have found that enhancing the Na+/H+ antiporter activity in a bacterium results in efficient production of an L-amino acid, and thereby the present invention has been accomplished. [0009]
It is an object of the present invention to provide a L-amino acid-producing bacterium belonging to the Enterobacteriaceae family, wherein said bacterium has been modified so that Na+/H+ antiporter activity is enhanced.
It is a further object of the present invention to provide the bacterium as described above, wherein said Na+/H+ antiporter activity is enhanced by modifying said bacterium so that expression of a gene selected from the group consisting of nhaAgene, nhaB gene, nhaR gene, chaA gen, mdfA gene, and combinations thereof, is enhanced.
It is a further object of the present invention to provide the bacterium as described above, wherein said expression is enhanced by increasing the copy number of the gene or genes, or by modifying an expression regulatory sequence of the gene or genes.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaA gene encodes a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and
(B) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 2, and has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaB gene encodes a protein selected from the group consisting of:
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4, and
(D) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 4, and has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaR gene encodes a protein selected from the group consisting of:
(E) a protein comprising the amino acid sequence of SEQ ID NO: 6; and (F) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 6, and has an activity to increase expression of Na+/H+ antiporter.
It is a further object of the present invention to provide the bacterium as described above, wherein said chaA gene encodes a protein selected from the group consisting of:
(G) a protein comprising the amino acid sequence of SEQ ID NO: 24; and (H) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 24, and has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said mdfA gene encodes a protein selected from the group consisting of:
(I) a protein comprising the amino acid sequence of SEQ ID NO: 26; and
(J) a protein comprising an amino acid sequence including substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 26, and has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaA gene is a DNA selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 ; and
(b) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaB gene is a DNA selected from the group consisting of:
(c) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 3; and
(d) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 3 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said nhaR gene is a DNA selected from the group consisting of:
(e) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 5; and
(f) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 5 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has an activity to increase expression of Na+ZH+ antiporter.
It is a further object of the present invention to provide the bacterium as described above, wherein said chaA gene is a DNA selected from the group consisting of:
(g) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 23; and
(h) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 23 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na4VH+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said mdfA gene is a DNA selected from the group consisting of:
(i) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 25; and
(j) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 25 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na4VH+ antiporter activity.
It is a further object of the present invention to provide the bacterium as described above, wherein said L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
It is a further object of the present invention to provide the bacterium as described above, wherein said bacterium belongs to the genus Escherichia or Pantoea. [0010]
It is a further object of the present invention to provide a method of producing an L-amino acid comprising culturing the bacterium as described above in a medium to produce and accumulate an L-amino acid in the medium or cells of the bacterium and collecting the L-amino acid from the medium or the cells.
It is a further object of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
Description of the Preferred Embodiments [0011] Hereinafter, the present invention will be described in detail. [0012] <1> Bacterium of the present invention
The bacterium of the present invention belongs to the Enterobacteήaceae family and has an L-amino acid-producing ability. This bacterium also is modified so that the NaVH+ antiporter activity is enhanced. In the present invention, the term "L-amino acid-producing ability" means an ability to produce and accumulate an L-amino acid in a medium or bacterial cells when the bacterium of the present invention is cultured in the medium. The bacterium of the present invention may produce a plurality of L-amino acids. The ability may be native to the bacterium, or the ability may be obtained by modifying the bacterium by mutation and or with recombinant DNA techniques. [0013]
The kind of L-amino acid is not particularly limited, but examples thereof include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline, aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and L-glycine, hydroxy monoaminocarboxylic acid such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine, and acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine, and L-asparagine. The bacterium of the present invention may produce two or more kinds of amino acids. [0014] <1-1> Imparting L-amino acid-producing ability
Hereinafter, examples of methods of imparting an L-amino acid-producing ability and examples of the bacteria to which an L-amino acid-producing ability can be imparted will be described. However, the bacterium is not limited thereto, as long as it has an L-amino acid-producing ability.
Bacteria belonging to the Enterobacteriaceae family, including those belonging to the genus Escherichia or Pantoea, can be used as the parent strain of the bacterium of the present invention. Other examples of bacteria belonging to the Enterobacteriaceae family include y-Proteobacteria such as Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, and Morganella. [0015]
Escherichia bacteria reported in Neidhardt et al. ((Backmann, B.J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K- 12, p. 2460-2488. Table 1. In F. D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, D. C), such as Escherichia coli can be utilized. Examples of a wild-type strain of Escherichia coli include the K- 12 strain or derivatives thereof, Escherichia coli MG 1655 strain (ATCC No. 47076), and W3110 strain (ATCC No. 27325). These strains are available from the American Type Culture Collection (ATCC) (Address: P.O. Box 1549, Manassas, VA 20108, 1, United States of America). [0016]
Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes, and an example of Pantoea bacteria is Pantoea ananatis. In recent years, Enterobacter agglomerans has been reclassifϊed in some cases as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like based on an analysis of the nucleotide sequence of 16S rRNA. Therefore, bacteria to be used in the present invention may belong to either the genus Enterobacter or the genus Pantoea as long as they are classified in the Enterobacteriaceae family. When Pantoea ananatis is bred using genetic engineering techniques, Pantoea ananatis AJ 13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ 13601 strain (FERM BP-7207), derivatives thereof, and the like, may be used. These strains were identified and deposited as Enterobacter agglomerans when they were isolated, but as described above, these strains have been reclassified as Pantoea ananatis based on an analysis of the nucleotide sequence of 16S rRNA. [0017]
The L-amino acid-producing ability can be imparted to a parent strain as described above, as follows.
In order to impart the L-amino acid-producing ability, methods which have been conventionally adopted in the breeding of Escherichia bacteria or the like, may be used, such as by acquiring nutrient-auxotrophic mutant strains, analogue resistant strains, or metabolic regulation mutant strains, or by creating a recombinant strain in which expression of an L-amino acid biosynthetic enzyme is enhanced (Amino Acid Fermentation, Japan Scientific Societies Press, first edition publication: May 30, 1986, p.77 to 100). In the present invention, properties such as nutrient-'auxotrophy, analogue-resistance, and metabolic regulation may be imparted alone or in combination with the imparting of L-amino acid-producing ability. Furthermore, expression of one or more L-amino acid biosynthetic enzymes may be enhanced. Furthermore, imparting of such properties as nutrient-auxotrophy, analogue-resistance and metabolic regulation mutation may be combined with enhancing the expression of L-amino acid biosynthetic enzymes. [0018]
Nutrient-auxotrophic mutant strains, L-amino acid-analogue resistant strains, and metabolic regulation mutant strains that have an L-amino acid-producing ability can be obtained as follows. A parent strain or a wild-type strain is mutated by a typical mutation treatment, such as irradiation with X-ray or ultraviolet ray or by treating with a mutagen, including N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS), followed by selection of strains that exhibit nutrient-auxotrophy, analogue-resistance or a metabolic regulation mutation and have an L-amino acid-producing ability. [0019]
Examples of an L-lysine analogue include oxalysine, lysinehydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and norleucine. Examples of an L-arginine analogue include arginine hydroxamate, homoarginine, D-arginine, and canavanine. [0020]
Specific examples of an L-lysine analogue resistant strain or metabolic regulation mutant strain having an L-lysine-producing ability include Escherichia coli AJl 1442 strain (FERM BP-1543, NRRL B-12185; JP 56-18596 A and U.S. Patent No. 4346170) and Escherichia coli VL611 strain (JP 2000- 189180 A). WC 196 strain (WO 96/17930) may be used as an L-lysine producing strain of Escherichia coli. WC 196 strain has been obtained by imparting AEC (S-(2-aminoethyl)-cysteine)-resistance to W3110 strain which was derived from Escherichia coli K- 12 strain. The WC 196 strain was named Escherichia coli AJ 13069 strain and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan) on December 6, 1994 and given an accession number of FERM P- 14690, and the deposit was then converted to an international deposit under the provisions of Budapest Treaty on September 29, 1995 and given an accession number of
FERM BP-5252.
[0021]
An L-amino acid-producing ability can also be imparted by enhancing the expression of a gene encoding an L-amino acid biosynthetic enzyme.
For example, as described below, an L-lysine-producing ability may be imparted by enhancing the activities of dihydrodipicolinate synthase and aspartokinase. That is, a gene fragment encoding dihydrodipicolinate synthase and a gene fragment encoding aspartokinase are ligated to a vector which functions in a host bacterium to be used for L-lysine production. The vector is preferably a multi-copy vector, and the obtained vector DNA is used to transform a host bacterium. The transformation results in increased copy numbers of the gene encoding the dihydrodipicolinate synthase and the gene encoding aspartokinase in a host cell, thereby enhancing the activities of these enzymes. Hereinafter, dihydrodipicolinate synthase, aspartokinase, and aspartokinase III are abbreviated as DDPS, AK, and AKIIl, respectively. [0022]
The gene encoding DDPS and the gene encoding AK are not particularly limited as long as the DDPS and AK activities are expressed in the host bacterium, and examples thereof include the genes of Escherichia coli, Methylophilus methylotrophus, Corynebacterium glutamicum, and the like. Nucleotide sequences of the DDPS gene derived from an Escherichia bacterium (dapA, Richaud, F. et al. J. Bacterid., 297 (1986)) and the AKIII gene derived from an Escherichia bacterium (lysC, Cassan, M., Parsot, C, Cohen, GN. and Patte, J.C., J. Biol. Chem., 261, 1052 (1986)) have been identified, so these genes can be obtained by PCR using primers synthesized based on their nucleotide sequences and the chromosomal DNA of a bacterium such as Escherichia coli K- 12 as a template. Hereinafter, dapA and lysC derived from Escherichia coli will be taken as an example, but a gene encoding DDPS and a gene encoding AK are not limited thereto. [0023]
It is known that wild-type DDPS derived from Escherichia coli is regulated by feedback inhibition by L-lysine, while wild-type AKIII derived from Escherichia coli is regulated by suppression and feedback inhibition by L-lysine. Therefore, when using dapA and lysC, mutated forms of these genes are preferably used so that the genes are not subject to feedback inhibition. Hereinafter, the mutant DDPS which is not subject to feedback inhibition by L-lysine is referred to as "mutant DDPS", and the DNA encoding the mutant DDPS is referred to as "mutant dapA or dapA*". Similarly, the mutant AKIII derived from Escherichia coli which is not subject to feedback inhibition by L-lysine is referred to as "mutant AKIN", and the DNA encoding the mutant AKIII is referred to as "mutant lysC". However, the DDPS and AK which can be used in the present invention are not necessarily these type of mutants since DDPS derived from Corynebacterium bacterium is not subject to feedback inhibition. [0024]
An example of a DNA encoding mutant DDPS not subject to feedback inhibition by L-lysine includes a DNA encoding a DDPS which has an amino acid sequence in which the histidine at position 118 is substituted with tyrosine. Meanwhile, an example of a DNA encoding mutant AKIII not subject to feedback inhibition by L-lysine includes a DNA encoding an AKIII having an amino acid sequence in which the threonine at position 352, the glycine at position 323, and the methionine at position 318 are substituted with isoleucine, asparagine and isoleucine, respectively (U.S. Patent No. 5661012 and U.S. Patent No. 6040160). Such mutant DNAs can be obtained by a site-specific mutation using PCR or the like.
Enhancing expression of the L-lysine biosynthetic genes as described above can be attained by transformation or homologous recombination using a plasmid or the like, in the same way as the nhaA gene, nhaB gene, nhaR gene, chaA gene, and mdfA gene described below. [0025]
Wide host-range plasmids RSFD80, pCABl, and pCABD2 are known to contain a mutant dapA gene encoding a mutant DDPS and a mutant lysC gene encoding a mutant AKIII (U.S. Patent No. 6040160). Escherichia coli JM109 strain transformed with RSFD80 was named AJ12396 (U.S. Patent No. 6040160), and the strain was deposited at the 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) on October 28, 1993 and given an accession number of FERM P- 13936, and the deposit was then converted to an international deposit under the provisions of Budapest Treaty on November 1, 1994 and given an accession number of FERM BP-4859. RSFD80 can be obtained from AJl 2396 strain by a conventional method.
[0026]
An L-lysine-producing ability can also be imparted by enhancing expression of genes encoding enzymes, other than DDPS and AK, which are involved in biosynthesis of L-lysine. Examples of such enzymes include enzymes in the diaminopimelate pathway such as dihydrodipicolinate reductase (dapB: hereinafter, the words in parentheses represent the gene names), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (WO96/40934), phosphoenolpyruvate carboxylase (pepC) (JP 60-87788 A), aspartate aminotransferase (aspC) (JP 06-102028 B), diaminopimelate epimerase gene (dapF) (JP 2003-135066), aspartate semialdehyde dehydrogenase (asd) (WO 00/61723), tetrahydrodipicolinate succinylase (dapD), and succinyl-diaminopimelate deacylase (dapE); or enzymes in the aminoadipic acid pathway such as homoaconitate hydratase (JP 2000-157276 A). The documents indicated in parentheses disclose L-lysine-producing strains in which expression of a gene encoding each enzyme is enhanced. The enhancement of expression of a gene encoding each enzyme may be combined with the enhancement of expression of the DDPS gene and AK gene. [0027]
Expression of genes other than L-lysine biosynthetic genes may also be enhanced to impart L-lysine-producing ability, and examples of such genes include those encoding enzymes involved in sugar uptake, sugar metabolism (glycolytic pathway), the TCA cycle, the pentose phosphate cycle, complementary pathway, and energy metabolism. Moreover, expression of genes that impart an amino acid-resistance to a host bacterium, genes encoding amino acid-export enzymes, and genes encoding enzymes involved in uptake of by-products may be enhanced. [0028]
Genes involved in sugar metabolism include genes encoding enzymes in the glycolytic pathway or enzymes involved in sugar uptake. Examples thereof include the glucose-6-phosphate isomerase gene (pgi; WO 01/02542), phosphoenolpyruvate synthase gene (pps; EP 877090 A), phosphoglucomutase gene (pgm; WO 03/04598), fructose bisphosphate aldolase gene (fbp; WO 03/04664), pyruvate kinase gene (pykF; WO 03/008609), transaldolase gene (talB; WO 03/008611), fumarase gene (fum; WO 01/02545), phosphoenolpyruvate synthase gene (pps; EP 877090 A), non-PTS sucrose uptake gene (esc; EP 149911 A), sucrose-assimilating gene (scrAB operon; WO 90/04636), PTS glucose uptake gene (ptsG, ptsH, ptsl, err; WO 03/04670, WO 03/04674, and EP 1254957 A), and a gene involved in maltose transport (malK; EP 1254957). [0029]
Examples of genes encoding TCA cycle enzymes include citrate synthase gene (git A; WO 03/008607), isocitrate dehydrogenase gene (icd; WO 03/008607), 2-ketoglutarate dehydrogenase gene (sucAB; WO 03/008614), and succinate dehydrogenase gene (sdh; WO 01/02544), glutamate dehydrogenase gene (gdh; U.S.Patent No 6,004,773). [0030]
Examples of genes encoding pentose phosphate cycle enzymes include glucose-6-phosphate dehydrogenase gene (zwf; WO 03/008607) and ribose-5-phosphate isomerase gene (rpiB; WO 03/008607). [0031]
Examples of genes encoding anaplerotic pathway include the phosphoenolpyruvate carboxylase gene (pepC; U.S. Patent No. 5,876,983), pyruvate carboxylase gene (pyc; EP 1092776), malate dehydrogenase gene (mdh; WO 01/02546), and pyruvate decarboxylase gene (pckA; WO 04/090125). [0032]
Examples of genes encoding enzymes involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and cytochromoe bo type oxidase gene (cyoB; EP 1070376).
Examples of genes that impart L-amino acid-resistance include the rhtB gene (U.S. Patent No. 6,887,691), rhtC gene (EP 1013765), yedA gene (EP 1449917), yddG gene (EP 1449918), ygaZH gene (EP 1239041), yahN, yfiK, and yeaS genes (EP 1016710), rhtA gene (Res Microbiol. 2003 Mar; 154(2): 123-35.), and ybjE gene (WO 2005/073390). [0033]
Furthermore, in the bacterium of the present invention, an activity of an enzyme that catalyzes a reaction which branches off from L-lysine biosynthetic pathway and produces a compound other than L-lysine may be decreased or may be made deficient. Examples of such an enzyme include homoserine dehydrogenase, lysine decarboxylase, and malic enzyme, and strains in which activities of such enzymes are decreased or deficient are described in WO 95/23864, WO 96/17930, WO 2005/010175, and the like. In Escherichia coli, lysine decarboxylases are encoded by a cadA gene (Genbank Accession No. NP_418555, SEQ ID NO: 19) and ldcC gene (Genbank Accession No. NP_414728, SEQ ID NO: 21) (WO 96/17930), so these genes may be disrupted to enhance L-lysine-producing ability. DNA molecules homologous to the cadA gene and ldcC gene may be used as long as they can cause homologous recombination with the cad A gene and ldcC gene on the chromosome of a host bacterium. For example, a DNA molecule homologous to the cadA gene may hybridize to a complementary strand of SEQ ID NO: 19 under stringent conditions, and a DNA molecule homologous to the ldcC gene may hybridize to a complementary strand of SEQ ID NO: 21 under stringent conditions. [0034]
Activities of these enzymes can be decreased or eliminated by introducing a mutation to the genes encoding the enzymes on the chromosome using a known mutation treatment, to thereby decrease or eliminate the activities of the enzymes in a cell. For example, decreasing or eliminating the activities of the enzymes can be attained by disrupting the genes encoding the enzymes on the chromosome by gene recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. In addition, this can also be attained by introducing an amino acid substitution (missense mutation) to the region encoding the enzymes on the chromosome, introducing a stop codon (nonsense mutation), introducing a frameshift mutation that adds or deletes one or two nucleotides, or deleting part of the gene (Journal of biological Chemistry 272: 8611-8617 (1997). Meanwhile, the activities of the enzymes can also be decreased or eliminated by constructing a mutant gene encoding a mutant enzyme which has a deletion in the coding region, and then replacing the normal gene on the chromosome with the mutant gene by homologous recombination, or introducing the mutant gene using a transposon or an IS factor. [0035]
For example, the following gene recombination method can be used to introduce a mutation that decreases or eliminates the activities of the above-mentioned enzymes. A mutant gene is prepared by modifying a partial sequence of a target gene so that it does not encode an enzyme that can function normally. Then, a bacterium belonging to the Enterobacteriaceae family is transformed with a DNA containing the mutant gene to cause recombination of a gene on the bacterial chromosome with the mutant gene, thereby substituting the target gene on the chromosome with the mutant gene. Examples of this type of gene substitution using homologous recombination include the method using a linear DNA called "Red-driven integration" (Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000), a combination of Red-driven integration and a cleavage system derived from λ phage (Cho, E.H., Gumport, R.I., Gardner, J.F.J. Bacteriol. 184: 5200-5203 (2002)) (WO 2005/010175), a method using a plasmid containing a temperature-sensitive replication origin (Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA. 97: 6640-6645 (2000); U.S. Patent No. 6303383; JP 05-007491 A), and the like. Meanwhile, a site-specific mutation by gene substitution using homologous recombination can also be performed by using a plasmid which is not able to replicate in a host cell. [0036]
The above-described methods for enhancing the expression of the L-lysine biosynthetic enzymes' genes and for decreasing the activities of enzymes can also be applied to genes encoding other L-amino acid synthetic enzymes so that the ability to produce another L-amino acid is imparted to a bacterium of the Enterobacteriaceae family. [0037]
Hereinafter, a bacterium to which an ability to produce an L-amino acid other than L-lysine is imparted will be exemplified. [0038] L-threonine-producing bacteria
Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like. [0039]
The strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSFlOlO-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Nagatinskaya Street 3 -A, 117105 Moscow, Russian Federation) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on April 7, 1987 under the accession number B-3996. [0040]
E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain to derive L-threonine-producing bacteria of the present invention. The strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage Cl repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on May 3, 1990 under accession number of VKPM B-5318. [0041]
Preferably, the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes:
-the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
-the thrB gene which codes for homoserine kinase;
-the thrC gene which codes for threonine synthase;
-the rhtA gene which codes for a putative transmembrane protein;
-the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and
-the aspC gene which codes for aspartate aminotransferase (aspartate transaminase). [0042]
The sequence of the thrA gene of Escherichia coli which encodes aspartokinase homoserine dehydrogenase I has been elucidated (nucleotide positions 337 to 2799, GenBank accession NC 000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12. The nucleotide sequence of the thrB gene of Escherichia coli which encodes homoserine kinase has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K- 12. The nucleotide . sequence of the thrC gene of Escherichia coli which encodes threonine synthase has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function together as a single threonine operon. To enhance the expression of the threonine operon, the attenuator region which affects the transcription can be removed from the operon (WO2005/049808, WO2003/097839). [0043]
• A thrA gene which has been mutated so that it encodes aspartokinase homoserine dehydrogenase I which is resistant to feedback inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40, which is present in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371. [0044]
The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORFl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and located between the pexB and ompX genes. The sequence expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A). [0045]
The nucleotide sequence of the asd gene of E.coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) by utilizing primers based on the nucleotide sequence of the gene. The asd genes from other microorganisms can be obtained in a similar marine (US 2005-0124048) . [0046]
Also, the nucleotide sequence of the aspC gene of E. col '/ has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC_000913.1, gi:16128895), and can be obtained by PCR. The aspC genes from other microorganisms can be obtained in a similar manner (WO03/072786). [0047] L-cysteine-producing bacteria
Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM 15 which has been transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601), E. coli W3110 which over-expresses genes which encode proteins suitable for secreting toxic substances (U.S. Patent No. 5,972,663), E. coli strains with decreased cysteine desulfohydrase activity (JPl 1155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for the cysteine regulon encoded by the cysB gene (WO0127307A1), and the like. [0048] L-leucine-producing bacteria
Examples of parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the genetic engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like. [0049]
The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples of these genes include those of the leu ABCD operon, which preferably include a leuA gene which has been mutated so that it encodes isopropylmalate synthase which is resistant to feedback inhibition by L-leucine (US Patent 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acids from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2). [0050] L-histidine-producing bacteria
Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U,S. Patent No. 6,258,554) and the like. [0051]
Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of these L-histidine-biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisl), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-arninoirnidazole carboxamide ribotide isomerase (his A), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth. [0052]
It is known that the genes encoding the L-histidine biosynthetic enzyme (hisG, hisBHAFI) are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation which induces resistance to the feedback inhibition into ATP phosphoribosyltransferase (hisG) (Russian Patent Nos. 2003677 and 2119536).
Specific examples of strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E.coli strains transformed with rht, a gene for an amino acid-export (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL- 1 ,2,4-triazole-3 -alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), and so forth. [0053]
L-glutamic acid-producing bacteria
Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC+ (EP 1172433). E. coli VL334 (VKPM B- 1641) is auxotrophic for L-isoleucine and L-threonine and is mutated in thrC and ilvA genes (U.S. Patent No. 4,278,765). A wild-type allele of the thrC gene was transferred by general transduction using a bacteriophage Pl grown on the wild-type E. coli strain Kl 2 (VKPM B-7). As a result, an L-isoleucine auxotrophic strain VL334thrC+ (VKPM B-8961) was obtained. This strain is able to produce L-glutamic acid. [0054]
Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of the enzymes involved in L-glutamic acid biosynthesis include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi). [0055]
Examples of strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A, EP955368A, and EP952221A. [0056]
Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid, and branches off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase, α-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, and glutamate decarboxylase. Bacteria belonging to the genus Escherichia deficient in the α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and
5,573,945.
[0057]
Specifically, these strains include the following:
E. coli W3110sucA::Kmr
E. co/z AJ12624 (FΕRM BP-3853)
E. coli AJ 12628 (FΕRM BP-3854)
E. coli AJ 12949 (FΕRM BP-4881)
[0058]
E. coli W3110sucA::Kmr is obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase. [0059]
Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FΕRM BP-5807) (U.S. Patent No. 5.908,768), FΕRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (FΕRM BP-5565) (U.S. Patent No. 6,110,714), and the like. [0060]
Examples of L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea which are deficient in α-ketoglutarate dehydrogenase activity or have a decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJl 3356 (U.S. Patent No. 6,331 ,419). Pantoea ananatis AJ 13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P- 16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in α-ketoglutarate dehydrogenase activity as a result of disruption of the αKGDH-El subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomercms AJl 3356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ 13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis. [0061] L-phenylalanine-producing bacteria
Examples of parent strains for deriving L-phenylalanine-prodύcing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E.coli AJ12739 (tyrA::Tnl0, tyrR) (VKPM B-8197); £.cø/z HW1089 (ATCC 55371) harboring the pheA34 gene (U.S. Patent No. 5,354,672); E.coli MWEC 101 -b (KR8903681); E.coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia which have an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al). [0062] L-tryptophan-producing bacteria
Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SV 164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase resistant to feedback inhibition by serine and a trpE allele encoding anthranilate synthase resistant to feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGXl 7 (pGX44) (NRRL B-12263) and AGX6(ρGX50)aroP (NRRL B- 12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli AGXl 7/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. Furthermore, L-tryptophan producing bacteria belonging to the genus Escherichia which have an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al). [0063]
Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and tryptophan synthase (trpAB) are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so a mutation which results in desensitizes the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include a E. coli SV 164 which harbors desensitized anthranilate synthase and a strain obtained by transforming the plasmid pGH5 into E. coli SV 164 (WO 94/08031), which contains a serA gene which has been mutated so that it encodes feedback-desensitized phosphoglycerate dehydrogenase. [0064]
Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains transformed with the tryptophan operon which contains a gene encoding desensitized anthranilate synthase (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of α and β subunits which are encoded by trpA and trpB, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275). [0065] L-proline-producing bacteria
Examples of parent strains for deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline (EP 1172433). [0066]
The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such preferred genes for L-proline producing bacteria include the proB gene coding for glutamate kinase which is desensitized to feedback inhibition by L-proline (DE Patent 3127361). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from the bacterial cell. Such genes are exemplified by the b2682 and b2683 genes (ygaZH genes) (EP1239041 A2). [0067]
Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B- 12403 and NRRL B- 12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15th Miami winter symposium, 1983, p.34), and the like. [0068] L-arginine-producing bacteria
Examples of parent strains for deriving L-arginine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPl 170361 Al), and the like. [0069]
Examples of parent strains for deriving L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of the L-arginine biosynthetic enzymes include N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argj), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase (carAB).
[0070]
L-valine-producing bacteria
Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased. Examples of parent strains for deriving L-valine-producing bacteria of the present invention also include mutants of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766). For example, E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. E coli VL 1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1 Dorozhny Proezd.) on June 24, 1988 under accession number VKPM B-4411. [0071]
Furthermore, mutants requiring lipoic acid for growth and/or lacking H+-ATPase can also be used as parent strains (WO96/06926). [0072] L-isoleucine-producing bacteria
Examples of parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Patent No. 5,998,178). [0073] <l-2> Enhancement of the NaVH+ antiporter activity
The bacterium of the present invention can be obtained by modifying a bacterium having an L-amino acid-producing ability as described above so that the Na4VH+ antiporter activity is enhanced. However, L-amino acid-producing ability may be imparted after the bacterium is modified so that the Na+/H+ antiporter activity is enhanced. The Na+/H+ antiporter activity may be enhanced by increasing the expression of a gene encoding a Na+/H+ antiporter or a gene that positively regulates the NaVH+ antiporter activity. Gene expression can be enhanced by modifying an expression regulatory sequence such as a promoter of an endogenous gene, or enhancing expression of an exogenous gene by introducing a plasmid containing the gene, or the like. These methods may be combined. [0074]
In the present invention, the term "NaVH+ antiporter" refers to a membrane protein involved in uptake of H+ into a cytoplasm and export of Na+, and the phrase "modifying so that the NaVH+ antiporter activity is enhanced" includes when the number of NaVH+ antiporter molecules per cell increases and when the Na+/H+ antiporter activity per molecule is increased as compared to a wild-type strain or an unmodified strain. The bacterium is modified so that NaVH+ antiporter activity is preferably not less than 150%, more preferably not less than 200%, further more preferably not less than 300% per bacterial cell as compared to a wild-type strain or an unmodified strain. In the present invention, examples of a wild-type bacterium belonging to the Enterobacteriaceae family to be used as a control include Escherichia coli MGl 655 strain (ATCC No. 47076), W3110 strain (ATCC No. 27325), and Pantoea ananatis AJ13335 strain (FERM BP-6615). [0075]
The NaVH+ antiporter activity can be enhanced increasing expression of a gene encoding a NaVH+ antiporter or a gene encoding a protein that positively regulates Na+/H+ antiporter activity. The increased NaVH+ antiporter activity can be confirmed by measuring the increase in expression of the gene encoding the NaVH+ antiporter. The increased expression can be confirmed by comparing the amount of NaVH+ antiporter mRNA in the bacterium of the present invention with a wild-type or unmodified strain. Examples of a method of measuring the amount of expression include Northern hybridization and RT-PCR (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The expression may be at any level as long as the expression is increased as compared to a wild-type or unmodified strain, and for example, the amount of expression is preferably increased not less than 1.5 -fold, more preferably not less than 2-fold, and more preferably not less than 3-fold as compared to a wild-type or unmodified strain. Meanwhile, enhancement of the NaVH+ antiporter activity can also be confirmed by an increase in the amount of Na+/H+ antiporter protein as compared to a wild-type or unmodified strain, and the amount of the protein can be evaluated, for example, by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). [0076]
Examples of a gene that encodes the Na+/H+ antiporter include the nhaA gene, nhaB gene, chaA gene, and mdfA gene, and homologues thereof. In the present invention, examples of these genes from Escherichia coli include the nhaA gene of SEQ ID NO: 1 (nucleotide numbers 17489-18655 of GenBank Accession No. NC 000913), nhaB gene of SEQ ID NO: 3 (a complementary strand of nucleotide numbers 1232399-1233940 of GenBank Accession No. NC 000913), chaA gene of SEQ ID NO: 23 (a complementary strand of nucleotide numbers 1269972..1271072 of GenBank Accession No. NC 000913.2), and mdfA gene of SEQ ID NO: 25 (nucleotide numbers 882896..884128 of GenBank Accession No. NC_000913.2). Meanwhile, the valine at position 1 in the amino acid sequence encoded by SEQ ID NO: 1 corresponds to the codon gtg, but the codon may also be translated as methionine. [0077]
Examples of nhaA gene and nha B gene derived from other bacteria include a gene that is derived from Yersinia pestis strain CO92 and encodes a NhaA protein having the amino acid sequence of SEQ ID NO: 7, and a gene that is derived from Salmonella enterica CTl 8 strain and encodes a NhaB protein having the amino acid sequence of SEQ ID NO: 8.
Homologues of the above-mentioned genes can be obtained by cloning, based on homologies to the above-listed genes, from coryneform bacteria such as Corynebacterium glutamicum and Brevibacterium lactofermentum, y-Proteobacteria such as those belonging to the genus Escherichia, Enterobacter, Klebsiella, Serratia, Erwinia, or Yersinia, Pseudomonas bacteria such as Pseudomonas aeruginosa, and Mycobacterium bacteria such as Mycobacterium tuberculosis. Homologue genes may be amplified by using, for example, synthetic oligonucleotides shown in SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, or SEQ ID NOS: 13 and 14 as primers. The homologies of amino acid sequences and nucleotide sequences can be determined by using the algorithm BLAST created by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or the algorithm FASTA created by Pearson (Methods Enzymol., 183, 63 (1990)). Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (www.ncbi.nlm.nih.gov). [0078]
Examples of a gene that encodes a protein that positively regulates the Na'/tT antiporter activity include a nhaR gene or a homologue thereof, and examples of nhaR gene from Escherichia coli include the nhaR gene of SEQ ID NO: 5 (nucleotide numbers 18715-19620 of GenBank Accession No. NC_000913). [0079]
Homologues of a gene encoding a Na+/H+ antiporter or of a gene encoding a protein that positively regulates Na+/H+ antiporter activity include mutant genes derived from another bacteria or a natural or artificial mutant genes, which show high structural similarity to a gene such as nhaA, nhaB, chaA, mdfA, and nhaR of Escherichia coli and functions to enhance the Na4VH+ antiporter activity when introduced or amplified in a host. Each of the homologue genes encoding the Na+/H+ antiporter (homologues of nhaA gene, nhaB gene, cha A gene, or mdfA gene) include genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 98% to each of the entire amino acid sequences of SEQ ID NO: 2, 4, 24 or 26, and encode a protein which functions as a Na+/H+ antiporter. Whether a protein functions as a Na+/H+ antiporter can be confirmed by expressing the genes in a host cell and examining the transport of Na+ and H+ across a membrane (Biochim Biophys Acta. 2005 Sep 30; 1709(3): 240-50.).
Homologues of a gene encoding a protein that positively regulates the Na+/H+ antiporter activity (homologue of nhaR gene) include genes that have homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 98% to the entire amino acid sequence of SEQ ID NO: 6, and encode a protein capable of increasing expression amount of Na+/H+ antiporter gene, in particular, nhaA gene. The increase in the expression of the Na+/H+ antiporter gene can be confirmed by expressing the nhaR gene in a host cell and determining the transcription amount of the nhaA gene (EMBO J. 1997 Oct 1 ; 16(19): 5922-9.). [0080]
Meanwhile, the nhaA gene, nhaB gene, chaA gene, and mdfA gene to be used in the present invention are not limited to wild-type genes and may be mutant or artificially modified genes that encode a protein having an amino acid sequence of SEQ ID NO: 2, 4, 24, or 26 and which may include substitution, deletion, insertion, addition of one or several amino acids at one or a plurality of positions, as long as the function of the NhaA protein, NhaB protein, ChaA protein, or MdfA protein encoded by these genes, that is, a Na+/H+ antiporter function, is maintained.
In addition, the nhaR gene to be used in the present invention is not limited to a wild-type gene and may be a mutant or an artificially modified gene that encodes a protein having an amino acid sequence of SEQ ID NO: 6 including substitution, deletion, insertion, addition of one or several amino acids at one or a plurality of positions as long as the function of NhaR protein encoded by the gene, that is, the ability to increase expression of the Na+/H+ antiporter is maintained.
In the present invention, , the term "one or several" specifically means 1 to 20, preferably 1 to 10, and more preferably 1 to 5, although this determination depends on the position in the protein's tertiary structure or the types of amino acid residues in the protein. The above-mentioned substitution is preferably a conservative substitution, which may include substitutions between aromatic amino acids such as substitution among Phe, Trp and Tyr, substitution between hydrophobic amino acids such as substitution among Leu, He and VaI, substitution between polar amino acids such as substitution between GIn and Asn, substitution between basic amino acids such as substitution among Lys, Arg and His, substitution between acidic amino acids such as substitution between Asp and GIu, substitution between hydroxyl group-containing amino acids such as substitution between Ser and Thr. Examples of conservative substitutions include substitution of Ser or Thr with ala; substitution of GIn, His or Lys with Arg; substitution of GIu, GIn, Lys, His or Asp with Asn; substitution of Asn, GIu or GIn with Asp; substitution of Ser or Ala with Cys; substitution of Asn, GIu, Lys, His, Asp or Arg with GIn; substitution of GIy, Asn, GIn, Lys or Asp with GIu; substitution of Pro with GIy; substitution of Asn, Lys, GIn, Arg or Tyr with His; substitution of Leu, Met, VaI or Phe with He; substitution of He, Met, VaI or Phe with Leu; substitution of Asn, GIu, GIn, His or Arg with Lys; substitution of He, Leu, VaI or Phe with Met; substitution of Trp, Tyr, Met, He or Leu with Phe; substitution of Thr or Ala with Ser; substitution of Ser or Ala with Thr; substitution of Phe or Tyr with Trp; substitution of His, Phe or Trp with Tyr; and substitution of Met, He or Leu with VaI. Meanwhile, the above-mentioned amino acid substitution, deletion, insertion, addition or inversion may be a result of a naturally-occurring mutation (mutant or variant) due to an individual difference, a difference of species of a bacterium harboring the nhaA gene, nhaB gene, chaA gene, mdfA gene, or nhaR gene. Meanwhile, each of the nhaA gene, nhaB gene, chaA gene and mdfA gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 1, 3, 23, or 25 or a probe that can be prepared from each of the sequences under stringent conditions and encodes a protein having the Na+/H+ antiporter activity. The nhaR gene may hybridize with a complementary strand of the nucleotide sequence of SEQ ID NO: 5 or a probe that can be prepared from the sequence under stringent conditions and encodes a protein having a function to increase expression of Na+/H+ antiporter. In the present invention, the term "stringent conditions" refers to conditions where a so-called specific hybrid is formed and a non-specific hybrid is not formed. It is difficult to clearly define the conditions by numerical value, but examples thereof include conditions where DNAs having high homology, for example, at least 80%, preferably 90%, more preferably 95%, and further more preferably 98% homology hybridize with each other and DNAs having homology less than the value do not hybridize with each other; and specifically include washing typical of Southern hybridization, e.g., washing at 60°C, I xSSC, 0.1% SDS, preferably 60°C, 0.1 *SSC, 0.1% SDS, more preferably 68°C, 0.1 xSSC, 0.1% SDS, once or preferably twice or three times. [0082]
Expression of the above-mentioned nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be increased by, for example, increasing the copy number of the genes in a cell using a gene recombination technique. For example, a DNA fragment containing the gene is ligated to a vector that functions in the host bacterium, preferably a multi-copy vector, to thereby prepare a recombinant DNA, and the recombinant DNA is used to transform the host bacterium. [0083]
When using the nhaA gene, nhaB gene, and nhaR gene of Escherichia coli, the nhaA gene, nhaB gene, and nhaR gene can be obtained by PCR (polymerase chain reaction; White, T.J. et al., Trends Genet. 5, 185 (1989)) using primers based on the nucleotide sequence of SEQ ID NOS: 1, 3 or 5, for example, primers of SEQ ID NOS: 9 and 10, 11 and 12 or 13 and 14 and a chromosomal DNA of Escherichia coli as a template. The nhaA gene, nhaB gene, and nhaR gene from another bacterium can also be obtained by PCR from the chromosomal DNA or genomic DNA library of the bacterium using, as primers, oligonucleotides prepared based on the known sequences of the nhaA gene, nhaB gene or nhaR gene of the bacterium or of the nhaA gene, nhaB gene or nhaR gene of another kind of bacterium, or the amino acid sequence of the NhaA protein, NhaB protein, or NhaR protein; or hybridization using an oligonucleotide prepared based on the sequence as a probe. A chromosomal DNA can be prepared from a bacterium that serves as a DNA donor by the method of Saito and Miura (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual for Biotechnology, edited by The Society for Biotechnology, Japan, p97-98, Baifukan Co., Ltd., 1992) or the like.
The chaA gene and mdf gene can also be obtained in the same way. [0084]
Then, a recombinant DNA is prepared by ligating the nhaA gene, nhaB gene, chaA gene, mdfA gene or nhaR gene which have been amplified by PCR to a vector DNA which is capable of functioning in the host bacterium. Examples of the vector capable of functioning in the host bacterium include vectors autonomously replicable in the host bacterium. [0085]
Examples of a vector which is autonomously replicable in Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSFlOlO (Gene vol. 75(2), p271-288, 1989), pBR322, pMW219, pMW119 (pMW is available form Nippon Gene Co., Ltd.), pSTV28, and pSTV29 (Takara Bio Inc.). A phage DNA vector can also be used. [0086]
To prepare ligate the gene to the above-mentioned vector, the vector is digested with a restriction enzyme corresponding to a recognition site in the terminus of a DNA fragment containing the nhaA gene, nhaB gene, chaA gene, mdfA gene or nhaR gene. Ligation is generally performed using a ligase such as T4 DNA ligase. Methods of digesting and ligating DNA, preparation of a chromosomal DNA, PCR, preparation of a plasmid DNA, transformation, design of oligonucleotides to be used as primers are methods well known to a person skilled in the art. These methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Sprig Harbor Laboratory Press, (1989), and the like. [0087]
The thus-prepared recombinant DNA is introduced into a bacterium in accordance with a conventional transformation method. Examples of the method include electroporation (Canadian Journal of Microbiology, 43. 197 (1997)). It is also possible to increase the DNA permeability by treating recipient cells with calcium chloride, which has been reported with Escherichia coli K-12 (Mandel, M. and Higa, A., J. MoI. Biol., 53, 159 (1970), and introduce a DNA into a competent cell prepared from a cell at proliferation stage, which has been reported with Bacillus subtilis (Duncan, C. H., Wilson, GA and Young, RE, Gene, 1, 153 (1977)). [0088]
The copy number of the nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can also be increased by introducing multiple copies of the genes into the chromosomal DNA of a bacterium. Introduction of multiple copies of the genes into the chromosomal DNA of a bacterium can be attained by homologous recombination using a target sequence present on the chromosomal DNA in multiple copies. Such a sequence present on a chromosomal DNA in multiple copies may be a repetitive DNA or an inverted repeat present on the edge of a transposing element. Alternatively, as disclosed in JP 2-109985 A, multiple copies of nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be introduced into the chromosomal DNA by inserting the genes into a transposon, and transferring it so that multiple copies of the gene are integrated into the chromosomal DNA. Integration of these genes into the chromosome can be confirmed by Southern hybridization using a portion of the genes as a probe. [0089]
Furthermore, expression of the nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene may be enhanced by, as described in WO 00/18935, substituting an expression regulatory sequence such as a promoter of the genes on a chromosomal DNA or of the genes on a plasmid with a stronger promoter, amplifying a regulator that increases expression of the genes, or deleting or attenuating a regulator that decreases expression of the genes. Examples of known strong promoters include the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and tet promoter.
The nhaR gene encodes a factor that positively regulates expression of the nhaA gene, so expression of the nhaA gene can be enhanced by amplifying expression of the nhaR gene.
Meanwhile, a promoter of nhaA gene, nhaB gene, chaA gene, mdfA gene, and/or nhaR gene can be made stronger so that expression of the genes are enhanced by introducing nucleotide substitution into it. Examples of a method of evaluating the strength of a promoter and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the like. In addition, it is known that a spacer sequence between the ribosome binding site (RBS) and translation initiation codon, especially, several nucleotides just upstream of the initiation codon, has a great influence on translation efficiency. Therefore, this sequence may be modified. [0090]
In addition, to enhance the activity of a protein encoded by the nhaA gene, nhaB gene, chaA gene, and/or mdfA gene, a mutation that increases the activity of the Na4VH+ antiporter may be introduced into the genes. Examples of such a mutation include a mutation in a promoter sequence to increase the transcription level of nhaA gene, nhaB gene, chaA gene, and/or mdfA gene, and a mutation in the coding region of these genes to increase the specific activities of the NhaA, NhaB, ChaA, or MdfA proteins. In addition, a mutation to enhance the activity of the NhaR protein that positively regulates expression of these genes may be introduced into the nhaR gene. [0091] <2> Method of producing L-amino acid
The method of producing an L-amino acid of the present invention comprises culturing the bacterium of the present invention in a medium to produce and accumulate an L-amino acid in the medium or bacterial cells, and collecting the L-amino acid from the medium or the bacterial cells. [0092]
A conventional medium to be used for fermentative production of an L-amino acid using a bacterium can be used. That is, a general medium containing a carbon source, nitrogen source, inorganic ion, and if necessary, other organic components can be used. In the present invention, examples of the carbon source include sugars such as glucose, sucrose, lactose, galactose, fructose and a starch hydrolysate; alcohols such as glycerol and sorbitol; and organic acids such as fumaric acid, citric acid and succinic acid. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; an organic nitrogen such as a soybean hydrolysate; ammonia gas; and aqueous ammonia. As organic trace nutrients, auxotrophic substances such as vitamin B 1 and L-homoserine, yeast extract, and the like are preferably contained in the medium in an appropriate amount. Besides such substances, if necessary, potassium phosphate, magnesium sulfate, iron ion, manganese ion, or the like may be added in small amounts. The medium to be used in the present invention may be a natural medium or a synthetic medium as long as it contains a carbon source, nitrogen source, inorganic ion, and if necessary, other organic trace nutrients. [0093]
The culture is preferably performed under aerobic conditions for 1 to 7 days at a temperature of 24°C to 37°C and a pH of 5 to 9. The pH can be adjusted with an inorganic or organic acidic or alkaline substance, ammonia gas or the like. The L-amino acid can be collected from the fermentation liquid by a conventional method such as ion-exchange resin, precipitation, and other known methods. Whenthe L-amino acid accumulates in the bacterial cells, the L-amino acid can be collected, for example, by disrupting the bacterial cells by ultrasonication or the like to release L-amino acid into the supernatant fraction, and then the bacterial cells are removed by centrifugation, followed by subjecting the resulting supernatant to an ion-exchange resin or the like. [0094]
When producing a basic amino acid, fermentation may be performed while controlling the pH of the medium during culture to 6.5-9.0 and controlling the pH of the medium after completion of the culture to 7.2-9.0, as well as controlling the pressure in the fermentation tank during fermentation so that it is positive. Alternatively, carbon dioxide or a mixed gas containing carbon dioxide may be added to the medium so that a bicarbonate ion and/or carbonate ion are present in an amount of at least 2 g/L in the culture medium during the culture period. These ions then function as counter ions against the cation of the basic amino acids, and the target basic amino acid may be collected(JP 2002-065287 A).
Examples [0095]
Hereinafter, the present invention will be described in more detail by referring to the following non-limiting examples. If not otherwise specified, reagents were purchased from Wako Pure Chemical Industries, Ltd. or Nacalai Tesque, Inc. The compositions of media used in Examples are shown below. The pH of each medium was adjusted with NaOH5KOH or HCl. [0096]
L medium:
Bacto-tryptone (Difco) 10 g/L
Yeast extract (Difco) 5 g/L
Sodium chloride 10 g/L pH 7.0
The medium was sterilized by steam at 120°C for 20 minutes.
[0097]
L agar medium:
L medium
Bacto-agar 15 g/L
The medium was sterilized by steam at 120°C for 20 minutes.
[0098]
L-lysine production medium for Escherichia bacterium:
Glucose 40 g/L
Ammonium sulfate 24 g/L
Potassium dihydrogen phosphate 1.0 g/L
Magnesium sulfate heptahydrate 1.0 g/L
Iron sulfate heptahydrate 0.01 g/L
Manganese sulfate heptahydrate 0.01 g/L
Yeast extract 2.0 g/L
Calcium carbonate (official grade) 50 g/L (separately sterilized)
The medium was adjusted to pH 7.0 with potassium hydroxide and sterilized by steam at
115°C for 10 minutes.
Glucose and MgSO4-7H2O were separately sterilized.
Calcium carbonate (official grade) was separately sterilized by dry heat at 180°C.
Chloramphenicol (25 mg/L) was added as an antibiotic.
[0099]
L-threonine production medium for Escherichia bacterium:
Glucose 40 g/L
Ammonium sulfate 16 g/L
Potassium dihydrogen phosphate 1.0 g/L Magnesium sulfate heptahydrate 1.0 g/L
Iron sulfate heptahydrate 0.01 g/L
Manganese sulfate heptahydrate 0.01 g/L
Calcium carbonate (official grade) 30 g/L (separately sterilized)
The medium was adjusted to pH 7.5 with potassium hydroxide and sterilized by steam at
115°C for 10 minutes.
Glucose and MgSO4-7H2O were separately sterilized.
Calcium carbonate (official grade) was separately sterilized by dry heat at 180°C.
Streptomycin (100 mg/L) and chloramphenicol (25 mg/L) were added as antibiotics.
[0100]
Example 1 :
<1> Construction of the plasmids used to amplify the nhaA gene, nhaB gene, or nhaR gene
To investigate the effects of amplifying the nhaA gene, nhaB gene, and nhaR gene on L-lysine production, vectors for amplifying each of these genes were constructed. The entire nucleotide sequence of the genome of Escherichia coli (Escherichia coli K- 12 strain) has been disclosed (Science, 277, 1453-1474 (1997)), and primers to amplify each of the genes (SEQ ID NOS: 9 and 10 for the nhaA gene, NOS: 11 and 12 for the nhaB gene, and SEQ ID NOS: 13 and 14 for the nhaR gene) were designed based on the known nucleotide sequences of these genes, which are disclosed in the publication. These primers were used to perform PCR using a chromosomal DNA of Escherichia coli MG 1655 strain as a template. The chromosomal DNA was obtained using Bacterial Genomic DNA purification kit (Edge Bio Systems). PCR was performed using Pyrobest DNA polymerase (Takara Bio Inc.) such that a cycle of 960C for 20 seconds, 65°C for 20 seconds and 72°C for 2 minutes was repeated 25 times.
Each of the PCR products was ligated to a Smal-digested vector pSTV28 (Takara Bio Inc.), to construct a plasmid for amplifying nhaA (pSnhaA), a plasmid for amplifying nhaB (pSnhaB), and a plasmid for amplifying nhaR (pSnhaR). Thereby, plasmids in which each of the above-mentioned genes was arranged downstream of a lac promoter in the forward direction were obtained. [0101] Example 2: Construction of a strain in which the lysine decarboxylase-encoding genes (cadA and ldcC) are disrupted
A strain which produces no lysine decarboxylase was constructed. The Lysine decarboxylases are encoded by the cadA gene (Genbank Accession No. NP 418555, SEQ ID NO: 19) and the ldcC gene (Genbank Accession No. NP_414728, SEQ ID NO: 21) (WO 96/17930). WC196 (FERM BP-5252) strain was used as the parent strain. [0102]
Disruption of the cadA gene and the ldcC gene were performed by the method developed by Datsenko and Wanner, which is called "Red-driven integration" (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) and by an excision system derived from λ phage (J. Bacteriol. 2002 Sep; 184(18): 5200-3. Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex. Cho EH, Gumport RI, Gardner JF.). "Red-driven integration" makes it possible to construct a gene-disrupted strain in one step by employing a PCR product obtained by using as primers synthetic oligonucleotides designed to have a part of the targeted gene on the 5 '-ends and a part of an antibiotic-resistance gene on the 3'-ends. Combining the λ phage-derived excision system permits the removal of the antibiotic-resistance gene that has been incorporated into the gene-disrupted strain ( WO2005/010175). [0103] (1) Disruption of the cadA gene
A plasmid pMWl 18-attL-Cm-attR (WO2005/010175) was used as a template for PCR. The plasmid pMW 118-attL-Cm-attR was obtained by inserting attL gene and attR gene, which are attachment sites of λ phage, and the cat gene, which is an antibiotic resistance gene, into pMWl 18 (Takara Bio Inc.) The genes are arranged in the following order: attL-cat-attR. [0104]
PCR was performed using, as primers, the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16, which have sequences corresponding to attL and attR on the 3'-terminals and a sequence corresponding to a part of the targeted cadA gene on the 5 '-terminals. [0105]
The amplified PCR product was purified on an agarose gel and introduced into Escherichia coli WC 196 strain by electroporation. This strain contains plasmid pKD46 which has temperature-sensitive replicability. The plasmid pKD46 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645) contains a DNA fragment of 2,154 nucleotides derived from λ phage which contains the Red recombinase-encoding genes (γ, β, and exo genes) of the λ Red homologous recombination system which is controlled by an arabinose-inducible ParaB promoter (GenBank/EMBL Accession No. J02459, nucleotide numbers 31088 to 33241). The plasmid pKD46 is necessary to integrate the PCR product into the chromosome of the WC 196 strain. [0106]
Competent cells for electroporation were prepared as follows. That is, cells of the Escherichia coli WC 196 strain were cultured overnight at 30°C in LB medium containing 100 mg/L ampicillin, and then diluted 100-fold with 5 mL of SOB medium (Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) containing ampicillin (20 mg/L) and L-arabinose (1 mM). The diluted cells were grown with aeration at 30°C until OD600 reached about 0.6, and then concentrated 100-fold and washed three times with 10% glycerol so that the cells were available for electroporation. The electroporation was performed with 70 μL of the competent cells and about 100 ng of the PCR product. After the electroporation, 1 mL of SOC medium (Molecular Cloning: Laboratory manual, 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) was added to the cells, cultured at 37°C for 2.5 hours, and then subjected to plate culture onto L-agar medium containing Cm (chloramphenicol) (25 mg/L), to thereby select Cm-resistant recombinant strains. Subsequently, to remove the plasmid pKD46, the cells were subcultured twice at 42°C on L-agar medium containing Cm, and ampicillin resistance of the resultant colonies were examined, to thereby yield ampicillin-sensitive strains in which pKD46 was removed. [0107]
Deletion of the cadA gene in the mutant strain, which had been identified by the chloramphenicol resistance gene, was confirmed by PCR. The cadA-disrupted strain was named WC196ΔcadA::att-cat strain. [0108]
Subsequently, the above-mentioned helper plasmid pMW-intxis-ts (JP 2005-058827 A) was used to remove the art-cat gene which had been introduced into the cadA gene. The plasmid pMW-intxis-ts carries the gene encoding integrase (Int) of λ phage, and the gene encoding excisionase (Xis), and has temperature-sensitive replicability. [0109]
Competent cells of the WC196ΔcadA::att-cat strain were prepared by a conventional method, and were then transformed with the helper plasmid pMW-intxis-ts, and then subjected to plate culture at 30°C onto L-agar medium containing 50 mg/L ampicillin, to thereby select ampicillin-resistant strains.
Subsequently, to remove the plasmid pMW-intxis-ts, the cells were subcultured twice at 42°C on L-agar medium, and ampicillin resistance and chloramphenicol resistance of the resulting colonies were examined, to thereby yield a chloramphenicol and ampicillin-sensitive strain, in which the cadA gene was disrupted, and art-cat and pMW-intxis-ts were removed. The strain was named WC196ΔcadA. [0110] (2) Disruption of the ldcC gene in the WC196ΔcadA strain
Disruption of the ldcC gene in the WC196ΔcadA strain was performed by using oligonucleotides of SEQ ID NOS: 17 and 18 as primers in the same way as described above. In this way, a cadA and ldcC-disrupted strain, WC 196ΔcadAΔldcC was obtained. [0111] Example 3 :
Effect of amplification of the nhaA gene, nhaB gene, and nhaR gene in an L-lysine-producing strain of Escherichia bacterium
Introduction of a plasmid for lysine production into the WC 196ΔcadAΔldcC strain
WC196ΔcadAΔldcC strain was transformed with a plasmid for lysine production, pCABD2 (WO 01/53459), which carries the dapA gene, dapB gene, and lysC gene, to thereby yield WC 196Δcad AΔldcC/pC ABD2 strain (WC196LC/pCABD2).
WC 196LC/pCABD2 strain was transformed with each of the plasmids prepared in Example 1: i.e., pSnhaA for amplifying nhaA; pSnhaB for amplifying nhaB; pSnhaR for amplifying nhaR; and a control plasmid pSTV28 (Takara Bio Inc.), to thereby yield chloramphenicol-resistant strains. Introduction of each of the plasmids was confirmed, and the pSnhaA-introduced strain, pSnhaB-introduced strain, pSnhaR-introduced strain, and pSTV28-introduced strain were named WC196LC/pCABD2/pSnhaA strain, WC196LC/pCABD2/pSnhaB strain, WC 196LC/ρC ABD2/pSnhaR strain, and WC196LC/pCABD2/pSTV28 strain, respectively.
WC 196LC/pCABD2/pSnhaA strain, WC196LC/pCABD2/pSnhaB strain, WC196LC/pCABD2/pSnhaR strain, and WC196LC/pCABD2/pSTV28 strain were each cultured at 37°C in L-medium containing 50 mg/L chloramphenicol until the final OD600 reached about 0.6, and an equal volume of 40% glycerol solution was added to each culture, followed by stirring. Then, the resulting solution was dispensed in appropriate amounts and stored at -80°C as glycerol stocks.
The glycerol stocks of the strains were thawed, and 100 μL of each stock was uniformly applied to an L-plate containing 25 mg/L chloramphenicol and 20 mg/L streptomycin and cultured at 37°C for 24 hours. The bacterial cells which grew on the plate were suspended in 3 mL of a fermentation medium, and 1 mL of the suspension whose OD620 was about 13.5 was inoculated into 20 mL of a fermentation medium (L-lysine production medium for Escherichia bacterium) containing 25 mg/L chloramphenicol and 20 mg/L streptomycin in a 500 mL-Sakaguchi flask and cultured at 37°C using a reciprocal shaker, followed by determination of the amount of L-lysine which had accumulated in the medium at 24 hours after beginning the culture. The amounts of L-lysine which accumulated in the medium were determined using Biotech Analyzer AS210 (Sakura Seiki Co,. Ltd.).
Table 1 shows the amounts of L-lysine which had accumulated in the medium. In the case of WC196LC/pCABD2/pSnhaA strain, WC196LC/pCABD2/ρSnhaB strain, and WC196LC/pCABD2/pSnhaR strain, the amounts of L-lysine accumulated in the medium were higher as compared to the control WCl 96LC/pCABD2/pSTV28 strain, which revealed that the lysine-producing ability was improved by amplifying the nhaA gene, nhaB gene, and nhaR gene. [0112]
Table 1
Amount of accumulated
Bacterial strain L-lysine (g/L)
24 hours later
WC 196LC/pC ABD2/pSTV28 8.7
WC 196LC/pCABD2/pSnhaA 11.8
WC 196LC/pCABD2/pSnhaB 11.6
WC 196LC/pCABD2/pSnhaR 14.9
[0113] Example 4
Effect of amplification of the nhaA gene, nha B gene, and nhaR gene in an L-threonine-producing strain of Escherichia bacterium
B-5318 strain was used as an L-threonine-producing strain. B-5318 strain was deposited in the Russian National Collection of Industrial Bacteria (VKPM), GNII Genetika) on May 3, 1990, under accession No. VKPM B-5318. The nhaA gene, nhaB gene, and nhaR gene were each amplified in the L-threonine-producing bacterium using the plasmids prepared in Example 1. [0114]
The B-5318 strain was transformed with the plasmids prepared in Example 1 : i.e., the pSnhaA for amplifying nhaA gene, pSnhaB for amplifying nhaB gene, and pSnhaR for amplifying nhaR, to thereby yield three different chloramphenicol-resistant strains. Introduction of the plasmids was confirmed, and the pSnhaA-introduced strain, pSnhaB-introduced strain, and pSnhaR-introduced strain were named B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain, respectively.
The B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain were cultured at 37°C in an L-medium containing 25 mg/L chloramphenicol and 100 mg/L streptomycin and the control B5318 strain was cultured in an L-medium containing 100 mg/L streptomycin until the final OD600 reached about 0.6. Then, an equal volume of a 40% glycerol solution was added to the culture, followed by stirring. Then, the solution was dispensed in appropriate amounts and stored at -80°C as glycerol stocks.
The glycerol stocks of the strains were thawed, and 100 μL of each of the plasmid-introduced strains was uniformly applied onto an L-plate containing 25 mg/L chloramphenicol and 100 mg/L streptomycin, while 100 μL of B5318 strain was uniformly applied on an L-plate containing 100 mg/L streptomycin, and they were cultured at 37°C for 24 hours. The bacterial cells which grew on the plate were suspended in 6 mL of physiological saline, and 1 mL of each of the suspensions whose OD620 was 22.0 was inoculated into 20 mL of a fermentation medium (L-threonine production medium for Escherichia bacterium) containing 25 mg/L chloramphenicol and 100 mg/L streptomycin (in the case of B-5318 strain, the medium contains only 100 mg/L streptomycin) in a 500 mL-Sakaguchi flask and cultured at 37°C for 24 hours using a reciprocal shaker. After the culture, the amounts of L-threonine which had accumulated in the medium were determined using high performance liquid chromatography.
Table 2 shows the amounts of L-threonine accumulated in the medium 6 and 24 hours after the beginning of the culture. In the case of B5318/pSnhaA strain, B5318/pSnhaB strain, and B5318/pSnhaR strain, the amounts of L-threonine which had accumulated after 6 and 24 hours were higher as compared to the control B5318 strain, which revealed that the L-threonine-producing ability was improved by amplifying nhaA gene, nhaB gene, and nhaR gene. [0115]
Table 2
Figure imgf000042_0001
Industrial Applicability [0116]
Use of the bacterium of the present invention enables efficient fermentative production of basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine and L-citrulline, aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine and L-glycine, hydroxy monoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acid such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, or acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine.

Claims

1. An L-amino acid-producing bacterium belonging to the Enterobacteriaceae family, wherein said bacterium has been modified so that Na+/H+ antiporter activity is enhanced.
2. The bacterium according to claim 1, wherein said Na+/H+ antiporter activity is enhanced by modifying said bacterium so that expression of a gene selected from the group consisting of nhaA gene, nhaB gene, nhaR gene, chaA gene, mdfA gene, and combinations thereof, is enhanced.
3. The bacterium according to claim 2, wherein said expression of is enhanced by increasing the copy number of the gene or genes, or by modifying an expression regulatory sequence of the gene or genes.
4. The bacterium according to claim 2 or 3, wherein said nhaA gene encodes a protein selected from the group consisting of:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and
(B) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 2, and has Na+/H+ antiporter activity.
5. The bacterium according to claim 2 or 3, wherein said nhaB gene encodes a protein selected from the group consisting of:
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4, and
(D) a protein comprising an amino acid sequence which includes substitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 4, and has Na+/H+ antiporter activity.
6. The bacterium according to claim 2 or 3, wherein said nhaR gene encodes a protein selected from the group consisting of:
(E) a protein comprising the amino acid sequence of SEQ ID NO: 6; and
(F) a protein comprising an amino acid sequence which includessubstitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 6, and has an activity to increase expression of Na+/H+ antiporter.
7. The bacterium according to claim 2 or 3, wherein said chaA gene encodes a protein selected from the group consisting of:
(G) a protein comprising the amino acid sequence of SEQ ID NO: 24; and (H) a protein comprising an amino acid sequence which includessubstitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 24, and has Na+/H+ antiporter activity.
8. The bacterium according to claim 2 or 3, wherein said mdfA gene encodes a protein selected from the group consisting of:
(I) a protein comprising the amino acid sequence of SEQ ID NO: 26; and (J) a protein comprising an amino acid sequence which includessubstitution, deletion, insertion or addition of one or several amino acids in SEQ ID NO: 26, and has NaVH+ antiporter activity.
9. The bacterium according to claim 2 or 3, wherein said nhaA gene is a DNA selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 ; and
(b) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
10. The bacterium according to claim 2 or 3, wherein said nhaB gene is a DNA selected from the group consisting of:
(c) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 3; and
(d) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 3 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
11. The bacterium according to claim 2 or 3, wherein said nhaR gene is a DNA selected from the group consisting of:
(e) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 5; and
(f) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 5 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has an activity to increase expression of Na+/H+ antiporter.
12. The bacterium according to claim 2 or 3, wherein said chaA gene is a DNA selected from the group consisting of:
(g) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 23; and
(h) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 23 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
13. The bacterium according to claim 2 or 3, wherein said mdfA gene is a DNA selected from the group consisting of:
(i) a DNA comprising a nucleotide sequence shown in SEQ ID NO: 25; and (j) a DNA that hybridizes with a complementary strand of the nucleotide sequence of SEQ ID NO: 25 or a probe prepared from the nucleotide sequence under stringent conditions, and wherein said DNA encodes a protein that has Na+/H+ antiporter activity.
14. The bacterium according to any one of claims 1 to 13, wherein said L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
15. The bacterium according to any one of claims 1 to 14, wherein said bacterium belongs to the genus Escherichia or Pantoea.
16. A method of producing an L-amino acid comprising culturing the bacterium according to any one of claims 1 to 15 in a medium to produce and accumulate an L-amino acid in the medium or cells of the bacterium and collecting the L-amino acid from the medium or the cells.
17. The method according to claim 16, wherein said L-amino acid is selected from the group consisting of L-lysine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-glutamic acid, and combinations thereof.
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