JP2010263789A - L-amino acid-producing microorganism and method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism which belongs to Enterobacter family and can efficiently produce an L-amino acid such as L-lysine, and to provide a method for producing an amino acid, by which the microorganism is used to efficiently produce the L-amino acid. <P>SOLUTION: The method for producing the L-amino acid comprises culturing a bacterium belonging to the Enterobacter family, which has productivity of the L-amino acid and has been modified to repress the expression of one or more genes selected from among ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW and yidQ, in a medium, thus accumulating the L-amino acid in the medium and then harvesting the L-amino acid from the medium. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing an L-amino acid using a microorganism, and more particularly to a method for producing an L-amino acid such as L-lysine, L-glutamic acid, L-threonine, L-tryptophan. L-glutamic acid is an industrially useful L-amino acid as a seasoning, and L-lysine, L-threonine, and L-tryptophan are industrially useful L-amino acids as animal feed additives, health food ingredients, amino acid infusions, and the like.

  L-amino acids are industrially produced by fermentation methods using various microorganisms. For example, L-glutamic acid is mainly produced by fermentation using so-called coryneform bacterium L-glutamic acid-producing bacteria belonging to the genus Brevibacterium, Corynebacterium, and Microbacterium, or mutants thereof ( For example, refer nonpatent literature 1). As a method for producing L-glutamic acid by fermentation using other microorganisms, microorganisms such as Bacillus genus, Streptomyces genus, Penicillium genus (for example, see Patent Document 1), Pseudomonas genus, Arthrobacter genus, Serratia genus, Microorganisms such as Candida (see, for example, Patent Document 2), microorganisms such as Bacillus, Pseudomonas, Serratia, Aerobacter Aerogenes (currently Enterobacter aerogenes) (see, for example, Patent Document 3), Escherichia coli A method using a mutant strain (for example, see Patent Document 1) or the like is known. Moreover, the manufacturing method of L-glutamic acid using the microorganisms which belong to Klebsiella genus, Erbinia genus or Pantoea genus, Enterobacter genus is also disclosed (for example, refer patent documents 2-4).

  In order to produce a target substance such as an L-amino acid by fermentation using a microorganism as described above, a method using a wild-type microorganism (wild strain), a method using an auxotrophic strain derived from a wild strain, a wild strain There are a method using metabolic control mutants derived from various drug-resistant mutants, and a method using strains having properties of both auxotrophic strains and metabolic control mutants.

  Furthermore, in recent years, recombinant DNA technology has been used for fermentative production of target substances. For example, enhancing the expression of a gene encoding an L-amino acid biosynthetic enzyme (Patent Literature 5, Patent Literature 6), or enhancing the inflow of a carbon source into the L-amino acid biosynthesis system (Patent Literature 7). ) To improve L-amino acid productivity of microorganisms.

The ydcU gene is presumed to encode the ABC transporter component (Non-patent Document 2) or the spermidine / putrescine transporter subunit (Non-patent Document 3) based on homology with the amino acid sequence encoded by the gene. However, no experimental proof has been made. There is no report on the relationship between the ydcU gene and L-amino acid production.
The smpA gene is presumed to encode small membrane protein A (Non-patent Document 4). The htrG gene is presumed to encode signal transduction proterin (SH3). The adiC gene is presumed to encode arginine: agmatine antiporte (Non-patent Document 5. The htrA gene encodes a serine endoprotease (protease Do) that is membrane-bound serine endoprotease (protease Do). (Non-patent document 6) The ydhK gene is presumed to encode a conserved inner membrane protein, and the bacA gene is presumed to encode undecaprenyl-diphosphatase. (Non-patent document 7) The yaiW gene is presumed to encode a predicted DNA-binding transcriptional regulator (non-patent document 8) The yidQ gene is conserved outer. It is presumed to encode membrane protein, but these genes and L-
There is no report on the relationship with amino acid production.
JP-A-5-244970 US Pat. No. 3,563,857 Japanese Patent Publication No.32-9393 JP 2000-189175 A US Pat. No. 5,168,056 US Pat. No. 5,776,736 US Pat. No. 5,906,925 Akashi Kunihiko et al. Amino Acid Fermentation, Academic Publishing Center, 195-215, Saurin, W. et al., J. Mol. Evol., 48 (1); 22-41 (1999) Riley, M. et al., Nucleic Acids Res., 34: 1-9 (2006) Proc. Natl. Acad. Sci. USA., 2007 Apr 10; 104 (15): 6400-5. Epub 2007 Apr 2. J. Bacteriol., 2003 Nov; 185 (22): 6556-61.) Arch. Biochem. Biophys., 2007 Aug 1; 464 (1): 80-9. Microbiology. 2007 Aug; 153 (Pt 8): 2518-29. FEMS Microbiol Lett., 2003 Aug 8; 225 (1): 1-7.

  An object of the present invention is to provide a microorganism belonging to the family Enterobacteriaceae that can efficiently produce an L-amino acid, and to provide a method for efficiently producing an L-amino acid using the microorganism. .

  As a result of intensive studies to solve the above problems, the present inventors have been modified so that the expression of ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ gene is weakened. It has been found that bacteria belonging to the family Enterobacteriaceae can efficiently produce L-amino acids, and the present invention has been completed.

That is, the present invention is as follows.
(1) L-amino acid producing ability and expression of one or more genes selected from ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, and yidQ are weakened Bacteria belonging to the family Enterobacteriaceae.
(2) The bacterium, wherein the expression of each gene is weakened by inactivation of ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ.
(3) The bacterium as described above, wherein the gene is ydcU, yfeK, or smpA.
(4) The bacterium as described above, wherein the ydcU gene is DNA encoding the amino acid sequence of SEQ ID NO: 2 or a variant thereof.
(5) The bacterium as described above, wherein the yfeK gene is DNA encoding the amino acid sequence of SEQ ID NO: 4 or a variant thereof.
(6) The bacterium as described above, wherein the smpA gene is DNA encoding the amino acid sequence of SEQ ID NO: 6 or a variant thereof.
(7) The bacterium as described above, wherein the htrG gene is DNA encoding the amino acid sequence of SEQ ID NO: 8 or a variant thereof.
(8) The bacterium as described above, wherein the adiC gene is DNA encoding the amino acid sequence of SEQ ID NO: 10 or a variant thereof.
(9) The bacterium as described above, wherein the htrA gene is DNA encoding the amino acid sequence of SEQ ID NO: 12 or a variant thereof.
(10) The bacterium as described above, wherein the ydhK gene is DNA encoding the amino acid sequence of SEQ ID NO: 14 or a variant thereof.
(11) The bacterium as described above, wherein the bacA gene is DNA encoding the amino acid sequence of SEQ ID NO: 16 or a variant thereof.
(12) The bacterium as described above, wherein the yaiW gene is DNA encoding the amino acid sequence of SEQ ID NO: 18 or a variant thereof.
(13) The bacterium as described above, wherein the yidQ gene is DNA encoding the amino acid sequence of SEQ ID NO: 20 or a variant thereof.
(14) The L-amino acid is L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L- The bacterium as described above, which is one or more L-amino acids selected from the group consisting of tryptophan and L-cysteine.
(15) The bacterium as described above, wherein the L-amino acid is L-lysine and the activity of lysine decarboxylase is weakened.
(16) The bacterium as described above, wherein the microorganism belonging to the family Enterobacteriaceae is an Escherichia bacterium, an Enterobacter bacterium, or a Pantoea bacterium.
(17) A method for producing an L-amino acid, comprising culturing the bacterium in a medium, accumulating the L-amino acid in the medium, and collecting the L-amino acid from the medium.
(18) The L-amino acid is L-glutamic acid, L-lysine, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L- The said method which is 1 type, or 2 or more types of L-amino acids selected from tryptophan and L-cysteine.
(19) The method as described above, wherein the L-amino acid is L-lysine.

  By using the microorganism of the present invention, L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-threonine, L- L-amino acids such as phenylalanine, L-tyrosine, L-tryptophan, or L-cysteine, L-glutamic acid can be produced by fermentation.

Hereinafter, the present invention will be described in detail.
<1> Microorganism of the Present Invention The bacterium of the present invention has L-amino acid-producing ability and is selected from ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, and yidQ. It is a bacterium belonging to the family Enterobacteriaceae that has been modified so that the expression of further genes is weakened. Here, the L-amino acid-producing ability means that when the bacterium of the present invention is cultured in a medium, the L-amino acid is produced in the medium or in the microbial cells and accumulated to such an extent that it can be recovered in the medium or from the microbial cells. Say. The bacterium of the present invention may be capable of producing a plurality of L-amino acids. As a bacterium having L-amino acid-producing ability, it may originally have L-amino acid-producing ability, but the following bacteria can be obtained by using a mutation method or recombinant DNA technology, It may be modified so as to have L-amino acid production ability.

Examples of the microorganism used in the present invention include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella Intestinal bacteria belonging to γ-proteobacteria such as Morganella, so-called coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium, Microbacterium, Alicyclobacillus, Bacillus ( Examples include microorganisms belonging to the genus Bacillus, the genus Saccharomyces, and the like. γ-Proteobacteria is NCBI (National Center for Biotechnology Information) taxonomy database http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=1236&lvl=3&p=mapview&p=has_linkout&p = blast_url & p = genome_blast & lin = f & keep = 1 & srchmode = 1 & unlock), those belonging to microorganisms can be used.

  Examples of bacteria belonging to the genus Escherichia include Escherichia coli. When breeding Escherichia coli using genetic engineering techniques, Escherichia coli K-12 strain and its derivatives, Escherichia coli MG1655 strain (ATCC 47076), and W3110 strain (ATCC 27325) may be used. it can. The Escherichia coli K-12 strain was isolated at Stanford University in 1922 and is a lysogen of λ phage and has F factor and can be used to create genetic recombinants such as conjugation. It is a highly bacterial strain. The genomic sequence of Escherichia coli K-12 has already been determined, and genetic information can be freely used. In order to obtain Escherichia coli K-12 strain and derivative strains, for example, they can be sold from the American Type Culture Collection (ATCC) (address ATCC, Address: PO Box 1549, Manassas, VA 20108, United States of America).

  In particular, Pantoea bacteria, Erwinia bacteria, and Enterobacter bacteria are bacteria classified as γ-proteobacteria, and are taxonomically closely related (J. Gen. Appl. Microbiol. 1997, 43 : 355-361; Int. J. Syst. Bacteriol. 1997, 47: 1061-1067). In recent years, bacteria belonging to the genus Enterobacter have been reclassified as Pantoea agglomerans or Pantoea dispersa by DNA-DNA hybridization experiments (Int. J. Syst. Bacteriol. 1989, 39: 337-345). Some bacteria belonging to the genus Erbinia have been reclassified as Pantoea ananas and Pantoea stealty (see Int. J. Syst. Bacteriol. 1993, 43: 162-173).

Enterobacter is an Enterobacter bacterium.
agglomerans) and Enterobacter aerogenes. Specifically, strains exemplified in European Patent Application Publication No. 952221 can be used. A representative strain of the genus Enterobacter is Enterobacter agglomerans ATCC12287.

Typical strains of the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specifically, the following strains are mentioned.
Pantoea Ananatis AJ13355 (FERM BP-6614) (European Patent Application Publication No. 0952221)
Pantoea Ananatis AJ13356 (FERM BP-6615) (European Patent Application Publication No. 0952221)

  In addition, although these strains are described as Enterobacter agglomerans in European Patent Application Publication No. 0952221, they are currently reclassified as Pantoea Ananatis by 16S rRNA nucleotide sequence analysis as described above. Has been.

Examples of the genus Erwinia include Erwinia amylobola and Erwinia carotobola, and examples of the genus Klebsiella include Klebsiella planticola. Specifically, the following strains are mentioned.
Elvinia Amilobola ATCC15580 Strain Elvinia Carotobola ATCC15713 Strain Klebsiella Planticola AJ13399 (FERM BP-6600) (European Patent Application Publication 955368)
(No. Description)
Klebsiella planticola AJ13410 strain (FERM BP-6617) (European Patent Application Publication No. 955368)

  In the present invention, the bacterium having L-amino acid-producing ability refers to a larger amount of L-amino acid, for example, preferably 0.5 g / in, than the wild strain or the parent strain of the bacterium when cultured in the medium or in the medium. It means a bacterium that accumulates L-amino acid of L or more, more preferably 1.0 g / L or more. Examples of the wild strain of bacteria include Escherichia coli such as Escherichia coli K-12.

  The type of L-amino acid is not particularly limited, but basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L -Aliphatic amino acids such as leucine, L-glycine, amino acids that are hydroxymonoaminocarboxylic acids such as L-threonine, L-serine, cyclic amino acids such as L-proline, L-phenylalanine, L-tyrosine, Aromatic amino acids such as L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine In particular, L-lysine, L-threonine, and L-tryptophan are preferable. L-lysine The most preferred. The bacterium of the present invention may be capable of producing two or more amino acids.

Hereinafter, a method for imparting L-amino acid-producing ability to the bacterium as described above or a method for enhancing the L-amino acid-producing ability of the bacterium as described above will be described.
In order to confer L-amino acid-producing ability, an auxotrophic mutant, an L-amino acid analog-resistant strain or a metabolically controlled mutant, and a recombinant strain with enhanced expression of an L-amino acid biosynthetic enzyme Can be applied to the breeding of amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Academic Publishing Center, Inc., May 30, 1986, first edition) Issue, see pages 77-100). Here, in the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be used singly or in combination of two or more. In addition, L-amino acid biosynthesis enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

  In order to obtain an auxotrophic mutant, an analog-resistant mutant, or a metabolically controlled mutant having L-amino acid production ability, the parent strain or the wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl Among the mutant strains obtained by treatment with a mutant such as -N'-nitro-N-nitrosoguanidine, auxotrophy, analog resistance, or metabolic control mutation is shown, and L-amino acid production ability is exhibited. It can be obtained by selecting what it has.

  The L-amino acid-producing ability can also be imparted or enhanced by enhancing enzyme activity by gene recombination. Examples of the enhancement of enzyme activity include a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, introducing an amplified plasmid in which a DNA fragment containing the gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication replication function of the plasmid in a microorganism, Alternatively, it can be achieved by making multiple copies of these genes on the chromosome by joining, transferring, etc., and introducing mutations into the promoter regions of these genes (see International Publication WO95 / 34672).

When the target gene is introduced onto the amplification plasmid or chromosome, the promoter for expressing these genes may be any promoter that functions in coryneform bacteria, and the promoter of the gene itself used. Or it may be modified. The expression level of the gene can also be controlled by appropriately selecting a promoter that functions strongly in coryneform bacteria, or by bringing the −35 and −10 regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like.

  Hereinafter, a method for imparting L-amino acid-producing ability to bacteria and a bacterium imparted with L-amino acid-producing ability will be exemplified.

L-Threonine-Producing Bacteria Preferred microorganisms having L-threonine-producing ability include bacteria having enhanced activity of one or more of L-threonine biosynthetic enzymes. Examples of the L-threonine biosynthesis enzyme include aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA) encoded by the thr operon, homoserine kinase (thrB), threonine synthase ( thrC), aspartate aminotransferase (aspartate transaminase) (aspC). The parentheses are abbreviations for the genes (the same applies to the following description). Of these enzymes, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase are particularly preferred. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of the Escherichia bacterium in which threonine degradation is suppressed include, for example, the TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

  The enzyme activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute the threonine operon, but the threonine operon forms an attenuator structure, and the expression of the threonine operon inhibits isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. This modification can be achieved by removing the leader sequence of the attenuation region or the attenuator. (Lynn, SP, Burton, WS, Donohue, TJ, Gould, RM, Gumport, RI, and Gardner, JFJ Mol. Biol. 194: 59-69 (1987); WO 02/26993 pamphlet; 2005/049808 pamphlet)

  A unique promoter is present upstream of the threonine operon, but it may be replaced with a non-natural promoter (see WO98 / 04715 pamphlet), and the expression of a gene involved in threonine biosynthesis is expressed by a lambda phage repressor and A threonine operon as governed by a promoter may be constructed. (See European Patent No. 0593792) In order to modify bacteria so that it is not subject to feedback inhibition by L-threonine, a strain resistant to α-amino-β-hydroxyvaleric acid (AHV) may be selected. Is possible.

  Thus, the threonine operon modified so as not to receive feedback inhibition by L-threonine has an increased copy number in the host or is linked to a strong promoter, and the expression level is improved. Is preferred. The increase in copy number can be achieved by transferring the threonine operon onto the genome by transposon, Mu-fuzzy, etc. in addition to amplification by plasmid.

In addition to the L-threonine biosynthetic enzyme, it is also preferable to enhance a glycolytic system, a TCA cycle, a gene related to the respiratory chain, a gene that controls gene expression, and a sugar uptake gene. These genes effective for L-threonine production include transhydronase (pntAB) gene (European Patent 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication No. 95/06114 pamphlet), phospho Examples include the enol pyruvate synthase gene (pps) (European Patent No. 877090), the pyruvate carboxylase gene of Coryneform bacteria or Bacillus bacteria (International Publication No. 99/18228, European Application Publication No. 1092776).

  It is also preferable to enhance the expression of a gene imparting resistance to L-threonine and a gene conferring resistance to L-homoserine, or imparting L-threonine resistance and L-homoserine resistance to the host. Examples of genes that confer resistance include the rhtA gene (Res. Microbiol. 154: 123-135 (2003)), the rhtB gene (European Patent Application Publication No. 0994190), and the rhtC gene (European Patent Application Publication No. 1013765). ), YfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, the methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

  Examples of L-threonine-producing bacteria or parent strains for inducing them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli FERM BP-3519 And FERM BP-3520 (US Pat.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) Strains belonging to the genus Escherichia such as, but not limited to.

  The TDH-6 strain lacks the thrC gene and is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 shares were deposited with the accession number RIA 1867 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) on November 19, 1987 . The strain was also deposited with the accession number B-3996 on 7 April 1987 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia). Has been.

  E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. Has been deposited internationally.

The thrA gene encoding aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide numbers 337 to 2799, GenBank accession NC — 000913.2, gi: 49175990). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913
.2, gi: 49175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding the threonine synthase of Escherichia coli has been elucidated (nucleotide numbers 3734-5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon. To increase the expression of the threonine operon, the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).

  The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.

The rhtA gene is present at 18 minutes of the E. coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is ORF1 (ybiF gene, nucleotide numbers 764 to 1651,
GenBank accession number AAA218541, gi: 440181), and located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called the rhtA gene (rht: resistant to homoserine and threonine). It has also been found that the rhtA23 mutation is a G → A substitution at position -1 relative 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 asd gene of E. coli has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the base sequence of the gene. Yes (see White, TJ et al., Trends Genet., 5, 185 (1989)). The asd gene of other microorganisms can be obtained similarly.

  In addition, the aspC gene of E. coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895) and can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to L-lysine analogs. L-lysine analogues inhibit the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and the like. . Mutant strains resistant to these lysine analogs can be obtained by subjecting bacteria belonging to the genus Escherichia to normal artificial mutation treatment. Specific examples of bacterial strains useful for the production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is released.

The WC196 strain can be used as an L-lysine-producing bacterium of Escherichia coli. This strain was bred by conferring AEC resistance to the W3110 strain derived from Escherichia coli K-12. This strain was named Escherichia coli AJ13069. On December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, Ibaraki, 305-8566, Japan Deposited as FERM P-14690 in Tsukuba City Higashi 1-chome 1 1 Central 6), transferred to the international deposit under the Budapest Treaty on 29 September 1995, and assigned the accession number FERM BP-5252 (US Pat. No. 5,827,698).

  Examples of L-lysine-producing bacteria or parent strains for deriving the same also include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced. Examples of such enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160). ), Phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase gene, diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (dapE) and aspartase (aspA) ( EP 1253195 A), but is not limited to these. Among these enzymes, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and Succinyl diaminopimelate deacylase is particularly preferred. In addition, the parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Pat.No. 5,830,716), a ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.

  Examples of L-lysine producing bacteria or parent strains for deriving the same include a decrease or loss of the activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine. There are also stocks. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175).

  A preferred L-lysine-producing bacterium is Escherichia coli WC196ΔcadAΔldc / pCABD2 (WO2006 / 078039). This strain is obtained by introducing the plasmid pCABD2 described in US Pat. No. 6,040,160 into the WC196 strain in which the cadA and ldcC genes encoding lysine decarboxylase are disrupted. WC196ΔcadAΔldc itself is also a preferred L-lysine-producing bacterium. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation in which feedback inhibition by L-lysine is released, and a mutation in which feedback inhibition by L-lysine is released. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes. Escherichia coli W3110 (tyrA) / pCABD2 carrying this plasmid was named AJ12604, and on January 28, 1991, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (address Tsukuba, Ibaraki, Japan 305-8566) Deposited under FERM P-11975 deposit number at 1-chome, 1-chome, 1-center, 6), transferred to an international deposit under the Budapest Treaty on September 26, 1991, and deposited under a deposit number of FERM BP-3579 ing.

Examples of L-cysteine producing bacteria L-cysteine producing bacteria or parent strains for deriving them include E. coli JM15 (US Pat. No. 6,218,168) transformed with a different cysE allele encoding a feedback inhibition resistant serine acetyltransferase. , Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfohydrase activity These include strains belonging to the genus Escherichia such as E. coli strains that have been reduced (JP11155571A2) and E. coli W3110 (WO0127307A1) that have increased the activity of the transcriptional regulator of the positive cysteine regulon encoded by the cysB gene. It is not limited.

Examples of L-leucine-producing bacteria L-leucine-producing bacteria or parent strains for inducing them include leucine-resistant E. coil strains (for example, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or β E. coli strains resistant to leucine analogs such as 2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (Japanese Patent Publication No. 62-34397 and JP-A-8-70879), Examples include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strains and E. coli H-9068 (JP-A-8-70879) obtained by the genetic engineering method described in WO96 / 06926. .

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis. As an example of such a gene, a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase preferably released from feedback inhibition by L-leucine is mentioned. . Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).

Examples of L-histidine-producing bacteria L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, RU2003677), E. coli 80 strain (VKPM B-7270, RU2119536) E. coli NRRL B-12116-B12121 (U.S. Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.No. 6,344,347), E. coli. Examples include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli H-9341 (FERM BP-6674) (EP1085087) and E. coli AI80 / pFM201 (US Pat. No. 6,258,554).

  Examples of L-histidine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.

  L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).

Specific examples of strains capable of producing L-histidine include E. coli FERM-P 5038 and 5048 into which a vector holding a DNA encoding an L-histidine biosynthetic enzyme has been introduced (Japanese Patent Laid-Open No. 56-005099). , E. coli strain introduced with a gene for amino acid transport (EP1016710A), sulfaguanidine,
Examples include E. coli strain 80 (VKPM B-7270, Russian Patent No. 2119536) imparted with resistance to DL-1,2,4-triazole-3-alanine and streptomycin.

Examples of L-glutamic acid-producing bacteria L-glutamic acid-producing bacteria or parent strains for inducing them 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 an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7). As a result, L-isoleucine-requiring L-glutamic acid producing bacterium VL334thrC + (VKPM B-8961) was obtained.

  Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, L-glutamic acid biosynthetic enzymes having one or two or more enhanced activities. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Methyl citrate synthase (prpC), phosphoenolpyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase ( eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase ( fbp), phosphofructokinase (pfkA) , pfkB), glucose phosphate isomerase (pgi) and the like. Of these enzymes, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase are preferred.

  Examples of strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A and EP952221A.

  As an example of an L-glutamic acid-producing bacterium or a parent strain for inducing the same, the activity of an enzyme that catalyzes the synthesis of a compound other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or deficient. Stocks are also mentioned. Examples of such enzymes include isocitrate triase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Examples include acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and the like. Bacteria belonging to the genus Escherichia lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. .

Specific examples include the following.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA :: Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter also referred to as “sucA gene”) of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.

  Other examples of L-glutamic acid-producing bacteria include those belonging to the genus Escherichia and having resistance to an aspartic acid antimetabolite. These strains may be deficient in α-ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5.908,768), and FFRM with reduced L-glutamate resolution. P-12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

  An example of an L-glutamic acid-producing bacterium of Pantoae ananatis is Pantoea ananatis AJ13355 strain. This strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from the soil of Kamata City, Shizuoka Prefecture. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Address: 1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki 305-8566, Japan) Deposited under the number FERM P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999 and given the deposit number FERM BP-6614. The strain was identified as Enterobacter agglomerans at the time of its isolation and was deposited as Enterobacter agglomerans AJ13355, but in recent years, it has been identified by Pantoea ananatis (Pantoea ananatis) by 16S rRNA sequence analysis. ).

  Examples of L-glutamic acid-producing bacteria of Pantoae ananatis include bacteria belonging to the genus Pantoea, which lack α-ketoglutarate dehydrogenase (αKGDH) activity or have reduced αKGDH activity. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the αKGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected as a mucus low production mutant strain from AJ13355 strain. There is SC17sucA (US Pat. No. 6,596,517) which is a gene-deficient strain. AJ13356 was founded on February 19, 1998, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-chome, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain was assigned the private number AJ417 and was deposited at the National Institute of Advanced Industrial Science and Technology as the deposit number FERM BP-08646 on February 26, 2004.

  Further, L-glutamic acid-producing bacteria of Pantoae ananatis include SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain. The SC17sucA / RSFCPG + pSTVCB strain is a plasmid RSFCPG containing the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain is a strain selected from this SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki 305-8566, Japan). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.

Examples of L-phenylalanine producing bacteria L-phenylalanine producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA :: Tn10, tyrR) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor ( VKPM B-8197) (WO03 / 044191), E. coli HW1089 (ATCC 55371) carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with released feedback inhibition (US Pat.No. 5,354,672), Strains belonging to the genus Escherichia such as E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat.No. 4,407,952) However, it is not limited to these. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K that retains the gene encoding chorismate mutase-prefenate dehydratase whose feedback inhibition is canceled -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 named AJ 12604 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) can also be used (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1, WO03 / 044192).

Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking the tryptophanyl-tRNA synthetase encoded by the mutant trpS gene (DSM10123) (U.S. Pat.No. 5,756,345), E. coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan. SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) Escherichia coli such as E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, US Pat.No. 6,319,696) with increased phosphoenolpyruvate production capacity Strains include belonging to Rihia genus, but is not limited thereto. L-tryptophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667).
A1).

  Examples of L-tryptophan-producing bacteria or parent strains for inducing them include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), 3-deoxy-D-arabinohepturonic acid-7-phosphorus Acid synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolate pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC ), Prephenate dehydratase, chorismate mutase and tryptophan synthase (trpAB). One or more strains having enhanced activity are also included. Prefenate dehydratase and chorismate mutase are encoded by the pheA gene as a bifunctional enzyme (CM-PD). Among these enzymes, phosphoglycerate dehydrogenase, 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase, 3-dehydroquinate synthase, shikimate dehydratase, shikimate kinase, 5-enolic acid Pyruvylshikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, chorismate mutase-prefenate dehydrogenase are particularly preferred. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase desensitized to feedback inhibition Examples include a transformant obtained by introducing plasmid pGH5 (WO 94/08031) into E. coli SV164.

  Examples of L-tryptophan-producing bacteria or parent strains for deriving them include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (JP-A 57-71397, JP-A 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of isocitrate triase-malate synthase operon (WO2005 / 103275).

Examples of L-proline-producing bacteria L-proline-producing bacteria or parent strains for deriving them include E. coli 702ilvA (VKPM B-8012) (EP 1172433), which lacks the ilvA gene and can produce L-proline. Strains belonging to the genus Escherichia, but are not limited thereto.

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP1239041 A2).

  Examples of bacteria belonging to the genus Escherichia having L-proline producing ability include NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), German Patent No. 3127361 And E. coli strains such as those described in Bloom FR et al (The 15th Miami winter symposium, 1983, p. 34).

Examples of L-arginine producing bacteria L-arginine producing bacteria or parent strains for inducing them include E. coli 237 strain (VKPM B-7925) (US Patent Application Publication 2002/058315 A1) and mutant N- Derivatives carrying acetylglutamate synthase (Russian patent application No. 2001112869), E. coli 382 strain (VKPM B-7926) (EP1170358A1), arginine producing strain introduced with argA gene encoding N-acetylglutamate synthetase Examples include, but are not limited to, strains belonging to the genus Escherichia, such as (EP1170361A1).

  Examples of L-arginine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoyl phosphate synthetase gene (carAB).

Examples of L-valine-producing bacteria L-valine-producing bacteria or parent strains for inducing them include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. L is produced by removing the ilvGMEDA operon area required for attenuation.
-It is preferable that the expression of the operon is not attenuated by valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.

  Examples of L-valine-producing bacteria or parent strains for deriving the same also include mutant strains having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was accepted on June 24, 1988 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) under the accession number VKPM B-4411. It has been deposited.

Furthermore, a mutant strain (WO96 / 06926) that requires lipoic acid for growth and / or lacks H ⁺ -ATPase can be used as a parent strain.

Examples of L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing them include mutants having resistance to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as the above, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882), but are not limited thereto. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

  The bacterium of the present invention has the ability to produce L-amino acids as described above, and one or more selected from ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, and yidQ. It has been modified so that gene expression is weakened.

  The bacterium of the present invention is a bacterium belonging to the family Enterobacteriaceae having L-amino acid-producing ability, wherein ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW or yidQ gene is expressed by genetic recombination It can be obtained by modifying to weaken. In the breeding of bacteria belonging to the family Enterobacteriaceae of the present invention, either modification of imparting L-amino acid-producing ability or modification that weakens the expression of each gene may be performed first.

  “Modified to attenuate the expression of one or more genes selected from ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ” means an unmodified strain, such as a parent strain or YdcU, YfeK, SmpA, HtrG, AdiC, HtrA, YdhK, BacA, YaiW or ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ compared to the wild type This means that each protein of YidQ has been modified so that it does not function normally. For example, by modifying the ydcU gene by genetic recombination, the number of YdcU protein molecules per cell relative to the parent strain or wild strain decreases, or when no YdcU protein molecules are produced, or YdcU This includes cases where the activity per molecule of the protein is reduced or lost. The number of YdcU protein molecules can be decreased by decreasing the transcription amount of the ydcU gene or the translation amount of the ydcU mRNA. The same applies to genes other than the ydcU gene. Examples of wild strains to be compared include Escherichia coli K12 strain, Pantoea ananatis AJ13355 strain, and Klebsiella planticola AJ13399 strain.

  Inactivation of ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ gene (hereinafter also referred to as “target gene”) refers to each of the genes encoded by each modified gene. It means that the protein loses its function completely.

  Specifically, each target gene is altered by deleting part or all of each target gene coding region on the chromosome, or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Etc. are achieved. In addition, the expression level of a gene can also be reduced by modifying an untranslated region other than the expression regulatory sequence. Furthermore, you may delete the whole target gene including the arrangement | sequence before and behind the target gene on a chromosome. In addition, a gene recombination introduces an amino acid substitution (missense mutation) into the coding region of the target gene on the chromosome, introduces a stop codon (nonsense mutation), or a frame in which one or two bases are added or deleted. It can also be achieved by introducing a shift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 266, 20833-20839 ( 1991)).

  The modification of each gene is preferably performed by gene recombination. Specifically, the gene recombination method uses homologous recombination to delete the expression regulatory sequence of the target gene on the chromosome, for example, the promoter region, the coding region, or a part or all of the non-coding region. Or insertion of other sequences into these regions.

  The modification of the expression regulatory sequence is preferably 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. When the coding region is deleted, if the function of the protein produced by each gene is reduced or deleted, the region to be deleted can be any of the N-terminal region, internal region, and C-terminal region. It may be the entire code area. Usually, the longer region to be deleted can surely inactivate the target gene. Further, it is preferable that the reading frames upstream and downstream of the region to be deleted do not match.

  Even when another sequence is inserted into the coding region, the insertion position may be in any region of the target gene, but the longer the inserted sequence, the more reliably the target gene can be inactivated. The sequences before and after the insertion site preferably do not match the reading frame. Other sequences are not particularly limited as long as they reduce or eliminate the function of the protein encoded by the target gene. Examples include antibiotic resistance genes and transposons carrying genes useful for L-glutamic acid production. It is done.

  In order to modify a target gene on a chromosome as described above, for example, a deletion type gene is prepared by deleting a partial sequence of the target gene and modifying it so as not to produce a protein that functions normally. This can be accomplished by replacing the target gene on the chromosome with the deleted gene by transforming bacteria with the contained DNA and causing homologous recombination between the deleted gene and the target gene on the chromosome. Even if the protein encoded by the deletion-type target gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function decreases or disappears. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), or Red-driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 ( 2002)) and the like (see WO2005 / 010175), a method using linear DNA, a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, and a replication origin in a host. For example, there is a method using a suspension vector that does not have (US Pat. No. 6,303,383 or Japanese Patent Laid-Open No. 05-007491).

Confirmation that the transcription amount of the target gene has decreased can be confirmed by comparing the amount of mRNA transcribed from the target gene with a wild type strain or an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular c
loning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The decrease in the amount of transcription may be any as long as it is reduced compared to the wild strain or the unmodified strain, but for example, at least 75% or less, 50% or less, 25% or less, compared to the wild strain or the unmodified strain, Alternatively, it is desirable that the concentration is reduced to 10% or less, and it is particularly preferable that no expression occurs.

  Confirmation that the amount of the protein encoded by the target gene has decreased can be confirmed by Western blotting using an antibody that binds to the protein (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001). )). The decrease in the amount of protein may be any as long as it is lower than that of the wild strain or non-modified strain, but for example, at least 75% compared to the wild strain or non-modified strain compared to the wild strain or non-modified strain. In the following, it is desirable to decrease to 50% or less, 25% or less, or 10% or less, and it is particularly preferable that no protein is produced (the activity is completely lost).

  The protein encoded by the ydcU gene (YdcU protein) is presumed to be a component of the ABC transporter or a subunit of the spermidine / putrescine transporter. “Function of YdcU protein” refers to a function as one element of such a putative transporter. SmpA is small membrane protein A, HtrG is signal transduction proterin (SH3), AdiC is arginine: agmatine antiporter, HtrA is membrane-bound serine endoprotease (protease Do), YdhK is conserved inner membrane protein, BacA is undecaprenyl Phosphatase, YaiW are predicted DNA-binding transcriptional regulators, and the YidQ gene is presumed to be a conserved outer membrane protein. Refers to the function.

  If the protein encoded by each gene does not function normally, the expression of the gene expressed in a strain in which the protein functions normally, for example, a wild strain, is expected to decrease or disappear. Confirmation of a decrease in the function of each protein in the cell can be performed by detection by means of Southern blotting or the like of mRNA transcribed from each gene, or by detection of gene products by Western blotting or the like.

Here, the YdcU protein of Enterobacteriaceae includes a protein (SEQ ID NO: 2) encoded by the ydcU gene (SEQ ID NO: 1) of Escherichia coli. The YdcU protein is speculated to encode a component of the ABC transporter or a subunit of the spermidine / putrescine transporter.
Examples of the YfeK protein include a protein (SEQ ID NO: 4) encoded by the Escherichia coli yfeK gene (GenBank Annotation NP — 416914.1 GI: 16130345, SEQ ID NO: 3).
Examples of the SmpA protein include a protein (SEQ ID NO: 6) encoded by an Escherichia coli smpA gene (GenBank NP_417107.2 GI: 90111468, SEQ ID NO: 5). The SmpA protein is estimated to be small membrane protein A.
Examples of the HtrG protein include a protein (SEQ ID NO: 8) encoded by the Escherichia coli htrG gene (GenBank Annotation NP_417527.1 GI: 16130951, SEQ ID NO: 7). HtrG is presumed to be a membrane-bound serine endoprotease (protease Do).
Examples of the AdiC protein include a protein (SEQ ID NO: 10) encoded by an Escherichia coli adiC gene (also called yjdE, yjdD, yjdD12. GenBank Annotation NP_418539.1 GI: 16131941, SEQ ID NO: 9). AdiC protein is presumed to be arginine: agmatine antiporter, and HtrA is a membrane-bound serine endoprotease (protease Do).
As the HtrA protein, the htrA gene of Escherichia coli (also called degP. Genb
ank annotation NP_414703.1 GI: 16128154, SEQ ID NO: 11) is the protein encoded by (SEQ ID NO: 12). The HtrA protein is presumed to be a membrane-bound serine endoprotease (protease Do) serine endoprotease (protease Do).
Examples of the YdhK protein include a protein (SEQ ID NO: 14) encoded by the Escherichia coli ydhK gene (GenBank NP — 416162.1 GI: 16129603, SEQ ID NO: 13). YdhK protein is presumed to be a conserved inner membrane protein.
Examples of the BacA protein include a protein (SEQ ID NO: 16) encoded by the bacA gene of Escherichia coli (GenBank NP — 417529.1 GI: 16130953, SEQ ID NO: 15). The BacA protein is presumed to be undecaprenyl-diphosphatase.
Examples of the YaiW protein include a protein (SEQ ID NO: 182) encoded by the Escherichia coli yaiW gene (GenBank NP_414912.1 GI: 16128363, SEQ ID NO: 17). YaiW protein is presumed to be a predicted DNA-binding transcriptional regulator.
Examples of the YidQ protein include a protein (SEQ ID NO: 20) encoded by the Escherichia coli yidQ gene (GenBank NC — 000913.2 (3865751..3866083), SEQ ID NO: 19). YidQ protein is presumed to be a conserved outer membrane protein.
Moreover, since there may be a difference in the base sequence of the gene encoding each protein depending on the species or strain to which the Enterobacteriaceae belongs, each gene has SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 , 15, 17, or 19 variants of the gene. Variants of each gene can be searched by BLAST etc. with reference to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 (http: //blast.genome .jp /). In addition, each gene variant is a base of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 using each gene homolog, for example, Enterobacteriaceae chromosome as a template. It includes genes that can be amplified by PCR using primers that can be generated based on the sequence.

  YdcU, SmpA, HtrG, AdiC, HtrA, YdhK, BacA, YaiW, or YidQ protein each have the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Although the codon used differs depending on the bacterial species and strain, and there may be differences in the base sequence of each gene, as long as the function of the encoded protein does not change, in these amino acid sequences, It may encode an amino acid sequence that includes one or several amino acid substitutions, deletions, insertions, or additions. Here, several means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5. The substitution, deletion, insertion or addition of one or several amino acids described above is a conservative mutation in which each protein production is normally performed. A conservative mutation is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Representative conservative mutations are conservative substitutions, and substitutions that are considered conservative substitutions include Ala to Ser or Thr substitution, Arg to Gln, His or Lys substitution, Asn to Glu, Gln, Lys, His or Asp substitution, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu Substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to His, Phe or Trp substitution, and Val to Met, Ile or Leu Substitution.

Variants of ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, and yidQ genes are the bases of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, respectively. Probe and string comprising not only a gene having a sequence but also a base sequence complementary to the base sequence consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 or a part thereof It may be DNA that hybridizes under gentle conditions. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, for example, DNAs having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more. Are hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C. Washing once at a salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS, more preferably 2-3 times The conditions to do are mentioned. The length of the probe is appropriately selected depending on the hybridization conditions, but is usually 100 bp to 1 Kbp.

  The probe may have a partial sequence of each gene. Such a probe can be prepared by a PCR reaction using an oligonucleotide prepared based on the base sequence of each gene as a primer and a DNA fragment containing each gene as a template by a method well known to those skilled in the art. . When a DNA fragment having a length of about 300 bp is used for the probe, washing conditions after hybridization under the above conditions are as follows: 50 ° C., 2 × SSC, 0.1% SDS once or 2 times. The condition of washing three times is mentioned.

  In addition, the description regarding the said variant and homolog is similarly applied also to the gene used for provision of the L-amino acid production ability to bacteria.

<2> Method for producing L-amino acid of the present invention The L-amino acid is obtained by culturing the microorganism of the present invention in a medium, producing and accumulating L-amino acid in the medium, and collecting the L-amino acid from the medium. Can be manufactured.

  As a medium used for the culture, a normal medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary can be used. Either synthetic or natural media can be used. Any type of carbon source and nitrogen source may be used as long as the strain to be cultured is available.

  As the carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses can be used. In addition, organic acids such as acetic acid and citric acid, and alcohols such as ethanol alone Or it can use together with another carbon source. As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates, and the like can be used. Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soybean protein breakdown products, etc. containing these, and auxotrophic mutations that require amino acids for growth. When using a strain, it is preferable to supplement the required nutrients.

  In particular, when using a liquid medium adjusted to conditions under which L-glutamic acid is precipitated, crystallization can be performed more efficiently by adding pantothenic acid to the medium (WO 2004/111258 pamphlet). As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.

The culture is preferably carried out with aeration while controlling the fermentation temperature at 20 to 45 ° C. and the pH at 3 to 9. When the pH is lowered during the culture, for example, calcium carbonate is added or neutralized with an alkali such as ammonia gas. Under such conditions, the target amino acid is accumulated in the culture medium by preferably culturing for about 10 to 120 hours.
Moreover, it can also culture | cultivate, making L-glutamic acid precipitate in a culture medium using the liquid culture medium adjusted to the conditions which L-glutamic acid precipitates. Examples of conditions for precipitation of L-glutamic acid include pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. Can do.

  In addition, when L-glutamic acid is precipitated in the medium, it can be crystallized more efficiently by adding crystals of L-glutamic acid and L-lysine as seed crystals in advance (European Patent No. 1233069, European Patent Application Published) 1624069).

  The method for collecting L-amino acid from the culture broth after completion of the culture may be performed according to a known recovery method. For example, it is collected by removing microbial cells from the culture solution and then concentrating crystallization or ion exchange chromatography. When culturing under conditions where L-glutamic acid is precipitated, L-glutamic acid precipitated in the culture solution can be collected by centrifugation or filtration. In this case, L-glutamic acid dissolved in the medium may be crystallized and then isolated together.

  In addition, when producing a basic amino acid, the pH during the cultivation is controlled to 6.5 to 9.0, and the pH of the medium at the end of the cultivation is 7.2 to 9.0. The fermenter pressure is controlled to be positive, or carbon dioxide or a mixed gas containing carbon dioxide is supplied to the culture medium, so that bicarbonate ions and / or carbonate ions in the culture medium are at least 2 g / L or more. And the fermentation is carried out by a method in which the bicarbonate ion and / or carbonate ion is used as a counter ion of a cation mainly composed of a basic amino acid, and the target basic amino acid is recovered. Alternatively, see JP-A-2002-065287, US Patent Application Publication No. 2002025564.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] Construction of L-lysine-producing bacterium and destruction of target gene Escherichia coli WC196ΔcadAΔldc described in WO 2006/078039 pamphlet was used as an L-lysine-producing bacterium. In this strain, the lysine decarboxylase genes cadA and ldc of Escherichia coli WC196 were transferred to the Red-driven integration method (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645). (2000)) and the λ phage-derived excision system.

Deletion of the target gene from the WC196ΔcadAΔldcC strain can be performed as follows. P1 transduction using KO collection strain (Baba, T. et al., (2006) Mol. Syst. Biol., 2, 2006 0008) in which the target gene is replaced with Kmr gene as donor and WC196ΔcadAΔldcC as recipient And a strain in which the target gene is replaced with the Kmr gene is obtained from WC196ΔcadAΔldcC. Next, a plasmid pCP20 carrying the FLP recombinase gene is introduced into these strains, and the Kmr gene is removed by Red-driven integration, whereby a strain lacking the target gene can be obtained from WC196ΔcadAΔldcC.
The KO collection strain is a collection of strains in which various genes of the Escherichia coli BW25113 [rrnB3 ΔlacZ4787 hsdR514 Δ (araBAD) 567 Δ (rhaBAD) 568 rph-1] strain have been replaced with the Kmr gene. @ chanko.lab.nig.ac.jp).

[Example 2] Disruption of ydcU, yfeK, and smpA genes of WC196ΔcadAΔldcC strain
P1 transduction was performed using the KO collection strain BW25113 ydcU :: Kmr as a donor and WC196ΔcadAΔldcC as a recipient to obtain WC196ΔcadAΔldcC ydcU :: Kmr. Next, plasmid pCP20 carrying the FLP recombinase gene was introduced into this strain, and the Kmr gene was removed by the Red-driven integration method to obtain WC196ΔcadAΔldcCΔydcU.
Using the BW25113 yfeK :: Kmr strain or the BW25113 smpA :: Kmr strain, strains WC196ΔcadAΔldcCΔyfeK and WC196ΔcadAΔldcCΔsmpA in which the yfeKo or smpA gene was disrupted were obtained in the same manner as described above.

[Example 3] L-lysine production by ydcU gene-disrupted strain WC196ΔcadAΔldcCΔydcU obtained in Example 2 and WC196ΔcadAΔldcC as a control were each applied to an L plate and cultured at 37 ° C for 24 hours. Approximately 1/8 volume of the cells on the plate was inoculated into a 500 mL Sakaguchi flask containing 20 mL of fermentation medium, and cultured at 37 ° C. for about 50 hours at 37 ° C. until the entire amount of glucose was consumed. After culture, the amount of L-lysine accumulated in the medium was measured using Biotech Analyzer AS210 (Sakura Seiki). The average value ± SD of L-lysine yield (yield relative to glucose) was calculated for 3 clones obtained independently and shown in Table 1.

[L plate composition]
Bacto Tryptone (Difco) 10g / L
East Extract (Difco) 5g / L
NaCl 5g / L
Agar 15g / L

[Fermentation medium composition]
Glucose 60g / L
(NH ₄ ) ₂ SO ₄ 30 g / L
KH ₂ PO ₄ 1.0g / L
MgSO ₄・ 7H ₂ 0 1.0g / L
FeSO ₄・ 7H ₂ 0 0.01g / L
MnSO ₄・ 4H ₂ 0 0.01g / L
East Extract (Difco) 2.0g / L
Calcium carbonate 30g / L
The pH was adjusted to 7.0 with potassium hydroxide, and autoclaved at 115 ° C. for 10 minutes. However, glucose and MgSO ₄ · 7H ₂ O were sterilized separately.

  As shown in Table 1, the strain in which the ydcU gene is disrupted has an improved L-lysine yield as compared to the parent strain.

[Example 4] L-lysine production by yfeK gene-disrupted strain WC196ΔcadAΔldcCΔyfeK obtained in Example 2 and WC196ΔcadAΔldcC as a control were cultured in the same manner as in Example 3, and the amount of L-lysine accumulated in the medium was determined. It was measured. The average value ± SD of the L-lysine yield (yield relative to glucose) was calculated for 3 clones obtained independently and shown in Table 2.

  As shown in Table 2, the strain in which the yfeK gene was disrupted has an improved L-lysine yield as compared to the parent strain.

[Example 5] L-lysine production by smpA gene disruption strain WC196ΔcadAΔldcCΔsmpA obtained in Example 2 and WC196ΔcadAΔldcC as a control were cultured in the same manner as in Example 3, and the amount of L-lysine accumulated in the medium was determined. It was measured. The average value ± SD of L-lysine yield (yield relative to glucose) was calculated for 4 clones obtained independently and shown in Table 3.

  As shown in Table 3, the strain in which the smpA gene was disrupted has an improved L-lysine yield as compared to the parent strain.

[Explanation of Sequence Listing]
SEQ ID NO: 1: nucleotide sequence of ydcU gene SEQ ID NO: 2: amino acid sequence of YdcU protein SEQ ID NO: 3: nucleotide sequence of yfeK gene SEQ ID NO: 4: amino acid sequence of YfeK protein SEQ ID NO: 5: nucleotide sequence of smpA gene SEQ ID NO: 6: SmpA Protein amino acid sequence SEQ ID NO: 7: nucleotide sequence of htrG gene SEQ ID NO: 8: amino acid sequence of HtrG protein SEQ ID NO: 9: nucleotide sequence of adiC gene SEQ ID NO: 10: amino acid sequence of AdiC protein SEQ ID NO: 11: nucleotide sequence of htrA gene No. 12: amino acid sequence of HtrA protein SEQ ID NO: 13: nucleotide sequence of ydhK gene SEQ ID NO: 14: amino acid sequence of YdhK protein SEQ ID NO: 15: nucleotide sequence of bacA gene SEQ ID NO: 16: amino acid sequence of BacA protein SEQ ID NO: 17: yaiW gene Nucleotide sequence of SEQ ID NO: 18: amino acid sequence of YaiW protein SEQ ID NO: 19: nucleotide sequence of yidQ gene SEQ ID NO: 20: YidQ tongue The amino acid sequence of click quality

Claims (19)

  L-amino acid-producing ability and modified so that expression of one or more genes selected from ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, and yidQ is weakened Bacteria belonging to the family Enterobacteriaceae.   2. The bacterium according to claim 1, wherein expression of each gene is weakened by inactivation of ydcU, yfeK, smpA, htrG, adiC, htrA, ydhK, bacA, yaiW, or yidQ.   The bacterium according to claim 1 or 2, wherein the gene is ydcU, yfeK, or smpA.   The bacterium according to any one of claims 1 to 3, wherein the ydcU gene is DNA encoding the amino acid sequence of SEQ ID NO: 2 or a variant thereof.   The bacterium according to any one of claims 1 to 3, wherein the yfeK gene is DNA encoding the amino acid sequence of SEQ ID NO: 4 or a variant thereof.   The bacterium according to any one of claims 1 to 3, wherein the smpA gene is DNA encoding the amino acid sequence of SEQ ID NO: 6 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the htrG gene is DNA encoding the amino acid sequence of SEQ ID NO: 8 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the adiC gene is DNA encoding the amino acid sequence of SEQ ID NO: 10 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the htrA gene is DNA encoding the amino acid sequence of SEQ ID NO: 12 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the ydhK gene is DNA encoding the amino acid sequence of SEQ ID NO: 14 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the bacA gene is DNA encoding the amino acid sequence of SEQ ID NO: 16 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the yaiW gene is DNA encoding the amino acid sequence of SEQ ID NO: 18 or a variant thereof.   The bacterium according to claim 1 or 2, wherein the yidQ gene is DNA encoding the amino acid sequence of SEQ ID NO: 20 or a variant thereof.   The L-amino acid is L-lysine, L-glutamic acid, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, and The bacterium according to any one of claims 1 to 13, which is one or more L-amino acids selected from the group consisting of L-cysteine.   The bacterium according to any one of claims 1 to 14, wherein the L-amino acid is L-lysine, and the activity of lysine decarboxylase is weakened.   The bacterium according to any one of claims 1 to 15, wherein the microorganism belonging to the family Enterobacteriaceae is an Escherichia bacterium, an Enterobacter bacterium, or a Pantoea bacterium.   A method for producing an L-amino acid, comprising culturing the bacterium according to any one of claims 1 to 16 in a medium, accumulating the L-amino acid in the medium, and collecting the L-amino acid from the medium.   The L-amino acid is L-glutamic acid, L-lysine, L-threonine, L-arginine, L-histidine, L-isoleucine, L-valine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, and The method according to claim 17, which is one or more L-amino acids selected from L-cysteine.   The method according to claim 18, wherein the L-amino acid is L-lysine.