Method for producing l-amino acid

Abstract

To provide a method for producing an L-amino acid such as L-glutamic acid.SOLUTION: An L-amino acid is produced by culturing in a medium a bacterium belonging to the Enterobacteriaceae family with an L-amino acid producing ability, which has been modified to have one or more modifications selected from the following modifications (A) to (F), and by collecting the L-amino acid from the medium and / or the bacterial cells. (A) Modification that reduces BudA protein activity; (B) modification that reduces BudB protein activity; (C) modification that reduces BudC protein activity; (D) modification that reduces the activity of PAJ_3461 protein; (E) modification that reduces the activity of PAJ_3462 protein; and (F) modification that reduces the activity of PAJ_3463 protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing L-amino acids such as L-glutamic acid by fermentation using bacteria. L-Amino acids are industrially useful as raw materials for seasonings, etc. [Background technology]

[0002] L-amino acids are industrially produced by fermentation using microorganisms such as bacteria capable of producing L-amino acids (Non-Patent Document 1). For example, strains isolated from nature or mutants thereof are used as such microorganisms. In addition, the L-amino acid producing ability of microorganisms can be improved by recombinant DNA technology.

[0003] The BudA protein encoded by the budA gene is known as acetolactate decarboxylase. The BudB protein encoded by the budB gene is known as acetolactate synthase. The BudC protein encoded by the budC gene is known as (S,S)-butanediol dehydrogenase, meso-2,3-butandiol dehydrogenase, or diacetyl reductase. It is known.

[0004] The PAJ_3461, PAJ_3462, and PAJ_3463 proteins encoded by the PAJ_3461, PAJ_3462, and PAJ_3463 genes are known as subunits of gluconate 2-dehydrogenase, respectively. It is being done.

[0005] A technique has been reported for producing substances such as 2-oxogluterate using carbon monoxide as a carbon source by utilizing a microorganism with reduced or no 2,3-butanediol-producing ability (Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2013 / 115659 [Non-patent literature]

[0007] [Non-Patent Document 1] Kunihiko Akashi et al., Amino Acid Fermentation, Academic Press, pp. 195-215, 1986 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to develop a novel technique for improving the L-amino acid producing ability of bacteria and to provide an efficient method for producing L-amino acids. [Means for solving the problem]

[0009] As a result of intensive research to achieve the above object, the present inventors have found that the L-amino acid producing ability of a bacterium belonging to the family Enterobacteriaceae can be improved by modifying the bacterium so that the bacterium has one or more modifications selected from the following modifications (A) to (F), thereby completing the present invention: (A) Modifications that reduce the activity of the BudA protein; (B) Modifications that reduce the activity of BudB protein; (C) Modification that reduces the activity of BudC protein; (D) Modifications that reduce the activity of the PAJ_3461 protein; (E) Modification that reduces the activity of the PAJ_3462 protein; (F) Modifications that reduce the activity of the PAJ_3463 protein.

[0010] That is, the present invention can be exemplified as follows. [1] A method for producing an L-amino acid, comprising the steps of: Cultivating a bacterium belonging to the family Enterobacteriaceae having an L-amino acid producing ability in a medium, and accumulating the L-amino acid in the medium and / or within the cells of the bacterium; collecting the L-amino acid from the medium and / or the bacterial cells; Including, the L-amino acid is a glutamic acid-based L-amino acid, The method, wherein the bacterium has one or more modifications selected from the following modifications (A) to (F): (A) Modifications that reduce the activity of the BudA protein; (B) Modifications that reduce the activity of BudB protein; (C) Modification that reduces the activity of BudC protein; (D) Modifications that reduce the activity of the PAJ_3461 protein; (E) Modification that reduces the activity of the PAJ_3462 protein; (F) Modifications that reduce the activity of the PAJ_3463 protein. [2] The method (specifically, the method described in [1]) above), wherein the bacterium has at least one or more modifications selected from the modifications (A) to (C) above. [3] The method (specifically, the method described in [1] or [2]) above, wherein the bacterium has at least the modifications (A) to (C) above. [4] The method (specifically, the method described in any one of [1] to [3]) wherein the bacterium has at least one or more modifications selected from the modifications (D) to (F). [5] The method (specifically, the method described in any one of [1] to [4]) above), wherein the bacterium has at least the modifications (D) to (F) above. [6] The method (specifically, the method described in any one of [1] to [5]) above), wherein the bacterium has the modifications (A) to (F) above. [7] The method (specifically, the method described in any one of [1] to [6]), The activity of the BudA protein is reduced by reducing the expression of the budA gene or by disrupting the gene; The activity of the BudB protein is reduced by reducing expression of the budB gene or by disrupting the gene; The activity of the BudC protein is reduced by reducing expression of the budC gene or by disrupting the gene; The activity of the PAJ_3461 protein is reduced by reducing the expression of the PAJ_3461 gene or by disrupting the gene; The activity of the PAJ_3462 protein is reduced by reducing the expression of the PAJ_3462 gene or by disrupting the gene; and / or A method, wherein the activity of the PAJ_3463 protein is reduced by reducing expression of the PAJ_3463 gene or by disrupting the gene. [8] The method (specifically, the method described in any one of [1] to [7]), The expression of the budA gene is reduced by modifying the expression regulatory sequence of the budA gene; The expression of the budB gene is reduced by modifying the expression regulatory sequence of the budB gene; The expression of the budC gene is reduced by modifying the expression regulatory sequence of the budC gene; The expression of the PAJ_3461 gene is reduced by modifying the expression regulatory sequence of the PAJ_3461 gene; The expression of the PAJ_3462 gene is reduced by modifying the expression regulatory sequence of the PAJ_3462 gene; and / or A method in which the expression of the PAJ_3463 gene is reduced by modifying an expression regulatory sequence of the PAJ_3463 gene. [9] The method (specifically, the method described in any one of [1] to [8]), the activity of the BudA protein is reduced by deletion of the budA gene; the activity of the BudB protein is reduced by deletion of the budB gene; the activity of the BudC protein is reduced by deletion of the budC gene; the activity of the PAJ_3461 protein is reduced by deletion of the PAJ_3461 gene; the activity of the PAJ_3462 protein is reduced by deletion of the PAJ_3462 gene; and / or A method, wherein the activity of the PAJ_3463 protein is reduced by deletion of the PAJ_3463 gene.

[10] The method (specifically, the method described in any one of [1] to [9]), The BudA protein is a protein described in the following (1a), (1b), or (1c): (1a) a protein comprising the amino acid sequence shown in SEQ ID NO:2; (1b) a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and having acetolactate decarboxylase activity; (1c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:2 and having acetolactate decarboxylase activity; The BudB protein is a protein described in the following (2a), (2b), or (2c): (2a) a protein comprising the amino acid sequence shown in SEQ ID NO:4; (2b) a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 4, and having acetolactate synthase activity; (2c) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4 and having acetolactate synthase activity; The BudC protein is a protein described in the following (3a), (3b), or (3c): (3a) a protein comprising the amino acid sequence shown in SEQ ID NO:6; (3b) an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 6, and having (S,S)-butanediol dehydrogenase activity, meso-2,3-butanediol dehydrogenase activity, and / or is a protein with diacetyl reductase activity; (3c) a peptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:6, and having (S,S)-butanediol dehydrogenase activity and meso-2,3-butanediol dehydrogenase activity; proteins with diacetyl dehydrogenase activity, and / or diacetyl reductase activity; The PAJ_3461 protein is a protein described in the following (4a), (4b), or (4c): (4a) a protein comprising the amino acid sequence shown in SEQ ID NO:8; (4b) Substitution or deletion of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 8. a protein comprising an amino acid sequence containing deletions, insertions, and / or additions and having gluconate-2-dehydrogenase activity; (4c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 8 and having gluconate-2-dehydrogenase activity; The PAJ_3462 protein is a protein described in the following (5a), (5b), or (5c): (5a) a protein comprising the amino acid sequence shown in SEQ ID NO: 10; (5b) a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 10, and having gluconate 2-dehydrogenase activity; (5c) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 10 and having gluconate-2-dehydrogenase activity; and / or The method, wherein the PAJ_3463 protein is a protein described in (6a), (6b), or (6c) below: (6a) a protein comprising the amino acid sequence shown in SEQ ID NO: 12; (6b) a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 12, and having gluconate 2-dehydrogenase activity; (6c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 12 and having gluconate-2-dehydrogenase activity;

[11] The method (specifically, the method according to any one of [1] to

[10] ) wherein the bacterium is a bacterium of the genus Pantoea or Escherichia.

[12] The method (specifically, the method described in any one of [1] to

[11] ) wherein the bacterium is Pantoea ananatis or Escherichia coli.

[13] The method (specifically, the method described in any one of [1] to

[12] ) wherein the glutamic acid-based L-amino acid is one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-arginine, L-citrulline, and L-ornithine.

[14] The method (specifically, the method according to any one of [1] to

[13] ) wherein the glutamic acid type L-amino acid is L-glutamic acid.

[15] The method (specifically, the method described in

[13] or

[14] ) above, wherein the L-glutamic acid is ammonium L-glutamate or monosodium L-glutamate. Effect of the Invention

[0011] According to the present invention, the L-amino acid producing ability of a bacterium can be improved, and L-amino acids can be produced efficiently. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below.

[0013] The method of the present invention comprises culturing a bacterium belonging to the Enterobacteriaceae family having an ability to produce an L-amino acid in a medium, accumulating the L-amino acid in the medium and / or within the bacterial cells of the bacterium, and collecting the L-amino acid from the medium and / or the bacterial cells. The bacterium used in the method is also referred to as the "bacterium of the present invention".

[0014] <1> The bacteria of the present invention The bacterium of the present invention is a bacterium belonging to the family Enterobacteriaceae that has been modified to have specific properties and is capable of producing an L-amino acid.

[0015] <1-1> Bacteria capable of producing L-amino acids In the present invention, the term "bacteria capable of producing an L-amino acid" refers to bacteria capable of producing a target L-amino acid when cultured in a medium and accumulating the target L-amino acid in the medium and / or in the bacterial cells to an extent that the target L-amino acid can be recovered. The bacterium capable of producing an L-amino acid may be a bacterium capable of accumulating a larger amount of the target L-amino acid in the medium and / or in the bacterial cells than a non-modified strain. The term "non-modified strain" refers to a control strain that has not been modified to have a specific property. In other words, examples of non-modified strains include wild-type strains and parent strains. The bacterium capable of producing an L-amino acid may be a bacterium capable of accumulating a target L-amino acid in a medium at a level of preferably 0.5 g / L or more, more preferably 1.0 g / L or more.

[0016] The L-amino acids produced in the present invention are L-amino acids of the glutamate family. "Glutamic acid L-amino acids" is a general term for L-glutamic acid and L-amino acids biosynthesized using L-glutamic acid as an intermediate. Examples of L-amino acids biosynthesized using L-glutamic acid as an intermediate include L-glutamine, L-proline, L-arginine, L-citrulline, and L-ornithine. Examples of glutamic acid L-amino acids include, in particular, L-glutamic acid. The bacterium of the present invention may have the ability to produce only one type of L-amino acid, or may have the ability to produce two or more types of L-amino acids.

[0017] In the present invention, the term "amino acid" refers to an L-amino acid unless otherwise specified. In addition, in the present invention, the term "L-amino acid" refers to an L-amino acid in a free form, a salt thereof, or a mixture thereof unless otherwise specified. Salts will be described later.

[0018] Bacteria belonging to the Enterobacteriaceae family include the genus Escherichia, Enterobacter Enterobacter, Pantoea, Klebsiella, and S. Serratia, Erwinia, Photorhabdus Examples of bacteria that can be used include bacteria belonging to the genera Bacillus subtilis, ...

[0019] Examples of Escherichia bacteria include, but are not limited to, bacteria classified into the genus Escherichia according to the classification known to experts in microbiology. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes Examples of mutant derivatives of Escherichia coli K-12 include those described in Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of Escherichia bacteria include Escherichia coli. Specific examples of Escherichia coli include Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as BL21(DE3) strain; and derivatives thereof.

[0020] Examples of bacteria belonging to the genus Enterobacter include, but are not limited to, bacteria classified into the genus Enterobacter according to classification known to experts in microbiology. Examples of bacteria belonging to the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Examples of Enterobacter agglomerans include Enterobacter agglomerans ATCC12287 strain. Examples of Enterobacter aerogenes include Enterobacter aerogenes ATCC13048 strain, NBRC12010 strain (Biotechonol Bioeng. 2007 Mar 27; 98(2) 340-348), and AJ110637 strain (FERM BP-10955). Examples of Enterobacter bacteria include those described in European Patent Application Publication No. EP 0 952 221. Enterobacter agglomerans also includes a species classified as Pantoea agglomerans.

[0021] Examples of the Pantoea bacteria include, but are not limited to, bacteria classified into the genus Pantoea according to classification known to experts in microbiology, such as Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples of Pantoea ananatis include Pantoea ananatis LMG20103 strain, AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), SC17 strain (FERM BP-11091), SC17(0) strain (VKPM B-9246), and SC17sucA strain (FERM BP-8646). Some Enterobacter and Erwinia bacteria have been reclassified into the Pantoea genus (Int. J. Syst. Bacteriol., 39, 337-345 (1989); Int. J. Syst. Bacteriol., 43, 162-173 (1993)). For example, some species of Enterobacter agglomerans have recently been reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc., based on the analysis of the 16S rRNA base sequence, etc. (Int. J. Syst. Bacteriol., 39, 337-345 (1989)). In the present invention, the genus Pantoea also includes bacteria that have been reclassified into the genus Pantoea in this way.

[0022] Erwinia bacteria include Erwinia amylovora, Erwinia Examples of bacteria belonging to the genus Klebsiella include Erwinia carotovora and Klebsiella planticola.

[0023] In recent years, bacteria belonging to the Enterobacteriaceae family have been reclassified into several families through comprehensive comparative genome analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599). However, in this specification, bacteria that were previously classified into the Enterobacteriaceae family are treated as bacteria belonging to the Enterobacteriaceae family. In other words, the above-listed bacteria, such as the genus Pantoea, are to be treated as bacteria belonging to the family Enterobacteriaceae, regardless of the current classification.

[0024] These strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). That is, a corresponding accession number is assigned to each strain, and the strains can be obtained using the accession number (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository institution where each strain was deposited.

[0025] The bacterium of the present invention may be one that inherently has an L-amino acid producing ability, or may be one that has been modified to have an L-amino acid producing ability. Bacteria having an L-amino acid producing ability can be obtained, for example, by imparting an L-amino acid producing ability to the above-mentioned bacteria, or by enhancing the L-amino acid producing ability of the above-mentioned bacteria.

[0026] The L-amino acid producing ability can be imparted or enhanced by a method that has been conventionally employed in breeding amino acid producing bacteria such as Corynebacterium or Escherichia bacteria (see Amino Acid Fermentation, Academic Press, first edition published May 30, 1986, pp. 77-100). Examples of such methods include obtaining auxotrophic mutants, obtaining L-amino acid analog-resistant strains, obtaining metabolically controlled mutants, and creating recombinant strains with enhanced activity of L-amino acid biosynthetic enzymes. In breeding L-amino acid-producing bacteria, the properties such as auxotrophy, analog resistance, metabolic control mutation, etc., that are imparted may be one, two, three or more. In breeding L-amino acid-producing bacteria, the activity of the L-amino acid biosynthetic enzymes that are enhanced may be one, two, three or more. Furthermore, imparting properties such as auxotrophy, analog resistance, metabolic control mutation, etc. may be combined with enhancing the activity of the biosynthetic enzymes.

[0027] Auxotrophic mutants, analogue-resistant mutants, or metabolically regulated mutants having L-amino acid production ability can be obtained by subjecting a parent strain or a wild-type strain to a conventional mutation treatment and selecting from the obtained mutants those that exhibit auxotrophy, analogue-resistant, or metabolically regulated mutation and have L-amino acid production ability. Conventional mutation treatments include irradiation with X-rays or ultraviolet rays, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0028] In addition, imparting or enhancing the ability to produce L-amino acids can also be achieved by enhancing the activity of enzymes involved in the biosynthesis of the target L-amino acid. Enhancement of enzyme activity can be carried out, for example, by modifying bacteria so that the expression of the gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935, EP1010755A, etc. Details of the techniques for enhancing enzyme activity will be described later.

[0029] In addition, imparting or enhancing the ability to produce L-amino acids can also be achieved by reducing the activity of enzymes that catalyze reactions branching from the biosynthesis pathway of the target L-amino acid to produce compounds other than the target L-amino acid. Here, the "enzymes that catalyze reactions branching from the biosynthesis pathway of the target L-amino acid to produce compounds other than the target L-amino acid" also include enzymes involved in the degradation of the target amino acid. Techniques for reducing enzyme activity will be described later.

[0030] Hereinafter, L-amino acid-producing bacteria and methods for imparting or enhancing the ability to produce L-amino acids will be specifically exemplified. Note that any of the properties of the L-amino acid-producing bacteria and the modifications for imparting or enhancing the ability to produce L-amino acids as exemplified below may be used alone or in appropriate combinations.

[0031] <L-glutamic acid-producing bacterium> As a method for imparting or enhancing the ability to produce L-glutamic acid, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from L-glutamic acid biosynthesis enzymes increases can be mentioned. Such enzymes are not particularly limited, but include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), pi Examples of the genes encoding the enzymes include pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase (pntAB). The genes in parentheses are examples of genes encoding the enzymes (the same applies in the following description). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methylcitrate synthase.

[0032] Examples of strains belonging to the family Enterobacteriaceae that have been modified to increase expression of the citrate synthase gene, the phosphoenolpyruvate carboxylase gene, and / or the glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A, and EP952221A, and examples of strains belonging to the family Enterobacteriaceae that have been modified to increase expression of the Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B.

[0033] Another method for imparting or enhancing L-glutamic acid producing ability is to modify a bacterium so as to reduce the activity of one or more enzymes selected from enzymes that catalyze a reaction that branches off from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid. Examples of such enzymes include, but are not limited to, isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), and succinate dehydrogenase (sdhABCD). Among these enzymes, for example, α- It is preferred to reduce or eliminate ketoglutarate dehydrogenase activity.

[0034] Escherichia bacteria with reduced or no α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Methods for reducing or deleting α-ketoglutarate dehydrogenase activity in enterobacteria such as Pantoea, Enterobacter, Klebsiella, and Erwinia are disclosed in U.S. Patent Nos. 6,197,559, 6,682,912, 6,331,419, 8,129,151, and WO2008 / 075483. Specific examples of Escherichia bacteria with reduced or no α-ketoglutarate dehydrogenase activity include the following strains. E. coli W3110sucA::Kmr E. coli AJ12624 (FERM BP-3853) E. coli AJ12628 (FERM BP-3854) E. coli AJ12949 (FERM BP-4881)

[0035] E. coli W3110sucA::Kmr expresses α-ketoglutarate dehydrogenase from E. coli W3110. This strain was obtained by disrupting the sucA gene encoding α-ketoglutarate dehydrogenase activity.

[0036] In addition, examples of L-glutamic acid producing bacteria or parent strains for deriving them include Pantoea ananatis strains such as Pantoea ananatis AJ13355 (FERM BP-6614), Pantoea ananatis SC17 (FERM BP-11091), and Pantoea ananatis SC17(0) (VKPM B-9246). The AJ13355 strain was isolated from soil in Iwata City, Shizuoka Prefecture, as a strain capable of growing in a medium containing L-glutamic acid and a carbon source at low pH. The SC17 strain was selected from the AJ13355 strain as a mutant with low mucilage production (U.S. Patent No. 6,596,517). The SC17 strain was deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (currently the Patent Organism Depositary of the National Institute of Technology and Evaluation, Japan; postal code: 292-0818; address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 4, 2009, and has been assigned the accession number FERM BP-11091. The AJ13355 strain was deposited on February 19, 1998 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently the Patent Organism Depositary Center, National Institute of Technology and Evaluation, Japan, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan, 292-0818, Japan) under the accession number FERM P-16644, and transferred to an international deposit under the Budapest Treaty on January 11, 1999, and assigned the accession number FERM BP-6614. The SC17(0) strain was deposited on September 21, 2005 at the Russian National Collection of Industrial Microorganisms (VKPM; FGUP GosNII Genetika, Russian Federation, 1st Dorozhny proezd, 117545 Moscow) under the accession number VKPM B-9246.

[0037] In addition, L-glutamic acid producing bacteria or parent strains for deriving them may also include bacteria of the genus Pantoea in which α-ketoglutarate dehydrogenase activity is reduced or deleted. Such strains include the AJ13355 strain, which is a strain that contains the E1 subunit of α-ketoglutarate dehydrogenase. Examples of such strains include the AJ13356 strain, which is a gene (sucA)-deficient strain (U.S. Patent No. 6,331,419), and the SC17sucA strain, which is a sucA gene-deficient strain of the SC17 strain (U.S. Patent No. 6,596,517). The AJ13356 strain was deposited on February 19, 1998 at the National Institute of Bioscience and Human-Technology (now the National Institute of Technology and Evaluation, Patent Organism Depositary Center, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, postal code: 292-0818, address: Room 120, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-16645, and was transferred to an international deposit based on the Budapest Treaty on January 11, 1999, and was assigned the accession number FERM BP-6616. The SC17sucA strain has been assigned private number AJ417 and deposited on February 26, 2004 at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (currently the Patent Organism Depositary of the National Institute of Technology and Evaluation, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number FERM BP-8646.

[0038] At the time of isolation, the AJ13355 strain was identified as Enterobacter agglomerans. However, it has been reclassified as Pantoea ananatis based on the analysis of the 16S rRNA base sequence, etc. Therefore, although the AJ13355 strain and the AJ13356 strain have been deposited at the above depository institution as Enterobacter agglomerans, they are described as Pantoea ananatis in this specification.

[0039] Examples of L-glutamic acid producing bacteria or parent strains for deriving them include bacteria of the genus Pantoea, such as Pantoea ananatis SC17sucA / RSFCPG+pSTVCB, Pantoea ananatis AJ13601, Pantoea ananatis NP106, and Pantoea ananatis NA1. The SC17sucA / RSFCPG+pSTVCB strain was obtained by introducing into the SC17sucA strain a plasmid RSFCPG containing the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, and a plasmid pSTVCB containing the citrate synthase gene (gltA) derived from Brevibacterium lactofermentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG+pSTVCB strain as a strain that exhibits resistance to high concentrations of L-glutamic acid at low pH. The NP106 strain was obtained by eliminating the plasmid RSFCPG+pSTVCB from the AJ13601 strain. The NA1 strain was obtained by introducing the plasmid RSFPPG into the NP106 strain (WO2010 / 027045). The plasmid RSFPPG has a structure in which the gltA gene of the plasmid RSFCPG is replaced with the methylcitrate synthase gene (prpC), i.e., it contains the prpC gene, the ppc gene, and the gdhA gene (WO2008 / 020654). The AJ13601 strain was deposited on August 18, 1999 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently the Patent Organism Depositary Center, National Institute of Technology and Evaluation, Japan; postal code: 292-0818; address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-17516, and transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the accession number FERM BP-7207.

[0040] In addition, the L-glutamic acid producing bacteria or parent strains for deriving them are preferably strains having both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity. Examples of such strains include strains in which the expression level is reduced or deleted (JP Patent Publication No. 2010-041920). Specifically, for example, a sucAsdhA double deletion strain of the Pantoea ananatis NA1 strain can be mentioned (JP Patent Publication No. 2010-041920).

[0041] Furthermore, L-glutamic acid producing bacteria or parent strains for deriving them may also include auxotrophic mutants. Specific examples of auxotrophic mutants include E. coli VL334thrC. + (VKPM B-8961; EP1172433). 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 (U.S. Patent No. 4,278,765). + is an L-isoleucine-requiring L-glutamic acid producer obtained by introducing the wild-type allele of the thrC gene into VL334. The wild-type allele of the thrC gene was introduced by general transduction method using bacteriophage P1 propagated in cells of wild-type E. coli K-12 strain (VKPM B-7).

[0042] In addition, L-glutamic acid producing bacteria or parent strains for deriving them may also include strains resistant to aspartic acid analogues. These strains may be, for example, deficient in α-ketoglutarate dehydrogenase activity. Specific examples of strains resistant to aspartic acid analogues and deficient in α-ketoglutarate dehydrogenase activity include E. coli AJ13199 (FERM BP-5807; U.S. Patent No. 5,908,768), and E. coli FERM P-12379 (U.S. Patent No. 5,393,671) and E. coli AJ13138 (FERM BP-5565; U.S. Patent No. 6,110,714) with reduced L-glutamic acid decomposition ability.

[0043] In addition, as a method for imparting or enhancing the ability to produce L-glutamic acid, for example, enhancing the expression of L-glutamic acid excretion genes such as the yhfK gene (WO2005 / 085419) and the ybjL gene (WO2008 / 133161) can be mentioned.

[0044] Incidentally, the method for imparting or enhancing the ability to produce L-glutamic acid may also be effective for imparting or enhancing the ability to produce L-amino acids (for example, L-glutamine, L-proline, L-arginine, L-citrulline, L-ornithine) biosynthesized using L-glutamic acid as an intermediate. That is, bacteria having the ability to produce L-amino acids biosynthesized using these L-glutamic acids as intermediates may appropriately have the properties of the above-described L-glutamic acid-producing bacteria. For example, bacteria having the ability to produce L-amino acids biosynthesized using these L-glutamic acids as intermediates may be modified so that the activities of α-ketoglutarate dehydrogenase and / or succinate dehydrogenase are decreased.

[0045] <L-glutamine-producing bacterium> As a method for imparting or enhancing the ability to produce L-glutamine, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from L-glutamine biosynthetic enzymes is increased can be mentioned. Such enzymes are not particularly limited, but include glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA). Incidentally, the activity of glutamine synthetase may be enhanced by disrupting the glutamine adenylyltransferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121).

[0046] In addition, as a method for imparting or enhancing the ability to produce L-glutamine, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from enzymes that catalyze reactions to produce compounds other than L-glutamine branching from the L-glutamine biosynthetic pathway is decreased can be mentioned. Such enzymes are not particularly limited, but include glutaminase. can be mentioned.

[0047] As an L-glutamine-producing bacterium or a parent strain for deriving the same, specifically, for example, a mutant glutamine synthetase in which the tyrosine residue at position 397 of glutamine synthetase is substituted with another amino acid residue, a strain belonging to the genus Escherichia having the mutant glutamine synthetase can be mentioned (US2003-0148474A).

[0048] <L-proline-producing bacterium> As a method for imparting or enhancing L-proline-producing ability, for example, a method of modifying a bacterium so that the activity of one or more enzymes selected from L-proline biosynthetic enzymes is increased can be mentioned. Such enzymes include glutamate-5-kinase (proB), γ-glutamyl-phosphate reductase, and pyrroline-5-carboxylate reductase (putA). For enhancing enzyme activity, for example, the proB gene encoding glutamate-5-kinase in which feedback inhibition by L-proline is released (German Patent No. 3127361) can be preferably used.

[0049] Further, as a method for imparting or enhancing L-proline-producing ability, for example, a method of modifying a bacterium so that the activity of an enzyme involved in L-proline degradation is decreased can be mentioned. Such enzymes include proline dehydrogenase and ornithine aminotransferase.

[0050] ​As the L - proline - producing bacterium or the parental strain for inducing the same, specifically, for example, E. coli NRRL B - 12403 and NRRL B - 12404 (British Patent No. 2075056), E. coli VKPM B - 8012 (Russian Patent Application No. 2000124295), the E. coli plasmid mutants described in German Patent No. 3127361, the E. coli plasmid mutants described in Bloom F.R. et al (The 15th Miami winter symposium, 1983, p.34), E. coli 702 strain (VKPM B - 8011) resistant to 3,4 - dehydroxyproline and azetidine - 2 - carboxylate, and E. coli 702ilvA strain (VKPM B - 8012; EP1172433) which is an ilvA gene - deficient strain of the 702 strain can be mentioned.

[0051] <L - arginine - producing bacterium> As a method for imparting or enhancing the L - arginine - producing ability, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from L - arginine biosynthetic enzymes is increased can be mentioned. Such enzymes are not particularly limited, and examples include N - acetylglutamate synthase (argA), N - acetylglutamate kinase (argB), N - acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthase (carAB). As the N - acetylglutamate synthase (argA) gene, for example, it is preferable to use a gene encoding a mutant N - acetylglutamate synthase in which the amino acid residues corresponding to positions 15 to 19 of the wild - type are substituted and the feedback inhibition by L - arginine is released (EP1170361A).

[0052] As a L-arginine-producing bacterium or a parent strain for deriving the same, specifically, for example, E. coli strain 237 (VKPM B-7925; US2002-058315A1), its derivative strain into which the argA gene encoding mutant N-acetylglutamate synthase has been introduced (Russian Patent Application No. 2001112869, EP1170361A1), E. coli strain 382 (VKPM B-7926; EP1170358A1) which is a strain derived from strain 237 with improved acetate assimilation ability, and E. coli 382ilvA+ strain which is a strain into which the wild-type ilvA gene derived from E. coli K-12 strain has been introduced into strain 382. E. coli strain 237 was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) (FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russia) under the accession number VKPM B-7925 on April 10, 2000 and transferred to an international deposit under the Budapest Treaty on May 18, 2001. E. coli strain 382 was deposited with the Russian National Collection of Industrial Microorganisms (VKPM) (FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russia) under the accession number VKPM B-7926 on April 10, 2000.

[0053] In addition, as a L-arginine-producing bacterium or a parent strain for deriving the same, strains having resistance to amino acid analogs etc. are also included. Such strains include, for example, E. coli mutant strains resistant to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamate, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thienylalanine, or sulfaguanidine (JP-A-56-106598).

[0054] <L-citrulline-producing bacteria and L-ornithine-producing bacteria> L-citrulline and L-ornithine are intermediates in the L-arginine biosynthesis pathway.Therefore, the method for imparting or enhancing the ability to produce L-citrulline and / or L-ornithine includes, for example, a method for modifying bacteria so that the activity of one or more enzymes selected from the L-arginine biosynthesis enzymes is increased.Such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), ornithine acetyltransferase (argJ), and carbamoylphosphate synthase (carAB) for L-citrulline. In addition, such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamylphosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), and ornithine acetyltransferase (argJ) for L-ornithine.

[0055] In addition, the L-citrulline producing bacteria may be, for example, any L-arginine producing bacteria (E. coli 382 strain (VKPM B-7926, etc.), argininosuccinate synthase encoded by the argG gene In addition, L-ornithine producing bacteria can be easily obtained by reducing the activity of ornithine carbamoyltransferase encoded by both the argF and argI genes from any L-arginine producing bacteria (such as E. coli 382 strain (VKPM B-7926)).

[0056] Specific examples of L-citrulline producing bacteria or parent strains for deriving them include E. coli 237 / pMADS11, 237 / pMADS12, and 237 / pMADS13 strains carrying mutant N-acetylglutamate synthase (Russian Patent No. 2215783, U.S. Patent No. 6,790,647, EP1170361B1), E. coli 333 (VKPM B-8084) and 374 (VKPM B-8086) carrying carbamoyl phosphate synthetase resistant to feedback inhibition (Russian Patent No. 2264459), and ... which have increased activity of α-ketoglutarate synthase and ferredoxin-NADP + Examples of the strains include strains belonging to the genus Escherichia, such as E. coli strains (EP2133417A1) in which the activities of reductase, pyruvate synthase, and / or α-ketoglutarate dehydrogenase have been further modified.Specific examples of L-citrulline-producing bacteria or parent strains for deriving the same include P. ananatis NA1sucAsdhA strains (US2009-286290A1) in which the activities of succinate dehydrogenase and α-ketoglutarate dehydrogenase have been reduced.

[0057] In addition, examples of methods for imparting or enhancing L-amino acid producing ability include methods for modifying bacteria so that the activity of secreting L-amino acids from bacterial cells is increased. The activity of excreting amino acids can be increased, for example, by increasing the expression of genes encoding proteins that excrete L-amino acids. Examples of genes encoding proteins that excrete various amino acids include the b2682 gene (ygaZ), the b2683 gene (ygaH), the b1242 gene (ychE), and the b3434 gene (yhgN) (JP Patent Publication 2002-300874).

[0058] Methods for imparting or enhancing L-amino acid producing ability include, for example, methods for modifying bacteria so as to increase the activity of proteins involved in sugar metabolism or energy metabolism.

[0059] Proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes. Genes encoding proteins involved in sugar metabolism include the glucose 6-phosphate isomerase gene (pgi; WO01 / 02542), the pyruvate carboxylase gene (PGI; WO01 / 02542), and the mitochondrial enzyme gene (MGE; WO01 / 02542). Examples of such genes include the pyc operon (WO99 / 18228, EP1092776A), phosphoglucomutase gene (pgm; WO03 / 04598), fructose bisphosphate aldolase gene (pfkB, fbp; WO03 / 04664), transaldolase gene (talB; WO03 / 008611), fumarase gene (fum; WO01 / 02545), non-PTS sucrose uptake gene (csc; EP1149911A), and sucrose utilization genes (scrAB operon; U.S. Pat. No. 7,179,623).

[0060] Examples of genes encoding proteins involved in energy metabolism include the transhydrogenase gene (pntAB; US Pat. No. 5,830,716) and the cytochrome bo type oxidase gene (cyoB; EP1070376A).

[0061] In addition, as a method for imparting or enhancing the ability to produce useful substances such as L-amino acids, for example, a method of modifying a bacterium so that the activity of phosphoketolase is increased (WO2006 / 016705). That is, the bacterium of the present invention may be modified so that the activity of phosphoketolase is increased. Increasing the activity of phosphoketolase may be particularly effective for imparting or enhancing the ability to produce glutamic acid-based L-amino acids such as L-glutamic acid. Examples of phosphoketolases include D-xylulose-5-phosphate-phosphoketolase and fructose-6-phosphate phosphoketolase. Either one of the D-xylulose-5-phosphate-phosphoketolase activity and the fructose-6-phosphate phosphoketolase activity may be enhanced, or both may be enhanced.

[0062] D-xylulose-5-phosphate-phosphoketolase activity means the activity of consuming phosphate to convert xylulose-5-phosphate to glyceraldehyde-3-phosphate and acetyl phosphate, and releasing one molecule of HO. This activity can be measured by the method described in Goldberg, M. et al. (Methods Enzymol., 9,515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). Examples of D-xylulose-5-phosphate phosphoketolase include those from bacteria of the genus Acetobacter, Bifidobacterium, Lactobacillus, Thiobacillus, Streptococcus, Methylococcus, Butyrivibrio, or Fibrobacter, and those from yeasts of the genus Candida, Rhodotorula, Rhodosporidium, Pichia, Yarrowia, Hansenula, Kluyveromyces, Saccharomyces, Trichosporon, or Wingea. Specific examples of D-xylulose-5-phosphate phosphoketolase and the gene encoding it are disclosed in WO2006 / 016705.

[0063] In addition, the fructose-6-phosphate phosphoketolase activity means the activity of consuming phosphate, converting fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate, and releasing one molecule of HO. This activity can be measured by the method described in Racker, E. (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J.Bacteriol. (2001) 183; 2929-2936). Examples of fructose-6-phosphate phosphoketolase include fructose-6-phosphate phosphoketolase of bacteria belonging to the Acetobacter, Bifidobacterium, Chlorobium, Brucella, Methylococcus, or Gardnerella genera, and yeasts belonging to the Rhodotorula, Candida, Saccharomyces, etc. genera. Specific examples of fructose-6-phosphate phosphoketolase and genes encoding same are disclosed in WO2006 / 016705.

[0064] It is possible that both phosphoketolase activities are possessed by a single enzyme (D-xylulose-5-phosphate / fructose-6-phosphate phosphoketolase).

[0065] The genes and proteins used for breeding L-amino acid producing bacteria may have the nucleotide sequence and amino acid sequence of known genes and proteins, such as the genes and proteins exemplified above. The genes and proteins used for breeding L-amino acid producing bacteria may also be conservative variants of known genes and proteins, such as the genes and proteins exemplified above. Specifically, for example, the genes used for breeding L-amino acid producing bacteria may be genes encoding proteins having an amino acid sequence in which one or several amino acids at one or several positions in the amino acid sequence of a known protein are substituted, deleted, inserted or added, as long as the original function is maintained. The genes used for breeding L-amino acid producing bacteria may be modified to have optimal codons depending on the codon usage frequency of the host used. Conservative variants of genes and proteins can be mutatis mutandis as described below regarding conservative variants of target genes and target proteins.

[0066] <1-2> Specific characteristics The bacterium of the present invention is modified to have a specific property. The bacterium of the present invention can be obtained by modifying a bacterium having an L-amino acid producing ability to have a specific property. The bacterium of the present invention can also be obtained by modifying a bacterium to have a specific property and then imparting or enhancing an L-amino acid producing ability. The bacterium of the present invention may be modified to have a specific property, thereby acquiring an L-amino acid producing ability. In addition to being modified to have a specific property, the bacterium of the present invention may appropriately have, for example, a property possessed by an L-amino acid producing bacterium as described above. The modifications for constructing the bacterium of the present invention can be carried out in any order.

[0067] By modifying a bacterium to have a specific property, the ability of the bacterium to produce an L-amino acid can be improved, i.e., the L-amino acid production by the bacterium can be increased, including an increase in the amount of L-amino acid accumulated and an increase in the yield of the L-amino acid.

[0068] Specific properties include the following modifications (A) to (F): (A) Modifications that reduce the activity of the BudA protein; (B) Modifications that reduce the activity of BudB protein; (C) Modification that reduces the activity of BudC protein; (D) Modifications that reduce the activity of the PAJ_3461 protein; (E) Modification that reduces the activity of the PAJ_3462 protein; (F) Modifications that reduce the activity of the PAJ_3463 protein.

[0069] The bacterium of the present invention may have, for example, one or more modifications selected from the modifications (A) to (F) above, for example, one, two, three, four, five, or all six of the modifications.

[0070] The bacterium of the present invention may have, for example, at least one or more modifications selected from the above modifications (A) to (C), for example, one, two, or all three modifications. The bacterium may, for example, have one or more modifications selected from the modifications (A) to (C) above, for example, one, two, or all three, and may further have one or more modifications selected from the modifications (D) to (F) above, for example, one, two, or all three.

[0071] The bacterium of the present invention may have, for example, at least one or more modifications selected from the above modifications (D) to (F), for example, one, two, or all three. The bacterium of the present invention may have, for example, one or more modifications selected from the above modifications (D) to (F), for example, one, two, or all three, and may further have one or more modifications selected from the above modifications (A) to (C), for example, one, two, or all three.

[0072] In one embodiment, the above modification (B) may not be selected alone. In the above, the bacterium of the present invention has at least one modification selected from the above (A) and (C) to (F). In one embodiment, the bacterium of the present invention may have one or more of the modifications selected from the above (A) and (C) to (F). The amino acid sequence may have one or more of the modifications selected from the above, for example, one, two, three, four, or all five of the modifications, and may further have the modification (B) above.

[0073] By modifying a bacterium to have one or more modifications selected from the above modifications (A) to (C) and one or more modifications selected from the above modifications (D) to (F), the L-amino acid producing ability of the bacterium may be further improved compared to when the bacterium only has one or more modifications selected from the above modifications (A) to (C) and / or when the bacterium only has one or more modifications selected from the above modifications (D) to (F). For example, by modifying a bacterium to have the above modifications (A) to (F), the L-amino acid producing ability of the bacterium may be further improved compared to when the bacterium only has the above modifications (A) to (C) and / or when the bacterium only has the above modifications (D) to (F).

[0074] "BudA protein" refers to a protein encoded by the budA gene. The BudA protein may be acetolactate decarboxylase. That is, "reduction in the activity of the BudA protein" may refer to reduction in acetolactate decarboxylase activity. "Acetolactate decarboxylase" may refer to a protein having an activity to catalyze a reaction of decarboxylating acetolactate to produce acetoin and / or the reverse reaction (EC 4.1.1.5, etc.). The same activity is also referred to as "acetolactate decarboxylase activity." The acetolactate decarboxylase activity may be, in particular, an activity to catalyze the following chemical reaction in either one or both directions: (2S)-2-hydroxy-2-methyl-3-oxobutanoate = (3R)-3-hydroxybutan-2-one + CO2

[0075] The nucleotide sequence of the budA gene of the bacterium to be modified and the amino acid sequence of the BudA protein encoded thereby can be obtained from a public database such as NCBI. The nucleotide sequence of the budA gene (PAJ_p0041) of Pantoea ananatis AJ13355 corresponds to the complementary sequence of the nucleotide sequence at positions 44299 to 45081 in the nucleotide sequence of the pEA320 plasmid of Pantoea ananatis AJ13355 registered under GenBank accession AP012033.1. The nucleotide sequence of the budA gene (PAJ_p0041) of Pantoea ananatis AJ13355 and the amino acid sequence of the BudA protein encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively.

[0076] "BudB protein" refers to a protein encoded by the budB gene. The BudB protein may be an acetolactate synthase. "Reduction in BudB protein activity" may mean a reduction in acetolactate synthase activity. "Acetolactate synthase" refers to the reaction that produces acetolactate from pyruvic acid and It may mean a protein having an activity to catalyze the reaction of acetolactate and / or the reverse reaction (EC 2.2.1.6, etc.). The activity is also called "acetolactate synthase activity". Specifically, the acetolactate synthase activity may be an activity to catalyze a reaction that produces one molecule of acetolactate and one molecule of carbon dioxide from two molecules of pyruvic acid and / or the reverse reaction. In particular, the acetolactate synthase activity may be an activity to catalyze the following chemical reaction in either one or both directions: 2 pyruvate = 2-acetolactate + CO2

[0077] The nucleotide sequence of the budB gene of the bacterium to be modified and the amino acid sequence of the BudB protein encoded thereby can be obtained from a public database such as NCBI. The nucleotide sequence of the budB gene (PAJ_p0040) of Pantoea ananatis AJ13355 corresponds to the complementary sequence of the nucleotide sequence at positions 42603 to 44282 in the nucleotide sequence of the pEA320 plasmid of Pantoea ananatis AJ13355 registered under GenBank accession AP012033.1. The nucleotide sequence of the budB gene (PAJ_p0040) of Pantoea ananatis AJ13355 and the amino acid sequence of the BudB protein encoded by the gene are shown in SEQ ID NOs: 3 and 4, respectively.

[0078] "BudC protein" refers to a protein encoded by the budC gene. The BudC protein may be (S,S)-butanediol dehydrogenase, meso-2,3-butandiol dehydrogenase, and / or diacetyl reductase. That is, "reduction in the activity of the BudC protein" may refer to reduction in (S,S)-butanediol dehydrogenase activity, meso-2,3-butandiol dehydrogenase activity, and / or diacetyl reductase activity.

[0079] "(S,S)-butanediol dehydrogenase" may refer to a protein having an activity of catalyzing a reaction in which (S,S)-butanediol is oxidized to produce (S)-acetoin and / or the reverse reaction (EC 1.1.1.76, etc.). This activity is also referred to as "(S,S)-butanediol dehydrogenase activity." Specifically, the (S,S)-butanediol dehydrogenase activity may be the activity of catalyzing the reaction of oxidizing (S,S)-butanediol to produce (S)-acetoin in the presence of an electron acceptor and / or the reverse reaction. Examples of the electron acceptor include NAD + In the reverse reaction, an electron donor may be used. Examples of the electron donor include NADH. The (S,S)-butanediol dehydrogenase activity may be, in particular, the activity of catalyzing the following chemical reaction in either or both directions: (2S,3S)-butane-2,3-diol + NAD + = (S)-acetoin + NADH + H +

[0080] "Meso-2,3-butandiol dehydrogenase" is an enzyme that oxidizes meso-2,3-butanediol to aldehydes. The term may refer to a protein having an activity of catalyzing a reaction to produce acetoin and / or the reverse reaction (EC 1.1.1.B20, etc.). The activity is also referred to as "meso-2,3-butandiol dehydrogenase activity." Specifically, meso-2,3-butandiol dehydrogenase activity may be activity of catalyzing a reaction to produce acetoin by oxidizing meso-2,3-butanediol in the presence of an electron acceptor and / or the reverse reaction. Examples of the electron acceptor include NAD + In the reverse reaction, an electron donor may be used. Examples of the electron donor include NADH. The meso-2,3-butandiol dehydrogenase activity may be, in particular, the activity of catalyzing the following chemical reaction in either one or both directions: (2R,3S)-butane-2,3-diol + NAD + = acetoin + NADH + H +

[0081] "Diacetyl reductase" may mean a protein having an activity to catalyze a reaction in which (S)-acetoin is oxidized to produce diacetyl and / or the reverse reaction (EC 1.1.1.304, etc.). The activity is also called "diacetyl reductase activity." Specifically, diacetyl reductase activity may be an activity to catalyze a reaction in which (S)-acetoin is oxidized to produce diacetyl in the presence of an electron acceptor and / or the reverse reaction. Examples of the electron acceptor include NAD + In the reverse reaction, an electron donor may be used. The electron donor may be NADH. The diacetyl reductase activity may be, in particular, the activity of catalyzing the following chemical reaction in either or both directions: (S)-acetoin + NAD + = diacetyl + NADH + H +

[0082] The nucleotide sequence of the budC gene of the bacterium to be modified and the amino acid sequence of the BudC protein encoded thereby can be obtained from a public database such as NCBI. The nucleotide sequence of the budC gene (PAJ_p0039) of Pantoea ananatis AJ13355 corresponds to the complementary sequence of the nucleotide sequence at positions 41805 to 42572 in the nucleotide sequence of the pEA320 plasmid of Pantoea ananatis AJ13355 registered under GenBank accession AP012033.1. The nucleotide sequence of the budC gene (PAJ_p0039) of Pantoea ananatis AJ13355 and the amino acid sequence of the BudC protein encoded by the gene are shown in SEQ ID NOs: 5 and 6, respectively.

[0083] "PAJ_3461 protein", "PAJ_3462 protein", and "PAJ_3463 protein" refer to proteins encoded by the PAJ_3461 gene, PAJ_3462 gene, and PAJ_3463 gene, respectively. The PAJ_3461 gene, PAJ_3462 gene, and PAJ_3463 gene are also collectively referred to as "GlcNDH genes". The PAJ_3461 protein, PAJ_3462 protein, and PAJ_3463 protein may all be gluconate 2-dehydrogenase. That is, "reduction in activity of PAJ_3461 protein", "reduction in activity of PAJ_3462 protein", and "reduction in activity of PAJ_3463 protein" may all mean reduction in gluconate 2-dehydrogenase activity. Specifically, the PAJ_3461 protein, the PAJ_3462 protein, and the PAJ_3463 protein may each be a subunit constituting gluconate 2-dehydrogenase. More specifically, the PAJ_3461 protein may be a subunit of unknown function constituting gluconate 2-dehydrogenase. More specifically, the PAJ_3462 protein may be a dehydrogenase subunit constituting gluconate 2-dehydrogenase. More specifically, the PAJ_3463 protein may be a cytochrome c subunit constituting gluconate 2-dehydrogenase. The PAJ_3461 protein, the PAJ_3462 protein, and the PAJ_3463 protein may, for example, form a complex composed of these proteins and function as gluconate 2-dehydrogenase. "Gluconate 2-dehydrogenase" may refer to a protein having an activity of catalyzing a reaction in which gluconic acid is oxidized to produce 2-dehydrogluconic acid and / or the reverse reaction (EC 1.1.99.3, etc.). The activity is also called "gluconate 2-dehydrogenase activity."Specifically, the gluconate 2-dehydrogenase activity may be the activity of catalyzing the reaction of oxidizing gluconic acid to produce 2-dehydrogluconic acid and / or the reverse reaction in the presence of an electron acceptor, such as oxidized quinone or NAD. + , NAD + , F.A.D. + For the reverse reaction, an electron donor may be used. Examples of electron donors include reduced quinones, NADH, NADPH, and FADH2. The gluconate 2-dehydrogenase activity may be, in particular, the activity of catalyzing the following chemical reaction in either or both directions: D-gluconate + electron acceptor = 2-dehydro-D-gluconate + reduced electron acceptor

[0084] In addition, "a protein has gluconate 2-dehydrogenase activity" means that the protein The present invention is not limited to cases where the protein has gluconate 2-dehydrogenase activity by itself, but includes cases where the protein has gluconate 2-dehydrogenase activity by itself. This includes cases where gluconate 2-dehydrogenase activity is present in combination with other subunits. "A protein has gluconate 2-dehydrogenase activity by itself" means that the protein This may mean that the protein functions as gluconate 2-dehydrogenase alone. The protein has gluconate 2-dehydrogenase activity in combination with other subunits. This means that the protein acts in combination with other subunits to form gluconate 2-dehydrogenase It may mean that the protein functions as gluconate 2-dehydrogenase (for example, the protein forms a complex with another subunit and functions as gluconate 2-dehydrogenase).

[0085] Other subunits for the PAJ_3461 protein include the PAJ_3462 protein and the PAJ_3463 protein. That is, with regard to the PAJ_3461 protein, "the protein has gluconate 2-dehydrogenase activity" is not limited to the case where the protein has gluconate 2-dehydrogenase activity alone, but also includes the case where the protein has gluconate 2-dehydrogenase activity in combination with the PAJ_3462 protein and / or the PAJ_3463 protein (particularly in combination with the PAJ_3462 protein and the PAJ_3463 protein).

[0086] Other subunits for the PAJ_3462 protein include the PAJ_3461 protein and the PAJ_3463 protein. That is, with regard to the PAJ_3462 protein, "the protein has gluconate 2-dehydrogenase activity" is not limited to the case where the protein has gluconate 2-dehydrogenase activity alone, but also includes the case where the protein has gluconate 2-dehydrogenase activity in combination with the PAJ_3461 protein and / or the PAJ_3463 protein (particularly in combination with the PAJ_3461 protein and the PAJ_3463 protein).

[0087] Other subunits for the PAJ_3463 protein include the PAJ_3461 protein and the PAJ_3462 protein. That is, with regard to the PAJ_3463 protein, "the protein has gluconate 2-dehydrogenase activity" is not limited to the case where the protein has gluconate 2-dehydrogenase activity alone, but also includes the case where the protein has gluconate 2-dehydrogenase activity in combination with the PAJ_3461 protein and / or the PAJ_3462 protein (particularly in combination with the PAJ_3461 protein and the PAJ_3462 protein).

[0088] The nucleotide sequences of the PAJ_3461 gene, the PAJ_3462 gene, and the PAJ_3463 gene contained in the bacterium to be modified and the amino acid sequences of the PAJ_3461 protein, the PAJ_3462 protein, and the PAJ_3463 protein encoded thereby can be obtained from a public database such as NCBI. The nucleotide sequence of the PAJ_3461 gene of Pantoea ananatis AJ13355 corresponds to the nucleotide sequence of positions 4149024 to 4149749 in the genome sequence of Pantoea ananatis AJ13355 registered under GenBank accession AP012032.2. The nucleotide sequence of the PAJ_3462 gene of Pantoea ananatis AJ13355 corresponds to the nucleotide sequence of positions 4149752 to 4151536 in the genome sequence of Pantoea ananatis AJ13355 registered under GenBank accession AP012032.2. The nucleotide sequence of the PAJ_3463 gene of Pantoea ananatis AJ13355 corresponds to the nucleotide sequence of positions 4151542 to 4152858 in the genome sequence of Pantoea ananatis AJ13355 registered under GenBank accession AP012032.2. The nucleotide sequence of the PAJ_3461 gene of Pantoea ananatis AJ13355 and the amino acid sequence of the PAJ_3461 protein encoded by the gene are shown in SEQ ID NOs: 7 and 8, respectively. The nucleotide sequence of the PAJ_3462 gene of Pantoea ananatis AJ13355 and the amino acid sequence of the PAJ_3462 protein encoded by the gene are shown in SEQ ID NOs: 9 and 10, respectively. The nucleotide sequence of the PAJ_3463 gene of Pantoea ananatis AJ13355 and the amino acid sequence of the PAJ_3463 protein encoded by the gene are shown in SEQ ID NOs: 11 and 12, respectively.

[0089] Methods for reducing protein activity will be described later. Protein activity can be reduced, for example, by reducing the expression of a gene encoding the protein or by disrupting the gene. Such methods for reducing protein activity can be used alone or in appropriate combination.

[0090] The BudA protein, the BudB protein, the BudC protein, the PAJ_3461 protein, the PAJ_3462 protein, and the PAJ_3463 protein are also collectively referred to as "target proteins." The budA gene, the budB gene, the budC gene, the PAJ_3461 gene, the PAJ_3462 gene, and the PAJ_3463 gene are also collectively referred to as "target genes."

[0091] The target gene may be, for example, a gene having the nucleotide sequence of the above-exemplified target gene (for example, the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11). The target protein may be, for example, a protein having the amino acid sequence of the above-exemplified target protein (for example, the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12). Unless otherwise specified, the expression "having an (amino acid or nucleotide) sequence" means "including the (amino acid or nucleotide) sequence" and also includes the case where the (amino acid or nucleotide) sequence is composed of the above-exemplified target protein.

[0092] The target gene may be a variant of the above-exemplified target gene (e.g., a gene having a nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11) as long as the original function is maintained. Similarly, the target protein may be a variant of the above-exemplified target protein (e.g., a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12) as long as the original function is maintained. Such a variant that maintains the original function may be referred to as a "conservative variant". The terms "budA gene", "budB gene", "budC gene", "PAJ_3461 gene", "PAJ_3462 gene", and "PAJ_3463 gene" respectively include the above-exemplified budA gene, budB gene, budC gene, PAJ_3461 gene, PAJ_3462 gene, and PAJ_3463 gene, as well as their conservative variants. Similarly, the terms "BudA protein", "BudB protein", "BudC protein", "PAJ_3461 protein", "PAJ_3462 protein", and "PAJ_3463 protein" respectively include the BudA protein, BudB protein, BudC protein, PAJ_3461 protein, PAJ_3462 protein, and PAJ_3463 protein exemplified above, as well as conservative variants thereof. Examples of conservative variants include homologs and artificially modified versions of the target genes and target proteins exemplified above. However, the target protein whose activity is reduced is a target protein possessed by the bacterium to be modified, in other words, a target protein encoded by a target gene possessed by the bacterium to be modified. The bacterium to be modified may have the target gene on a chromosome or on an extrachromosomal structure (e.g., a plasmid). "Chromosome" may be used interchangeably with "genome".

[0093] "Maintaining the original function" means that the variant of a gene or protein has a function (e.g., activity or property) corresponding to the function (e.g., activity or property) of the original gene or protein. "Maintaining the original function" for a gene means that the variant of the gene encodes a protein that maintains the original function. That is, "maintaining the original function" for each target gene may mean that the variant of the gene encodes a protein that has the activity of each target protein (which may be acetolactate decarboxylase activity for BudA protein; acetolactate synthase activity for BudB protein; (S,S)-butanediol dehydrogenase activity, meso-2,3-butandiol dehydrogenase activity, and / or diacetyl reductase activity for BudC protein; gluconate 2-dehydrogenase activity for PAJ_3461 protein, PAJ_3462 protein, and PAJ_3463 protein). Furthermore, "maintaining the original function" for each target protein may mean that the protein variant has the activity of each target protein (which may be acetolactate decarboxylase activity for BudA protein; acetolactate synthase activity for BudB protein; (S,S)-butanediol dehydrogenase activity, meso-2,3-butandiol dehydrogenase activity, and / or diacetyl reductase activity for BudC protein; and gluconate 2-dehydrogenase activity for PAJ_3461 protein, PAJ_3462 protein, and PAJ_3463 protein).

[0094] The acetolactate decarboxylase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., acetolactate) and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., acetoin). In the case of the reverse reaction, the acetolactate decarboxylase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., acetoin and carbon dioxide) and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., acetolactate).

[0095] Acetolactate synthase activity is, for example, the reaction of an enzyme with its corresponding substrate (e.g., pyruvate). In the case of the reverse reaction, the acetolactate synthase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., acetolactate and carbon dioxide) and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., pyruvate).

[0096] (S,S)-butanediol dehydrogenase activity can be measured, for example, by incubating an enzyme with a corresponding substrate (e.g., (S,S)-butanediol) in the presence of an electron acceptor and measuring the enzyme- and substrate-dependent production of a corresponding product (e.g., (S)-acetoin). In the case of the reverse reaction, (S,S)-butanediol dehydrogenase activity can be measured, for example, by incubating an enzyme with a corresponding substrate (e.g., (S)-acetoin) in the presence of an electron donor and measuring the enzyme- and substrate-dependent production of a corresponding product (e.g., (S,S)-butanediol).

[0097] Meso-2,3-butandiol dehydrogenase activity can be measured by, for example, incubating the enzyme with the corresponding substrate (e.g., meso-2,3-butanediol) in the presence of an electron acceptor, and determining the enzyme- and substrate-dependent In the reverse reaction, meso-2,3-butandiol dehydrogenase activity can be measured by, for example, incubating the enzyme with a corresponding substrate (e.g., acetoin) in the presence of an electron donor and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., meso-2,3-butanediol). It can be measured by:

[0098] Diacetyl reductase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., (S)-acetoin) in the presence of an electron acceptor and then catalyzing the enzyme- and substrate-dependent production of the corresponding product (e.g., (S)-acetoin). In the reverse reaction, diacetyl reductase activity can be measured by, for example, incubating the enzyme with a corresponding substrate (e.g., diacetyl) in the presence of an electron donor and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., (S)-acetoin).

[0099] Gluconate 2-dehydrogenase activity is, for example, the reaction of the enzyme with the corresponding substrate in the presence of an electron acceptor. The gluconate 2-dehydrogenase activity can be measured by incubating the enzyme with a substrate (e.g., gluconate) and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., 2-dehydrogluconate). In addition, in the case of the reverse reaction, gluconate 2-dehydrogenase activity can be measured by, for example, It can be measured by incubating the enzyme with a corresponding substrate (eg, 2-dehydrogluconic acid) and measuring the enzyme- and substrate-dependent production of the corresponding product (eg, gluconic acid).

[0100] Examples of conservative variants are given below.

[0101] A homologue of a target gene or a homologue of a target protein can be easily obtained from a public database by, for example, a BLAST search or a FASTA search using the base sequence of the target gene or the amino acid sequence of the target protein as a query sequence. Also, a homologue of a target gene can be easily obtained by, for example, using the chromosomes of various organisms as templates and oligonucleotides prepared based on the base sequences of these known target genes as primers. It can be obtained by PCR using the same method as described above.

[0102] The target gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12) have been substituted, deleted, inserted, and / or added, so long as the original function is maintained. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the above "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0103] The above-mentioned substitution, deletion, insertion, and / or addition of one or several amino acids is a conservative mutation that maintains the normal function of the protein. A representative conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid, Leu, Ile, and Val are substituted with each other when the substitution site is a hydrophobic amino acid, Gln and Asn are substituted with each other when the substitution site is a polar amino acid, Lys, Arg, and His are substituted with each other when the substitution site is a basic amino acid, Asp and Glu are substituted with each other when the substitution site is an acidic amino acid, and Ser and Thr are substituted with each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His, or Lys, substitutions of Asn to Glu, Gln, Lys, His, or Asp, substitutions of Asp to Asn, Glu, or Gln, substitutions of Cys to Ser or Ala, substitutions of Gln to Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu to Gly, Asn, Gln, Lys, or Asp, substitutions of Gly to Pro, substitutions of His to Asn, Lys, Gln, Arg, or Tyr, substitutions of Il Examples of such substitutions include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those that occur due to naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organism from which the gene is derived.

[0104] Furthermore, the target gene may be a gene encoding a protein having an amino acid sequence that has, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity to the entire amino acid sequence, so long as the original function is maintained.

[0105] In addition, the target gene may be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned base sequence (e.g., the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11), such as a sequence complementary to the entire or partial base sequence, as long as the original function is maintained. "Stringent conditions" refers to The condition refers to a condition under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, the condition can be a condition under which DNAs having a high identity, for example, DNAs having an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, hybridize with each other, and DNAs having a lower identity than that do not hybridize with each other, or a condition under which washing is performed once, preferably 2 to 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS, which is the washing condition for normal Southern hybridization.

[0106] As described above, the probe used in the hybridization is a complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the above-mentioned gene as a template. For example, a DNA fragment of about 300 bp in length can be used as a probe. When a DNA fragment of about 300 bp in length is used as a probe, The washing conditions for dye synthesis include 50° C., 2×SSC, and 0.1% SDS.

[0107] In addition, since the degeneracy of codons differs depending on the host, the target gene may be one in which any codon has been replaced with an equivalent codon, i.e., the target gene may be a variant of the target gene exemplified above due to the degeneracy of the genetic code.

[0108] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using the default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using the default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0109] The above descriptions regarding conservative variants of genes and proteins can be applied mutatis mutandis to any proteins such as L-amino acid biosynthetic enzymes and the genes encoding them.

[0110] <1-3> Methods for increasing protein activity Below, methods for increasing protein activity are described.

[0111] "The activity of a protein is increased" means that the activity of the protein is increased compared to that of a non-modified strain. "The activity of a protein is increased" specifically means that the activity of the protein per cell is increased compared to that of a non-modified strain. The "non-modified strain" as used herein means a control strain that has not been modified to increase the activity of the target protein. Examples of non-modified strains include wild-type strains and parent strains. Examples of non-modified strains include type strains of each bacterial species. Examples of non-modified strains include the strains exemplified in the explanation of bacteria. That is, in one embodiment, the activity of the protein may be increased compared to that of a type strain (i.e., a type strain of the species to which the bacterium of the present invention belongs). In another embodiment, the activity of the protein may be increased compared to that of the E. coli K-12 MG1655 strain. In another embodiment, the activity of the protein may be increased compared to that of the P. ananatis AJ13355 strain. In another embodiment, the activity of the protein may be increased compared to that of the P. ananatis NA1 strain. In addition, "protein activity is increased" is also referred to as "protein activity is enhanced." More specifically, "protein activity is increased" may mean that the number of molecules of the protein per cell is increased and / or the function of the protein per molecule is increased compared to a non-modified strain. That is, the "activity" in "protein activity is increased" may not be limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. "Number of molecules of the protein per cell" may mean the average number of molecules of the protein per cell. In addition, "protein activity is increased" includes not only increasing the activity of the target protein in a strain that originally has the activity of the target protein, but also imparting the activity of the target protein to a strain that does not originally have the activity of the target protein. In addition, as long as the activity of the protein is increased as a result, the activity of a suitable target protein may be imparted after reducing or eliminating the activity of the target protein originally possessed by the host.

[0112] The degree of increase in protein activity is not particularly limited as long as the activity of the protein is increased compared to that of the non-modified strain. For example, the activity of the protein may be increased by 1.5 times or more, or 2 times, compared to that of the non-modified strain. In addition, when the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein, and the protein may be produced to an extent that its activity can be measured, for example.

[0113] Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of a gene that codes for the protein. "Gene expression is increased" means that the expression of the gene is increased compared to a non-modified strain such as a wild type or a parent strain. "Gene expression is increased" specifically means that the expression level of the gene per cell is increased compared to a non-modified strain. "Expression level of the gene per cell" may mean the average expression level of the gene per cell. "Gene expression is increased" may more specifically mean that the transcription level (mRNA level) of the gene is increased and / or the translation level (protein level) of the gene is increased. "Gene expression is increased" is also referred to as "enhanced gene expression." The expression of the gene may be increased, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to a non-modified strain. "Gene expression is increased" includes not only increasing the expression level of the target gene in a strain in which the target gene is originally expressed, but also expressing the target gene in a strain in which the target gene is not originally expressed. In other words, "gene expression is increased" may mean, for example, introducing a target gene into a strain that does not harbor the gene, thereby expressing the gene.

[0114] Increased gene expression can be achieved, for example, by increasing the copy number of the gene.

[0115] The copy number of a gene can be increased by introducing the gene into a host chromosome. The introduction of a gene into a chromosome can be carried out, for example, by using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include a method using linear DNA such as Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, a method using a suicide vector that does not have a replication origin that functions in the host, and a transduction method using a phage. Only one copy of a gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a base sequence that has multiple copies on a chromosome. Examples of base sequences that exist in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of a transposon. Homologous recombination may also be performed by targeting an appropriate base sequence on a chromosome, such as a gene that is not necessary for the production of a target substance. Genes can also be randomly introduced onto a chromosome using transposons or Mini-Mu (JP Patent Publication 2-109985, US5,882,888, EP805867B1). Such a method of modifying a chromosome using homologous recombination is not limited to the introduction of a target gene, but can be used for any modification of a chromosome, such as modification of an expression regulatory sequence.

[0116] Introduction of the target gene onto the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0117] The copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene is linked to a vector that functions in a host to construct an expression vector for the gene, and the host is transformed with the expression vector, thereby increasing the copy number of the gene. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the target gene as a template. A vector capable of autonomously replicating in the host cell can be used as the vector. The vector is preferably a multicopy vector. In addition, in order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. The vector may also have a promoter or a terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomously replicating in bacteria of the Enterobacteriaceae family, such as Escherichia coli, include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (Qiagen), pCold TF DNA (Takara Bio), pACYC series vectors, and broad host range vector RSF1010.

[0118] When a gene is introduced, the gene may be retained in the host so as to be expressible. Specifically, the gene may be retained so as to be expressed under the control of a promoter that functions in the host. The promoter is not particularly limited as long as it functions in the host. The "promoter that functions in the host" refers to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a heterologous species. The promoter may be a promoter specific to the gene to be introduced or a promoter of another gene. For example, a stronger promoter as described below may be used as the promoter.

[0119] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a terminator derived from a heterologous species. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene. Specific examples of terminators include the T7 terminator, the T4 terminator, the fd phage terminator, the tet terminator, and the trpA terminator.

[0120] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0121] In addition, when two or more genes are introduced, each gene may be retained in the host in an expressible manner. For example, all of the genes may be retained on a single expression vector, or all of the genes may be retained on a chromosome. In addition, each gene may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. In addition, two or more genes may be introduced as an operon. Examples of "introducing two or more genes" include introducing genes that each code for two or more proteins (e.g., enzymes), introducing genes that each code for two or more subunits that compose a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0122] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene derived from the host or a gene derived from a heterologous species. The gene to be introduced may be obtained by PCR, for example, using primers designed based on the base sequence of the gene and using the genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. The gene to be introduced may also be totally synthesized based on the base sequence of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene may be used as is or after appropriate modification. That is, a variant of the gene may be obtained by modifying the gene. The gene may be modified by a known method. For example, a desired mutation may be introduced into a desired site of DNA by site-directed mutagenesis. That is, for example, a coding region of a gene may be modified by site-directed mutagenesis so that the encoded protein contains substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Examples of site-specific mutagenesis include a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and a method using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, a gene variant may be totally synthesized.

[0123] In addition, when a protein functions as a complex consisting of multiple subunits, all or only a part of the multiple subunits may be modified as long as the activity of the protein is increased as a result. That is, for example, when the activity of a protein is increased by increasing the expression of a gene, the expression of all or only a part of the multiple genes encoding the subunits may be enhanced. Usually, it is preferable to enhance the expression of all of the multiple genes encoding the subunits. In addition, each subunit constituting the complex may be derived from one organism, or from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, genes encoding multiple subunits derived from the same organism may be introduced into the host, or genes derived from different organisms may be introduced into the host.

[0124] Moreover, an increase in gene expression can be achieved by improving the transcription efficiency of the gene. An increase in gene expression can be achieved by improving the translation efficiency of the gene. An increase in gene transcription efficiency and translation efficiency can be achieved, for example, by modifying an expression regulatory sequence. "Expression regulatory sequence" is a general term for a site that affects gene expression. Examples of expression regulatory sequences include promoters, Shine-Dalgarno (SD) sequences (also called ribosome binding sites (RBS)), and spacer regions between the RBS and the start codon. Expression regulatory sequences can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression regulatory sequences can be modified, for example, by a method using a temperature-sensitive vector or the Red-driven integration method (WO2005 / 010175). Cut.

[0125] The improvement of gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a stronger promoter. A "stronger promoter" means a promoter that improves gene transcription more than the wild-type promoter that is originally present. Examples of stronger promoters include known high expression promoters such as T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter derived from Bifidobacterium, PR promoter, and PL promoter. In addition, as a stronger promoter, a highly active type of a conventional promoter may be obtained by using various reporter genes. For example, the activity of a promoter can be increased by bringing the -35 and -10 regions in the promoter region closer to the consensus sequence (WO 00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent Application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0126] The translation efficiency of a gene can be improved, for example, by replacing the Shine-Dalgarno (SD) sequence (also called the ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence. A "stronger SD sequence" refers to an SD sequence that improves the translation of mRNA more than the wild-type SD sequence that is originally present. An example of a stronger SD sequence is the RBS of gene 10 derived from phage T7 (Olins PO et al, Gene, 1988, 73, 227-235). In addition, substitutions or insertions of several nucleotides in the spacer region between the RBS and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), It is known that modifications such as deletions or deletions have a significant effect on mRNA stability and translation efficiency, and gene translation efficiency can also be improved by modifying these.

[0127] The improvement of gene translation efficiency can also be achieved, for example, by modifying codons. For example, the translation efficiency of a gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. That is, the gene to be introduced may be modified to have optimal codons depending on the codon usage frequency of the host to be used. Codon replacement can be performed, for example, by site-directed mutagenesis. Alternatively, gene fragments with replaced codons may be totally synthesized. The codon usage frequency in various organisms is disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).

[0128] Furthermore, increasing gene expression can also be achieved by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

[0129] The above-mentioned techniques for increasing gene expression may be used alone or in any combination.

[0130] Modifications that increase the activity of a protein can also be achieved by, for example, enhancing the specific activity of the protein. Enhancement of specific activity also includes desensitization to feedback inhibition. Proteins with enhanced specific activity can be obtained, for example, by searching for various organisms. Highly active proteins can also be obtained by introducing mutations into existing proteins. The mutations introduced can be, for example, substitution, deletion, insertion, and / or addition of one or several amino acids at one or several positions of the protein. Mutations can be introduced, for example, by the site-specific mutagenesis method described above. Mutations can also be introduced, for example, by mutation treatment. Examples of mutation treatments include irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Random mutations can also be induced by directly treating DNA with hydroxylamine in vitro. The enhancement of specific activity may be used alone or in any combination with the above-mentioned techniques for enhancing gene expression.

[0131] The method of transformation is not particularly limited, and a conventionally known method can be used. For example, a method of transformation can be used in which a recipient cell is transformed with calcium chloride, as reported for Escherichia coli K-12. Alternatively, a method of preparing competent cells from growing cells and introducing DNA into them, as reported for Bacillus subtilis, can be used (Duncan, CH, Wilson, GA and Young, FE, 1977. Gene 1: 153-167). Alternatively, a method of introducing recombinant DNA into a DNA recipient bacterium by converting the cell of the DNA recipient bacterium into a protoplast or spheroplast state that easily incorporates recombinant DNA, as known for Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933), can also be applied. Alternatively, an electric pulse method (JP Patent Publication 2-207791) as reported for coryneform bacteria can also be used.

[0132] The increase in the activity of a protein can be confirmed by measuring the activity of the protein.

[0133] Increased activity of a protein can also be confirmed by confirming increased expression of the gene encoding the protein. Increased expression of a gene can be confirmed by confirming increased transcription level of the gene or increased amount of the protein expressed from the gene.

[0134] The increase in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a wild-type strain or a non-modified strain such as a parent strain. Methods for evaluating the amount of mRNA include northern hybridization, RT-PCR, microarray, and RNA-seq. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., the number of molecules per cell) may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more compared to that of a non-modified strain.

[0135] The increase in the amount of the protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein (e.g., the number of molecules per cell) may be increased, for example, by 1.5 times or more, 2 times or more, or 3 times or more compared to the non-modified strain.

[0136] The above-mentioned methods for increasing protein activity can be used to enhance the activity of any protein or enhance the expression of any gene.

[0137] <1-4> Methods for reducing protein activity Below, methods for reducing protein activity are described.

[0138] "The activity of a protein is reduced" means that the activity of the protein is reduced compared to that of a non-modified strain. "The activity of a protein is reduced" specifically means that the activity of the protein per cell is reduced compared to that of a non-modified strain. The "non-modified strain" as used herein means a control strain that has not been modified to reduce the activity of the target protein. Examples of non-modified strains include wild-type strains and parent strains. Examples of non-modified strains include type strains of each bacterial species. Examples of non-modified strains include the strains exemplified in the explanation of bacteria. That is, in one embodiment, the activity of the protein may be reduced compared to that of a type strain (i.e., a type strain of the species to which the bacterium of the present invention belongs). In another embodiment, the activity of the protein may be reduced compared to that of the E. coli K-12 MG1655 strain. In another embodiment, the activity of the protein may be reduced compared to that of the P. ananatis AJ13355 strain. In another embodiment, the activity of the protein may be reduced compared to that of the P. ananatis NA1 strain. In addition, "the activity of a protein is reduced" also includes a case where the activity of the protein is completely lost. More specifically, "the activity of a protein is reduced" may mean that the number of molecules of the protein per cell is reduced and / or the function of the protein per molecule is reduced compared to a non-modified strain. That is, the "activity" in "the activity of a protein is reduced" is not limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of a gene encoding the protein. "The number of molecules of a protein per cell" may mean the average number of molecules of the protein per cell. In addition, "the number of molecules of a protein is reduced" also includes a case where the protein is not present at all. In addition, "the function of a protein per molecule is reduced" also includes a case where the function of the protein per molecule is completely lost. The degree of reduction in the activity of a protein is not particularly limited as long as the activity of the protein is reduced compared to a non-modified strain.The activity of the protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0139] Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of a gene encoding the protein. "Reduced gene expression" means that the expression of the gene is reduced compared to a non-modified strain such as a wild type strain or a parent strain. "Reduced gene expression" specifically means that the expression level of the gene per cell is reduced compared to a non-modified strain. "Expression level of the gene per cell" may mean the average expression level of the gene per cell. "Reduced gene expression" may more specifically mean that the transcription level (mRNA level) of the gene is reduced and / or the translation level (protein level) of the gene is reduced. "Reduced gene expression" also includes cases where the gene is not expressed at all. "Reduced gene expression" is also referred to as "weakened gene expression." The expression of the gene may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0140] The reduction in gene expression may be due to, for example, a reduction in transcription efficiency, a reduction in translation efficiency, or a combination thereof. The reduction in gene expression may be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter, a Shine-Dalgarno (SD) sequence (also called a ribosome binding site (RBS)), or a spacer region between the RBS and the start codon. When modifying an expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases of the expression regulatory sequence are modified. The reduction in gene transcription efficiency may be achieved, for example, by replacing a promoter of a gene on a chromosome with a weaker promoter. The term "weaker promoter" refers to a promoter that weakens the transcription of a gene compared to the wild-type promoter that is originally present. An example of a weaker promoter is an inducible promoter. That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (for example, in the absence of an inducer). In addition, a part or all of the region of the expression regulatory sequence may be deleted (deleted). In addition, the reduction in gene expression may also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Reduction of gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces the expression of the gene. For example, gene expression can be reduced by replacing a codon in the coding region of the gene with a synonymous codon that is used less frequently in the host. Furthermore, gene expression itself can be reduced, for example, by disrupting the gene as described below.

[0141] Modifications that reduce the activity of a protein can be achieved, for example, by disrupting the gene that codes for the protein. "The gene is disrupted" means that the gene is modified so that it does not produce a protein that functions normally. "Not producing a protein that functions normally" includes cases where no protein is produced from the gene at all, and cases where the gene produces a protein with reduced or lost function (e.g., activity or properties) per molecule.

[0142] The destruction of a gene can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Deletion of a gene" refers to the deletion of a part or the whole region of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on a chromosome. The sequences before and after the coding region of the gene may include, for example, an expression regulatory sequence of the gene. As long as the activity of the protein can be reduced, the region to be deleted may be any region, such as an N-terminal region (a region that codes for the N-terminal side of a protein), an internal region, or a C-terminal region (a region that codes for the C-terminal side of a protein). In general, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, a region with a length of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of the coding region of the gene. In addition, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. Reading frame mismatches can result in a frameshift downstream of the region to be deleted.

[0143] Gene disruption can also be achieved, for example, by introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or introducing an addition or deletion of 1 to 2 bases (frameshift 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 26 116, 20833-20839(1991)).

[0144] Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the base sequence to be inserted, the more reliably the gene can be inactivated. In addition, it is preferable that the sequences before and after the insertion site do not have the same reading frame. A mismatch in the reading frame may cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples of such sequences include marker genes such as antibiotic resistance genes and genes useful for producing target substances.

[0145] The gene may be disrupted so that the amino acid sequence of the encoded protein is deleted (defective). In other words, modification to reduce the activity of the protein can be achieved, for example, by deleting the amino acid sequence of the protein (a part or all of the region of the amino acid sequence), specifically by modifying the gene so as to code a protein from which the amino acid sequence (a part or all of the region of the amino acid sequence) is deleted. The term "deletion of the amino acid sequence of the protein" refers to the deletion of a part or all of the region of the amino acid sequence of the protein. The term "deletion of the amino acid sequence of the protein" refers to the absence of the original amino acid sequence in the protein, and also includes the case where the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region that has been changed to another amino acid sequence by frameshift may be considered as the deleted region. The deletion of the amino acid sequence of the protein typically shortens the full length of the protein, but there may be cases where the full length of the protein does not change or is extended. For example, the deletion of a part or all of the region of the coding region of the gene can delete the region coded by the deleted region in the amino acid sequence of the encoded protein. For example, by introducing a stop codon into the coding region of a gene, the region coded by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. For example, by frameshifting in the coding region of a gene, the region coded by the frameshift site can be deleted. The position and length of the region to be deleted in the deletion of the amino acid sequence are as follows: The same explanations as for the location and length of the region to be deleted in gene deletion can be applied mutatis mutandis.

[0146] The above modification of a gene on a chromosome can be achieved, for example, by preparing a disrupted gene modified so as not to produce a protein that functions normally, transforming a host with recombinant DNA containing the disrupted gene, and causing homologous recombination between the disrupted gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, the recombinant DNA can be easily manipulated if it contains a marker gene according to the host's characteristics such as nutritional requirements. Examples of disrupted genes include genes in which a part or all of the coding region of a gene has been deleted, genes in which a missense mutation has been introduced, genes in which a nonsense mutation has been introduced, genes in which a frameshift mutation has been introduced, and genes in which an insertion sequence such as a transposon or a marker gene has been inserted. Even if a protein encoded by a disrupted gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as it allows homologous recombination to occur in a desired manner. For example, a host can be transformed with a linear DNA containing a disrupted gene, the linear DNA having upstream and downstream sequences of a wild-type gene on a chromosome at both ends, and homologous recombination can be caused upstream and downstream of the wild-type gene, thereby replacing the wild-type gene with the disrupted gene.Such gene disruption by gene replacement using homologous recombination has already been established, and includes a method using linear DNA such as "Red-driven integration" (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), a method combining the Red-driven integration method with an excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, and a method using a suicide vector that does not have a replication origin that functions in the host (U.S. Pat. No. 6,303,383, JP-A-05-007491). Such a chromosome modification technique using homologous recombination is not limited to the disruption of a target gene, but can be used for any modification of a chromosome, such as modification of an expression regulatory sequence.

[0147] Furthermore, a modification that reduces the activity of a protein may be performed, for example, by a mutation treatment. Examples of the mutation treatment include irradiation with X-rays, irradiation with ultraviolet light, and N-methyl-N' Examples of such mutagens include treatment with mutagens such as N-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0148] In addition, when a protein functions as a complex consisting of multiple subunits, all of the multiple subunits may be modified, or only a part of them may be modified, as long as the activity of the protein is reduced as a result. That is, for example, all of the multiple genes encoding the subunits may be disrupted, or only a part of them may be disrupted, or the like. In addition, when a protein has multiple isozymes, the activities of all of the multiple isozymes may be reduced, or only a part of them may be reduced, as long as the activity of the protein is reduced as a result. That is, for example, all of the multiple genes encoding the isozymes may be disrupted, or only a part of them may be disrupted, or the like.

[0149] The above-mentioned methods for reducing the activity of a protein may be used alone or in any combination.

[0150] The decrease in the activity of a protein can be confirmed by measuring the activity of the protein.

[0151] The decrease in protein activity can also be confirmed by confirming the decrease in expression of the gene encoding the protein. This can be confirmed by confirming a decrease in the amount of transcription of the gene or a decrease in the amount of protein expressed from the gene.

[0152] The reduction in the amount of gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Methods for evaluating the amount of mRNA include northern hybridization, RT-PCR, microarrays, and RNA-seq (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., the number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0153] The reduction in the amount of the protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein (e.g., the number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0154] Depending on the means used for the disruption, gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene.

[0155] The above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein or reduce or destroy the expression of any gene. The above-mentioned methods for reducing protein activity can also be used when the gene to be reduced or destroyed is present on an extrachromosomal structure (e.g., a plasmid). In this case, the term "chromosome" in the above-mentioned methods for reducing protein activity can be read as "extrachromosomal structure" (e.g., a plasmid).

[0156] <2> The method for producing L-amino acids according to the present invention The method of the present invention is a method for producing an L-amino acid, comprising culturing the bacterium of the present invention in a medium, accumulating the L-amino acid in the medium and / or within the bacterial cells of the bacterium, and collecting the L-amino acid from the medium and / or the bacterial cells. The L-amino acids are as described above. In the present invention, one type of L-amino acid may be produced, or two or more types of L-amino acids may be produced.

[0157] The medium to be used is not particularly limited as long as the bacterium of the present invention can grow and the desired L-amino acid can be produced. As the medium, for example, a conventional medium used for culturing bacteria such as bacteria of the Enterobacteriaceae family can be used. As the medium, for example, a medium containing components selected from a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic components and inorganic components as necessary can be used. The type and concentration of the medium components may be appropriately set depending on various conditions such as the type of bacteria used.

[0158] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, fumaric acid, citric acid, and succinic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. Examples of carbon sources include sugars. In addition, plant-derived raw materials can be preferably used as the carbon source. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing them, and decomposition products of those plant organs. The form of use of plant-derived raw materials is not particularly limited, and can be used in any form, such as raw products, squeezed juice, pulverized products, and purified products. In addition, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in the plant biomass may be hydrolyzed in advance to liberate pentose, and then the cellulose may be hydrolyzed to produce hexose. Xylose may be supplied by conversion from hexose, such as glucose, by having the bacterium of the present invention possess a conversion pathway from hexose to xylose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0159] Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soy protein hydrolysate, ammonia, and urea. Ammonia gas and aqueous ammonia used for pH adjustment may be used as the nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

[0160] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0161] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0162] Specific examples of other various organic and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, calcium, zinc, copper, and cobalt; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. As the other various organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

[0163] When using an auxotrophic mutant strain that requires amino acids or the like for growth, it is preferable to supplement the medium with the required nutrients.

[0164] It is also preferable to limit the amount of biotin in the medium or to add a surfactant or penicillin to the medium.

[0165] The culture conditions are not particularly limited as long as the bacterium of the present invention can grow and produce the desired L-amino acid. The culture can be carried out under normal conditions used for culturing bacteria such as bacteria of the Enterobacteriaceae family. The culture conditions can be appropriately set depending on various conditions such as the type of bacteria used.

[0166] The culture can be carried out using a liquid medium. During the culture, the bacterium of the present invention may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the bacterium of the present invention may be seed-cultured in a liquid medium and then inoculated into the liquid medium for main culture. That is, the culture may be divided into a seed culture and a main culture. In this case, the culture conditions for the seed culture and the main culture may or may not be the same. There is no particular restriction on the amount of the bacterium of the present invention contained in the medium at the start of the culture. For example, the main culture may be inoculated with 1 to 50% (v / v) of the seed culture liquid into the medium for main culture. This may be done by

[0167] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination of these. The medium at the start of the culture is also called the "initial medium". The medium supplied to the culture system (fermenter) in fed-batch culture or continuous culture is also called the "fed-batch medium". Supplying a fed-batch medium to the culture system in fed-batch culture or continuous culture is also called "fed-batch". When the culture is divided into a seed culture and a main culture, for example, both the seed culture and the main culture may be performed by batch culture. For example, the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch culture or continuous culture.

[0168] In the present invention, each medium component may be contained in the initial medium, the feed medium, or both. The type of component contained in the initial medium may or may not be the same as the type of component contained in the feed medium. The concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feedings are intermittently performed, the type and / or concentration of components contained in the feed medium for each feeding may or may not be the same.

[0169] The concentration of the carbon source in the medium is not particularly limited as long as the bacterium of the present invention can grow and an L-amino acid can be produced. The concentration of the carbon source in the medium may be as high as possible within a range in which the production of an L-amino acid is not inhibited. The concentration of the carbon source in the medium may be, for example, 1 to 30 w / v%, preferably 3 to 10 w / v%, as the initial concentration (initial concentration in the medium). In addition, an additional carbon source may be appropriately added to the medium. For example, an additional carbon source may be added to the medium according to the consumption of the carbon source accompanying the progress of fermentation.

[0170] The culture can be carried out, for example, under aerobic conditions. The aerobic conditions refer to a state in which the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of an oxygen membrane electrode, and may be preferably 1.5 ppm or more. The oxygen concentration may be controlled, for example, to 5 to 50%, preferably about 10%, relative to the saturated oxygen concentration. Specifically, the culture under aerobic conditions can be carried out by aeration culture, shaking culture, stirring culture, or a combination thereof. The pH of the medium can be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature can be, for example, 20 to 40°C, preferably 25 to 37°C. The culture period can be, for example, 10 hours to 120 hours. Culturing may be continued, for example, until the carbon source in the medium is consumed or until the activity of the bacterium of the present invention is lost. By culturing the bacterium of the present invention under such conditions, L-amino acids are accumulated in the medium and / or within the cells.

[0171] In addition, when producing L-glutamic acid, the culture can be performed while precipitating L-glutamic acid in a liquid medium adjusted to the conditions for precipitating L-glutamic acid. The conditions for precipitating L-glutamic acid include, for example, pH 5.0 to 4.0, preferably pH 4.5. Preferably, the condition is pH 4.0 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0 (EP1078989A). When using a liquid medium adjusted to the conditions for precipitating L-glutamic acid, more efficient crystallization can be achieved by adding pantothenic acid to the medium (WO2004 / 111258). In addition, when a liquid medium adjusted to the conditions for L-glutamic acid precipitation is used, crystallization can be more efficient by adding L-glutamic acid crystals as seed crystals to the medium (EP1233069A).In addition, when a liquid medium adjusted to the conditions for L-glutamic acid precipitation is used, crystallization can be more efficient by adding L-glutamic acid crystals and L-lysine crystals as seed crystals to the medium (EP1624069A).

[0172] In addition, when producing a basic amino acid such as L-lysine, the culture step (fermentation step) may be carried out so that bicarbonate ions and / or carbonate ions serve as counter ions of the basic amino acid. Such a fermentation form is also called "carbonate fermentation". According to carbonate fermentation, it is possible to produce a basic amino acid by fermentation while reducing the amount of sulfate ions and / or chloride ions that have conventionally been used as counter ions of basic amino acids. Carbonate fermentation can be carried out, for example, as described in US2002-025564A, EP1813677A, and JP2002-65287A.

[0173] The fermentation liquid can be treated, for example, with a liquid cyclone. The liquid cyclone can be, for example, of a general shape with a cylindrical diameter of 10 to 110 mm, made of ceramic, stainless steel, or resin. The amount of the fermentation liquid fed to the liquid cyclone can be set, for example, according to the bacterial cell concentration and L-amino acid concentration in the fermentation liquid. The amount of the fermentation liquid fed to the liquid cyclone can be, for example, 2 to 1200 L / min.

[0174] The production of L-amino acids can be confirmed by known techniques used for detecting or identifying compounds, such as HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in appropriate combination.

[0175] The recovery of L-amino acids from the fermentation broth can be carried out by known methods used for separating and purifying compounds. Examples of such methods include the ion exchange resin method (Nagai, H. et al., 2001). al., Separation Science and Technology, 39(16), 3691-3710), precipitation method, membrane separation method (JP Patent Publication No. 9-164323, JP Patent Publication No. 9-173792), crystallization method (WO2008 / 078448, WO2008 / 078646). These methods can be used alone or in appropriate combination. When L-amino acids accumulate in the cells, for example, the cells can be disrupted by ultrasonic waves or the like, and the cells can be removed by centrifugation to obtain a supernatant, from which the L-amino acids can be recovered by an ion exchange resin method or the like. The recovered L-amino acids may be in the free form, a salt thereof, or a mixture thereof. Examples of salts include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts. In the case of producing L-glutamic acid, the recovered L-glutamic acid may be, specifically, for example, free L-glutamic acid, sodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or a mixture thereof. For example, sodium L-glutamate (MSG) can be obtained by adding an acid to the ammonium L-glutamate in the fermentation liquid to crystallize it, and adding an equimolar amount of sodium hydroxide to the crystals. Note that activated carbon may be added before or after the crystallization to decolorize it (see Industrial Crystallization of Sodium Glutamate, Journal of the Society of Sea Water Science of Japan, Vol. 56, No. 5, Tetsuya Kawakita). The sodium L-glutamate crystals can be used, for example, as an umami seasoning. The sodium L-glutamate crystals may be mixed with nucleic acids such as sodium guanylate and sodium inosinate, which also have an umami taste, and used as a seasoning.

[0176] When the L-amino acid precipitates in the medium, it can be collected by centrifugation, filtration, etc. The L-amino acid precipitated in the medium may be isolated together with the L-amino acid dissolved in the medium after crystallization.

[0177] The recovered L-amino acid may contain, in addition to the L-amino acid, bacterial cells, medium components, water, metabolic by-products of the bacteria, and other components. The L-amino acid may be purified to a desired degree. The purity of the recovered L-amino acid is, for example, 50% (w / w) or more, preferably 50% (w / w) or more. The content may be 85% (w / w) or more, and particularly preferably 95% (w / w) or more (JP1214636B, USP5,431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714, US2005 / 0025878). EXAMPLES

[0178] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0179] Example: L-glutamic acid production by budABC-deficient and GlcNDH gene (PAJ_3461, PAJ_3462, and PAJ_3463)-deficient strains of Pantoea ananatis (1) Materials and methods (1-1) Bacterial strain used Pantoea ananatis SC17(0) / RSF-Red-TER strain (WO2008 / 090770) Pantoea ananatis NA1 strain (WO2008 / 090770)

[0180] (1-2) Culture medium used LB medium (Table 1) Glu fermentation medium (Table 2) SOC medium (Table 3) LBGM9 medium (Table 4)

[0181] [Table 1]

[0182] [Table 2]

[0183] [Table 3]

[0184] [Table 4]

[0185] (1-3) Gene deletion by λ-red method Pantoea ananatis SC17(0) / RSF-Red-TER strain was inoculated into 4 ml of LB medium containing 25 mg / L Cm (chloramphenicol) in a test tube and cultured overnight at 34 °C with shaking at 120 rpm to obtain a preculture solution. Next, 0.5 mL of the preculture solution was inoculated into 50 mL of LB medium containing 1 mM IPTG and 25 mg / L Cm in a Sakaguchi flask and cultured for 3 hours. The resulting culture solution was harvested using a refrigerated centrifuge and washed three times with ice-cold 10% glycerol solution to prepare competent cells. Next, the PCR fragment for gene deletion was introduced into the competent cells by electroporation. After adding SOC medium and culturing for 2 hours at 34 °C, the culture solution was spread on LB agar medium containing 40 mg / L Km (kanamycin). The gene deletion strain was obtained as a Km resistant strain.

[0186] (1-4) Gene deletion by genome introduction The gene-deleted strain obtained in (1-3) above was extracted using the PurElute genome extraction kit. TM The genome was extracted using a Bacterial Genomic Kit (EdgeBio). The strain was cultured overnight on agar medium, and the cells were scraped off from the grown plate and soaked in 10% glycerol. Competent cells were prepared by washing three times with PBS. The genome of the gene-deficient strain was introduced into competent cells by the cloning method. SOC medium was added and the cells were incubated for 2 hours at 34°C for recovery, after which the culture was spread onto LBGM9 agar medium containing 40 mg / L Km. The gene-deficient strain was obtained as a Km-resistant strain.

[0187] (2) Construction of P. ananatis NA1△budABC::Km strain Pantoea ananatis SC17(0)hisD::(attL λ -Km R -attR λ )(Joanna I Katashkina et al., Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis, BMC Mol Biol, doi: 10.1186 / 1471-2199-10-34.) genome as a template. Using the primers in columns 13 and 14, an attL-Km-attR fragment for deleting the budABC genes was obtained by PCR, to which the upstream sequence of the budA ORF and the downstream sequence of the budC ORF were added. The gene was introduced into the SC17(0) / RSF-Red-TER strain by the λ-Red method described in (1-3), and the SC17(0)△budABC::Km / RSF-Red-TER strain was obtained as a Km-resistant strain. It was confirmed that the Km resistance gene was inserted in the SC17(0)△budABC::Km / RSF-Red-TER strain so that the budABC region ORF was deleted. Extracted from the SC17(0)△budABC::Km / RSF-Red-TER strain The genome thus obtained was introduced into the Glu-producing strain NA1 of Pantoea ananatis by the above-mentioned (1-4) genome introduction, and the NA1△budABC::Km strain was obtained as a Km-resistant strain. The PCR used revealed that the Km resistance gene was deleted from the budABC region ORF in the NA1△budABC::Km strain. I confirmed that it was inserted properly.

[0188] (3) L-Glutamic acid production by P. ananatis NA1△budABC::Km strain The P. ananatis NA1 strain was cultured on LBGM9 agar medium containing 12.5 mg / L Tet (tetracycline). The P. ananatis NA1△budABC::Km strain was cultured on LBGM9 agar medium containing 12.5 mg / L Tet and 40 mg / L Km. The bacteria were cultured on an agar medium. A quarter of the plate of bacteria was added to 5 mL of Glu fermentation medium containing 12.5 mg / L Tet. The medium was inoculated and cultured at 34°C for 24 hours. The culture was analyzed using a bioanalyzer BF5 (Oji Instruments). The residual sugar (RS) and L-glutamic acid contents in the liquid were measured.

[0189] The results are shown in Table 5. Deletion of the budABC genes improved L-glutamic acid production. This demonstrates that reduced activity of the BudA protein, BudB protein, and / or BudC protein improves production of L-amino acids such as L-glutamic acid.

[0190] [Table 5]

[0191] (4) Construction of P. ananatis NA1△GlcNDH::Km strain Pantoea ananatis SC17(0)hisD::(attL λ -Km R -attR λ )(Joanna I Katashkina et al., Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis, BMC Mol Biol, doi: 10.1186 / 1471-2199-10-34.) genome as a template. The primers in columns 17 and 18 were used to clone the upstream sequence of the PAJ_3461 ORF and the downstream sequence of the PAJ_3463 ORF. An attL-Km-attR fragment for deleting the GlcNDH gene (PAJ_3461, PAJ_3462, and PAJ_3463) with the added sequence was obtained by PCR. This fragment was introduced into SC17(0) / RSF-Red-TER by the λ-Red method described above in (1-3), and the SC17(0)△GlcNDH::Km / RSF-Red-TER strain was obtained as a Km-resistant strain. By PCR using the primers of SEQ ID NOs: 19 and 20, it was confirmed that the Km-resistant gene had been inserted in the SC17(0)△GlcNDH::Km / RSF-Red-TER strain so as to delete the GlcNDH region ORF. The genome extracted from the SC17(0)△GlcNDH::Km / RSF-Red-TER strain was introduced into the Glu-producing strain NA1 of Pantoea ananatis by the above (1-4) genome introduction, and the NA1△GlcNDH::Km strain was obtained as a Km-resistant strain. By PCR using the primers of SEQ ID NO: 19 and 20, it was confirmed that the Km-resistant gene was inserted in the NA1△GlcNDH::Km strain so that the GlcNDH region ORF was deleted.

[0192] (5) L-Glutamic acid production by P. ananatis NA1△GlcNDH::Km strain The P. ananatis NA1 strain was cultured on LBGM9 agar medium containing 12.5 mg / L Tet (tetracycline). The P. ananatis NA1△GlcNDH::Km strain was cultured on LBGM9 agar medium containing 12.5 mg / L Tet and 40 mg / L Km. The bacteria were cultured on an agar medium. A quarter of the plate of bacteria was added to 5 mL of Glu fermentation medium containing 12.5 mg / L Tet. The medium was inoculated and cultured at 34°C for 24 hours. The culture was analyzed using a bioanalyzer BF5 (Oji Instruments). The residual sugar (RS) and L-glutamic acid contents in the liquid were measured.

[0193] The results are shown in Table 6. L-glutamic acid production was improved by deleting the GlcNDH genes (PAJ_3461, PAJ_3462, and PAJ_3463). Therefore, it was revealed that the production of L-amino acids such as L-glutamic acid is improved by reducing the activity of the PAJ_3461 protein, the PAJ_3462 protein, and / or the PAJ_3463 protein.

[0194] [Table 6]

[0195] (6) Construction of P. ananatis NA1△GlcNDH strain In order to remove the antibiotic resistance marker introduced into the chromosome, a helper plasmid pMW-intxis-sacB(Spc) (WO2015 / 005405A1) for expressing the int-xis gene of λ phage was introduced into the gene deletion strain obtained in (4) above by electroporation, and colonies were formed at 30° C. using LB medium containing 100 mg / L Spc. Then, single colonies were isolated at 34° C. using LB medium containing 10% sucrose, M9 minimal salts, and 1 mM IPTG without Spc, and a strain in which pMW-intxis-sacB(Spc) and the antibiotic resistance marker gene on the chromosome had been lost was obtained based on sensitivity to antibiotics (antibiotics corresponding to Spc and the antibiotic resistance marker introduced into the chromosome).

[0196] (7) Construction of P. ananatis NA1△GlcNDH△budABC::Km strain The genome extracted from the SC17(0)△budABC::Km / RSF-Red-TER strain constructed in (2) above was The genome of (1-4) was introduced into the P. ananatis NA1△GlcNDH strain, and the NA1△GlcNDH△budABC::Km strain was obtained as a Km-resistant strain. By PCR using primers of SEQ ID NOs: 15 and 16, it was confirmed that the Km-resistant gene was inserted in the NA1△GlcNDH△budABC::Km strain so that the budABC region ORF was deleted.

[0197] (8) L-Glutamic acid production by P. ananatis NA1△GlcNDH△budABC::Km strain P. ananatis NA1 strain was cultured on LBGM9 agar medium containing 12.5 mg / L Tet (tetracycline). P. ananatis NA1△GlcNDH::Km strain, NA1△budABC::Km strain, and NA1△GlcNDH△budABC::Km strain were cultured on LBGM9 agar medium containing 12.5 mg / L Tet and 40 mg / L Km. A quarter of the plate of cells was inoculated into 5 mL of Glu fermentation medium containing 12.5 mg / L Tet and cultured at 34°C for 24 hours. The residual sugar (RS) and L-glutamic acid contents in the culture medium were measured using a Bioanalyzer BF5 (Oji Instruments).

[0198] The results are shown in Table 7. The deletion of the GlcNDH genes (PAJ_3461, PAJ_3462, and PAJ_3463) and the deletion of the budABC genes improved L-glutamic acid production per cell. In addition, the combination of deletion of the GlcNDH gene and the budABC gene further improved L-glutamic acid production per cell compared with the deletion of only the GlcNDH gene and the deletion of only the budABC gene. Thus, it was revealed that the combination of the reduction in activity of the PAJ_3461 protein, the PAJ_3462 protein, and / or the PAJ_3463 protein and the reduction in activity of the BudA protein, the BudB protein, and / or the BudC protein improves the production of L-amino acids such as L-glutamic acid per cell.

[0199] [Table 7]

[0200] [Explanation of sequence listing] SEQ ID NO: 1: Nucleotide sequence of the budA gene of P. ananatis AJ13355 SEQ ID NO: 2: Amino acid sequence of the BudA protein of P. ananatis AJ13355 SEQ ID NO: 3: Nucleotide sequence of the budB gene of P. ananatis AJ13355 SEQ ID NO: 4: Amino acid sequence of BudB protein of P. ananatis AJ13355 SEQ ID NO: 5: Nucleotide sequence of the budC gene of P. ananatis AJ13355 SEQ ID NO: 6: Amino acid sequence of the BudC protein of P. ananatis AJ13355 SEQ ID NO: 7: Nucleotide sequence of the PAJ_3461 gene of P. ananatis AJ13355 SEQ ID NO: 8: Amino acid sequence of the PAJ_3461 protein of P. ananatis AJ13355 SEQ ID NO: 9: Nucleotide sequence of the PAJ_3462 gene of P. ananatis AJ13355 SEQ ID NO: 10: Amino acid sequence of the PAJ_3462 protein of P. ananatis AJ13355 SEQ ID NO: 11: Nucleotide sequence of the PAJ_3463 gene of P. ananatis AJ13355 SEQ ID NO: 12: Amino acid sequence of the PAJ_3463 protein of P. ananatis AJ13355 SEQ ID NOs: 13 to 20: primers

Claims

1. A method for producing L-amino acids, Culturing bacteria belonging to the Enterobacteriaceae family that have the ability to produce L-amino acids in a culture medium, and accumulating L-amino acids in the culture medium and / or within the bacterial cells, and To collect the L-amino acid from the culture medium and / or the bacterial cells, Includes, The aforementioned L-amino acid is a glutamic acid-based L-amino acid, The method involves the bacteria having all of the following modifications (D) to (F): (D) Modifications that reduce the activity of the PAJ_3461 protein; (E) Modifications that reduce the activity of the PAJ_3462 protein; and (F) Modification that reduces the activity of the PAJ_3463 protein.

2. The method according to claim 1, wherein the bacterium further has one or more modifications selected from the following modifications (A) to (C): (A) Modifications that reduce the activity of the BudA protein; (B) Modifications that reduce the activity of the BudB protein; (C) Modification that reduces the activity of the BudC protein.

3. The method according to claim 2, wherein the bacteria have all of the modifications described in (A) to (C).

4. A method according to any one of claims 1 to 3, The activity of the BudA protein is reduced by decreasing the expression of the budA gene or by disrupting the gene; The activity of the BudB protein is reduced by decreasing the expression of the budB gene or by disrupting the gene; The activity of the BudC protein is reduced by decreasing the expression of the budC gene or by disrupting the gene; The activity of the PAJ_3461 protein is reduced by decreasing the expression of the PAJ_3461 gene or by disrupting the gene; The activity of the PAJ_3462 protein is reduced by reducing the expression of the PAJ_3462 gene or by disrupting the gene; and / or The activity of the PAJ_3463 protein reduces the expression of the PAJ_3463 gene, A method that reduces the gene by destroying it.

5. A method according to any one of claims 1 to 3, The expression of the budA gene is reduced by modification of the budA gene expression regulatory sequence; The expression of the budB gene is reduced by modification of the budB gene expression regulatory sequence; The expression of the budC gene is reduced by modification of the budC gene expression regulatory sequence; The expression of the PAJ_3461 gene is reduced by modification of the regulatory sequence of the PAJ_3461 gene; The expression of the PAJ_3462 gene is reduced by modification of the PAJ_3462 gene expression regulatory sequence; and / or A method by which the expression of the PAJ_3463 gene is reduced by modifying the expression regulatory sequence of the PAJ_3463 gene.

6. A method according to any one of claims 1 to 3, The activity of the BudA protein is reduced by the deletion of the budA gene; The activity of the BudB protein is reduced by the deletion of the budB gene; The activity of the BudC protein is reduced by the deletion of the budC gene; The activity of the PAJ_3461 protein is reduced by the deletion of the PAJ_3461 gene; The activity of the PAJ_3462 protein is reduced by deletion of the PAJ_3462 gene; and / or A method by which the activity of the PAJ_3463 protein is reduced by deletion of the PAJ_3463 gene.

7. A method according to any one of claims 1 to 3, The BudA protein is the protein described in (1a), (1b), or (1c) below: (1a) Proteins containing the amino acid sequence shown in Sequence ID No. 6; (1b) A protein having acetolactate decarboxylase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 6; (1c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 6, and that has acetolactate decarboxylase activity; The BudB protein is the protein described in (2a), (2b), or (2c) below: (2a) Proteins containing the amino acid sequence shown in Sequence ID No. 4; (2b) A protein having acetolactate synthase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 4; (2c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 4, and that has acetolactate synthase activity; The BudC protein is the protein described in (3a), (3b), or (3c) below: (3a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (3b) The amino acid sequence shown in Sequence ID No. 2 includes an amino acid sequence comprising substitutions, deletions, insertions, and / or additions of 1 to 10 amino acid residues, and exhibits (S,S)-butanediol dehydrogenase activity, meso-2,3-butanediol dehydrogenase activity, and / or This is a protein that possesses diacetyl reductase activity; (3c) Contains an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 2, and also has (S,S)-butanediol dehydrogenase activity, meso-2,3-butanediol Proteins having dihydrogenase activity and / or diacetyl reductase activity; The PAJ_3461 protein is the protein described in (4a), (4b), or (4c) below: (4a) Proteins containing the amino acid sequence shown in Sequence ID No. 8; (4b) A protein having gluconic acid-2-dehydrogenase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 8; (4c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 8, and that has gluconate-2-dehydrogenase activity; The PAJ_3462 protein is the protein described in (5a), (5b), or (5c) below: (5a) Proteins containing the amino acid sequence shown in Sequence ID No. 10; (5b) A protein having gluconic acid-2-dehydrogenase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 10; (5c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 10, and having gluconic acid-2-dehydrogenase activity; and / or The PAJ_3463 protein is the protein described in (6a), (6b), or (6c) below, in this method: (6a) Proteins containing the amino acid sequence shown in SEQ ID NO: 12; (6b) A protein having gluconic acid-2-dehydrogenase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 12; (6c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 12, and having gluconic acid-2-dehydrogenase activity.

8. The method according to any one of claims 1 to 3, wherein the bacteria are bacteria of the genus Pantoea or Escherichia.

9. The method according to any one of claims 1 to 3, wherein the bacterium is Pantoea ananatis or Escherichia coli.

10. The method according to any one of claims 1 to 3, wherein the glutamate-based L-amino acid is one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-arginine, L-citrulline, and L-ornithine.

11. The method according to claim 10, wherein the glutamic acid-based L-amino acid is L-glutamic acid.

12. The method according to claim 11, wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.