WO2012002486A1 - Method for producing l-amino acid - Google Patents

Method for producing l-amino acid Download PDF

Info

Publication number
WO2012002486A1
WO2012002486A1 PCT/JP2011/065027 JP2011065027W WO2012002486A1 WO 2012002486 A1 WO2012002486 A1 WO 2012002486A1 JP 2011065027 W JP2011065027 W JP 2011065027W WO 2012002486 A1 WO2012002486 A1 WO 2012002486A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
amino acid
activity
strain
protein
Prior art date
Application number
PCT/JP2011/065027
Other languages
French (fr)
Japanese (ja)
Inventor
佑介 萩原
裕梨 益満
Original Assignee
味の素株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 味の素株式会社 filed Critical 味の素株式会社
Publication of WO2012002486A1 publication Critical patent/WO2012002486A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)

Definitions

  • the present invention relates to a method for producing L-amino acids using bacteria, and more particularly, to a method for producing L-amino acids using ethanol as a raw material, and bacteria used in the method.
  • L-amino acids are industrially useful as additives for animal feeds, health food ingredients, amino acid infusions, and the like.
  • L-amino acids are industrially produced by fermentation using microorganisms belonging to the genera Brevibacterium, Corynebacterium, Escherichia and the like. In these production methods, strains isolated from nature, artificial mutants of the strains, and microorganisms modified so as to increase the activity of basic L-amino acid biosynthetic enzymes by recombinant DNA technology are used. It is used. (Patent Documents 1 to 9)
  • a saccharide is used as a carbon source as a main component, but ethanol can also be used as a carbon source in the same manner as a saccharide (Patent Document 10).
  • Ethanol taken up by cells is first converted to acetaldehyde by alcohol dehydrogenase.
  • the resulting acetaldehyde is thought to be converted to acetic acid by acetaldehyde dehydrogenase or acetyl-CoA by acetaldehyde CoA dehydrogenase.
  • AldB protein found in Escherichia coli is known to have NADP-dependent acetaldehyde dehydrogenase activity, and has similar properties to human mitochondrial aldehyde dehydrogenase, such as increased activity against various substrates in the presence of MgCl 2. (Non-Patent Document 1).
  • Non-patent Document 2 since the expression of AldB increases in the stationary culture phase in the presence of ethanol, it is considered to have a function of reducing stress caused by alcohol or aldehyde (Non-patent Document 2). However, it was not clear whether AldB converted acetaldehyde into acetic acid when actually assimilating ethanol. Furthermore, the relationship between AldB activity and L-amino acid production from ethanol was not known at all.
  • An object of the present invention is to provide a method for efficiently producing an L-amino acid using ethanol as a raw material and a bacterium belonging to the family Enterobacteriaceae, and a bacterium used in the method.
  • acetic acid is produced from acetaldehyde by the acetaldehyde dehydrogenase activity of AldB.
  • Acetic acid is then converted to acetyl-CoA by a reaction catalyzed by acetyl-CoA synthase (ACS) or phosphotransacetylase (PTA) and acetate kinase (ACK), both from ATP to AMP or ADP. With conversion.
  • AdhE another protein with acetaldehyde dehydrogenase activity
  • AdhE also has alcohol dehydrogenase activity and catalyzes the reaction of directly converting acetaldehyde to acetyl-CoA, so there is no loss of ATP and it is energetically I thought it was advantageous.
  • Xu, J. et al., J. Bacteriol., 177 (1995) 3166-3175 show that the presence of ethanol increases AldB expression in the stationary phase of culture. As a result, ethanol utilization may be reduced in aldb-deficient strains.
  • the present invention is as follows.
  • a method for producing an L-amino acid wherein a bacterium belonging to the family Enterobacteriaceae having L-amino acid producing ability is cultured in a medium containing ethanol, and the L-amino acid is collected from the medium, The method wherein the bacterium is modified so that the activity of the AldB protein is reduced.
  • the bacterium has a chromosomal aldB gene disrupted.
  • the AldB protein is any one of the following proteins (A) and (B).
  • the said method whose said aldB gene is DNA of the following (a) or (b).
  • the bacterium includes dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and The method, wherein the activity of one or more enzymes selected from the group consisting of succinyl diaminopimelate deacylase is enhanced and / or the activity of lysine decarboxylase is attenuated.
  • the bacterium used in the present invention is a bacterium belonging to the family Enterobacteriaceae having L-amino acid-producing ability and modified so that the activity of the AldB protein is reduced. .
  • the ability to produce L-amino acid refers to the ability to produce L-amino acid and accumulate it in the medium or in the cells when the bacterium used in the present invention (hereinafter also referred to as “the bacterium of the present invention”) is cultured in the medium. Say. Preferably, it refers to the ability to accumulate the target L-amino acid in the medium in an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more.
  • the bacterium having L-amino acid-producing ability may be inherently L-amino acid-producing bacterium, but a bacterium described later can be transformed into L-amino acid using a mutation method or recombinant DNA technology. It may be modified so as to have an amino acid-producing ability.
  • L-amino acid is not particularly limited, but basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L- Aliphatic amino acids such as leucine and glycine, amino acids that are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, and fragrances such as L-phenylalanine, L-tyrosine and L-tryptophan Amino acids, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, amino acids having an amide group in the side chain such as L-glutamine and L-asparagine Is mentioned.
  • the bacterium of the present invention may be capable of producing
  • L-amino acids include free L-amino acids and L-amino acid salts such as sulfates, hydrochlorides, and carbonates.
  • Bacteria belonging to the family Enterobacteriaceae used to obtain the bacterium of the present invention are not particularly limited, but Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Pectobacterium, Photorubadus, Providencia, Salmonella, Serratia, Includes bacteria belonging to genera such as Shigella, Morganella, and Yersinia.
  • the bacterium belonging to the genus Escherichia is not particularly limited, but means that the bacterium is classified into the genus Escherichia according to the classification known to experts in microbiology. For example, written by Knighthard et al. (Neidhardt, F. C. Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology / Second Edition pp. 2477-2483. Table 1. American Society for Microbiology Press, Washington The ones that are being used are included. Specific examples include Escherichia coli W3110 (ATCC 32525) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.
  • strains can be sold, for example, from the American Type Culture Collection (address P.O. Box 1549 Manassas, VA 20108, United States of America). That is, the registration number corresponding to each strain is given, and it can receive distribution using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. The same applies to other ATCC strains described below.
  • the bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to microbiologists. Certain types of Enterobacter agglomerans, recently based on 16S rRNA sequence analysis, etc., are based on Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii Or others (Int.tJ. Syst. Bacteriol., 43, 162-173 (1993)). In the present invention, the bacteria belonging to the genus Pantoea include bacteria that have been reclassified to the genus Pantoea in this way.
  • Pantoea ananatis Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans at the time of isolation, and deposited as Enterobacter agglomerans, but as described above, they were reclassified as Pantoea ananatis by 16S rRNA sequence analysis, etc. .
  • the Enterobacter bacterium is not particularly limited, but means that the bacterium is classified into the genus Enterobacter according to a classification known to microbiologists. Examples thereof include Enterobacter agglomerans and Enterobacter aerogenes. Specifically, strains exemplified in European Patent Application Publication No. EP952221 can be used. Representative strains of the genus Enterobacter include Enterobacter agglomerans ATCC 12287, Enterobacter aerogenes ATCC 13048, Enterobacter aerogenes NBRC 12010 (Biotechonol Bioeng.2007 Mar 27; 98 (2) 340-348), and Entero Examples include Bacter Aerogenes AJ110637 (FERM-10BP-10955).
  • Examples of the genus Erwinia include Erwinia amylovora, examples of the genus Klebsiella include Klebsiella planticola, and examples of the bacterium belonging to the genus Pectobacterium include: Atrocepticum (Pectobacterium atrosepticum) (former name Erwinia carotovora) and the like.
  • L-amino acid producing bacterium and imparting or enhancing L-amino acid producing ability
  • L-amino acid live bacteria belonging to the family Enterobacteriaceae and a method for imparting L-amino acid producing ability to bacteria, or bacteria A method for enhancing the L-amino acid-producing ability is described.
  • auxotrophic mutants In order to confer L-amino acid-producing ability, acquisition of auxotrophic mutants, L-amino acid analog resistant strains or metabolic control mutants, and recombinant strains with enhanced expression of L-amino acid biosynthetic enzymes
  • the method that has been conventionally used for breeding amino acid-producing bacteria such as Escherichia bacteria can be applied (Amino Acid Fermentation, Japan Society for Publishing Press, May 30, 1986, first edition published, 77th) See page 100).
  • the auxotrophy, analog resistance, metabolic control mutation and other properties imparted may be singly or may be two or more.
  • L-amino acid biosynthetic enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.
  • an auxotrophic mutant an analog resistant strain, or a metabolically controlled mutant having L-amino acid-producing ability
  • the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl.
  • -Treated with a treatment with a mutant such as -N'-nitro-N-nitrosoguanidine, among the obtained mutant strains shows auxotrophy, analog resistance, or metabolic control mutation, and has an ability to produce L-amino acid It can be obtained by selecting what it has.
  • the imparting or enhancing of the ability to produce L-amino acid can be performed by enhancing the enzyme activity by gene recombination.
  • the enzyme activity can be enhanced by, for example, a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced.
  • a method for enhancing the expression of a gene a recombinant plasmid in which a DNA fragment containing a target gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in a microorganism is introduced. Or making multiple copies of DNA fragments containing the gene of interest by joining, transferring, etc. on the chromosome, and introducing mutations into the promoter region of the gene of interest (International Publication Pamphlet WO95 / 34672). reference).
  • the promoter for expressing these genes may be any promoter that functions in the family Enterobacteriaceae, and is the promoter of the gene itself used. It may be modified or modified.
  • the expression level of the gene can also be controlled by appropriately selecting a promoter that functions strongly in the family Enterobacteriaceae, or by bringing the ⁇ 35 and ⁇ 10 regions of the promoter closer to the consensus sequence.
  • the method for enhancing the expression of the enzyme gene as described above is described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like.
  • L-lysine-producing bacteria such as Escherichia coli L-lysine-producing bacteria or parent strains for inducing the same include mutants having resistance to L-lysine analogs.
  • L-lysine analogs inhibit the growth of Escherichia coli, but this inhibition is completely or partially desensitized when L-lysine is present in the medium.
  • L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), ⁇ -methyllysine, ⁇ -chlorocaprolactam, and the like. .
  • Mutants having resistance to these lysine analogs can be obtained by subjecting Escherichia coli to normal artificial mutation treatment.
  • Specific examples of bacterial strains useful for the production of L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and E. coli VL611. In these strains, feedback inhibition of aspartokinase by L-lysine is released.
  • WC196 strain can be used as an L-lysine producing bacterium of E.coli.
  • This strain was obtained from the W3110 strain derived from E. coli K-12 and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine.
  • the wild type lysC gene on the chromosome of the W3110 strain was replaced with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance (US Pat. No. 5,827,698).
  • the stock was named Escherichia coli AJ13069.
  • L-lysine-producing bacteria or parent strains for inducing them include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced.
  • L-lysine biosynthetic enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160).
  • ppc Phosphoenolpyruvate carboxylase
  • aspartate aminotransferase aspartate aminotransferase
  • aspartate semialdehyde dehydrogenase aspartate semialdehyde dehydrogenase
  • dapF diaminopimelate epimerase
  • dapD tetrahydrodipicolinate succinylase
  • dapE succinyl diaminopimelate deacylase
  • aspartase aspA
  • the parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Patent No. 5,830,716), ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.
  • the parentheses are abbreviations for these genes.
  • Wild-type dihydrodipicolinate synthase derived from Escherichia coli is known to undergo feedback inhibition by L-lysine, and wild-type aspartokinase derived from Escherichia coli is subject to inhibition and feedback inhibition by L-lysine. It has been known. Therefore, when using the dapA gene and the lysC gene, these genes must be mutant genes that encode mutant enzymes that are not subject to feedback inhibition by L-lysine, or mutant genes that are not subject to suppression by L-lysine. Is preferred.
  • DNA encoding a mutant dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine examples include DNA encoding a protein having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue.
  • the threonine residue at position 352 is replaced with an isoleucine residue
  • the glycine residue at position 323 is replaced with an asparagine residue
  • 318 Examples include DNA encoding AKIII having a sequence in which the methionine at the position is replaced with isoleucine (see US Pat. Nos. 5,610,010 and 6,040,160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.
  • plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant mutant dihydrodipicolinate synthase and mutant lysC encoding mutant aspartokinase (USA) Patent No. 6040160).
  • Escherichia coli strain JM109 transformed with RSFD80 US Pat. No.
  • L-lysine-producing bacteria or parent strains for deriving the same include reduction or loss of the activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthetic pathway to produce compounds other than L-lysine. There are also stocks. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175).
  • strains in which the cadA gene and the ldcC gene are disrupted include Escherichia coli WC196LC (WC196 ⁇ cadA ⁇ ldcC) (US 5,827,698, US20060160191).
  • the WC196LC strain was named AJ110692 and was internationally established on October 7, 2008 by the National Institute of Advanced Industrial Science and Technology (AIST), the Patent Biological Deposit Center (1-6 Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, 305-8566). Deposited and given accession number FERM BP-11027.
  • L-threonine-producing bacteria examples include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371) E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli. coli FERM BP-3519 and FERM BP-3520 (US Pat.No.
  • E. coli MG442 Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)
  • E. coli VL643 and VL2055 EP 1149911 A
  • the TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine.
  • the B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine.
  • E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it.
  • the B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter.
  • VKPM B-5318 was assigned on May 3, 1990 to Lucian National Collection of Industrial Microorganisms (VKPM) (GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation). Deposited at B-5318.
  • the bacterium used in the present invention is further modified so that expression of one or more of the following genes is increased.
  • the thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990).
  • the thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12.
  • the thrB gene encoding E. ⁇ ⁇ ⁇ coli homoserine kinase has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 99049175990).
  • the thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12.
  • the thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990).
  • the thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon.
  • the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).
  • mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.
  • the rhtA gene is present on the 18th minute of the E. ⁇ coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system.
  • the rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene.
  • the unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant to homoserine and threonine).
  • the rhtA23 mutation conferring resistance to high concentrations of threonine or homoserine has been found to be a G ⁇ A substitution at position -1 relative to the ATG initiation codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).
  • the E. coli asd gene has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene. (See White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189). The asd gene of other microorganisms can be obtained similarly.
  • the aspC gene of E.coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895) and can be obtained by PCR.
  • the aspC gene of other microorganisms can be obtained similarly.
  • L-cysteine-producing bacteria examples include E. coli JM15 (US) each transformed with a plurality of cysE alleles encoding a feedback inhibition resistant serine acetyltransferase. (Patent No. 6,218,168, Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfide Examples include, but are not limited to, strains such as E. coli strains (JP11155571A2) that have reduced enzyme activity and E. coli W3110 (WO0127307A1) that have increased activity of the transcriptional regulator of the positive cysteine regulon encoded by the cysB gene. Not.
  • L-leucine-producing bacteria examples include leucine-resistant E. coli strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or ⁇ E. coli strains resistant to leucine analogs such as -2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP-B-62-34397 and JP-A-8-70879), Examples include, but are not limited to, E. coli strains obtained by the genetic engineering method described in WO96 / 06926, E. coli H-9068 (JP-A-8-70879), and the like.
  • the bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis.
  • a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase which is preferably desensitized to feedback inhibition by L-leucine can be mentioned.
  • the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).
  • L-histidine-producing bacteria examples include E. coli 24 strain (VKPM B-5945, RU2003677), E. coli 80 strain (VKPM B-7270, RU2119536) E. coli NRRL B-12116-B12121 (U.S. Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.No. 6,344,347), E. coli. Examples include, but are not limited to, E. coli H-9341 (FERM BP-6674) (EP1085087) and E. coli AI80 / pFM201 (US Pat. No. 6,258,554).
  • L-histidine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased.
  • genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.
  • L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).
  • strains capable of producing L-histidine include E. coli FERM P-5038 and FERM P-5048 introduced with a vector carrying a DNA encoding an L-histidine biosynthetic enzyme (JP-A-56-56). No. 005099), E.licoli strain (EP1016710A) introduced with a gene for amino acid transport, E. coli 80 strain (VKPM) with resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin B-7270, (Russian Patent No. 2119536) and the like.
  • L-glutamic acid-producing bacteria examples include, but are not limited to, E. coli VL334thrC + (EP 1172433).
  • E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765).
  • the wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7).
  • VKPM B-8961 L-isoleucine-requiring L-glutamic acid-producing bacterium VL334thrC +
  • L-glutamic acid-producing bacteria or parent strains for deriving the same include, but are not limited to, strains in which expression of one or more genes encoding L-glutamic acid biosynthetic enzymes are increased.
  • genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Phosphoenol pyruvate carbosylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenol pyruvate synthase (ppsA), enolase (eno), phosphoglyceromutas
  • strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A and EP952221A.
  • L-glutamic acid-producing bacteria or parent strains for deriving the same are those in which the activity of an enzyme that catalyzes the synthesis of compounds other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or absent Stocks are also mentioned.
  • Such enzymes include isocitrate triase (aceA), ⁇ -ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), ⁇ -glutamyltransferase (ggt), ⁇ -glutamylcysteine synthetase (gshA), ⁇ -glutamic acid And putrescine synthase (ycjK).
  • aceA isocitrate triase
  • succA ⁇ -ketoglutarate dehydrogenase
  • pta phosphotransacetylase
  • ack acetate kinase
  • ack ace
  • Escherichia coli lacking ⁇ -ketoglutarate dehydrogenase activity or having reduced ⁇ -ketoglutarate dehydrogenase activity and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945.
  • E. coli W3110sucA Kmr E. coli AJ12624 (FERM BP-3853) E. coli AJ12628 (FERM BP-3854) E. coli AJ12949 (FERM BP-4881)
  • E. coli W3110sucA is a strain obtained by disrupting the ⁇ -ketoglutarate dehydrogenase gene (hereinafter also referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in ⁇ -ketoglutarate dehydrogenase.
  • L-glutamic acid-producing bacteria include Escherichia coli that has resistance to an aspartic acid antimetabolite. Such a strain may be deficient in ⁇ -ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5.908,768) and further reduced L-glutamate resolution. Examples include strains such as FFRM P-12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714).
  • Pantoea ananatis AJ13355 strain An example of an L-glutamic acid-producing bacterium of Pantoae ananatis is Pantoea ananatis AJ13355 strain. This strain was isolated from the soil of Iwata City, Shizuoka Prefecture as a strain that can grow on a medium containing L-glutamic acid and a carbon source at a low pH. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center (address: 1st, 1st, 1st, 1-chome, Tsukuba, Ibaraki, Japan, 305-8566).
  • examples of L-glutamic acid-producing bacteria of Pantoae ananatis include bacteria belonging to the genus Pantoea in which ⁇ -ketoglutarate dehydrogenase ( ⁇ KGDH) activity is deficient or ⁇ KGDH activity is reduced.
  • Such strains include AJ13356 (US Pat. No. 6,331,419) in which the ⁇ KGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected from AJ13355 strain as a low mucus production mutant.
  • SC17sucA US Pat. No. 6,596,517) which is a gene-deficient strain.
  • AJ13356 was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616.
  • AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification.
  • the SC17sucA strain has been assigned the private number AJ417, and was deposited at the National Institute of Advanced Industrial Science and Technology as the accession number FERM BP-08646 on February 26, 2004.
  • SC17sucA / RSFCPG + pSTVCB strain As L-glutamic acid-producing bacteria of Pantoea ananatis, SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain can be mentioned.
  • the SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppsA), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum.
  • the AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH.
  • the NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain.
  • AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (305-1856, Ibaraki, Japan, 1st-chome, 1st-chome, 1st-chome, 1st-centre, 6th). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.
  • L-phenylalanine producing bacteria examples include E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197), E carrying a mutant pheA34 gene coli HW1089 (ATCC 55371) (US Pat.No. 5,354,672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patents) No. 4,407,952) and the like, but is not limited thereto. In addition, E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197), E carrying a mutant pheA34 gene coli HW1089 (ATCC 55371) (US Pat.No. 5,354,672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141,
  • Escherichia coli L-phenylalanine-producing bacteria having increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).
  • L-tryptophan-producing bacteria examples include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / which lack the function of tryptophanyl-tRNA synthetase encoded by the trpS gene.
  • pMU91 DSM10123 (U.S. Pat.No. 5,756,345), E having a serA allele encoding phosphoglycerate dehydrogenase that is not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase that is not subject to feedback inhibition by tryptophan.
  • E. coli SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614)
  • E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased phosphoenolpyruvate production capacity Strains including but not limited to.
  • Escherichia coli L-tryptophan-producing bacteria having increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).
  • L-tryptophan-producing bacteria or parent strains for inducing the same examples include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and a kind of activity of an enzyme selected from tryptophan synthase (trpAB) Also included are strains with increased above. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E.
  • coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase with desensitized feedback inhibition
  • Examples include a transformant obtained by introducing the plasmid pGH5 (WO 94/08031) into E.coli SV164.
  • L-tryptophan-producing bacteria or parent strains for deriving the same examples include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open Nos. 57-71397 and 1994 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of ⁇ and ⁇ subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO2005 / 103275).
  • L-proline-producing bacteria examples include E. coli 702ilvA (VKPM B-8012) (EP 1172433) that lacks the ilvA gene and can produce L-proline However, it is not limited to these.
  • the bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis.
  • An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline.
  • the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) gene (EP1239041 gene A2).
  • Escherichia coli having L-proline-producing ability examples include NRRL B-12403 and NRRL B-12404 ⁇ (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), and German Patent No. 3127361 Or the E. coli strain having the plasmid variant described in Bloom FR et al (The 15th Miami winter symposium, 1983, p.34).
  • L-arginine producing bacteria examples include E. coli strain 237 (VKPM B-7925) (US Patent Application Publication 2002/058315 A1) and mutant N- Derivatives carrying acetylglutamate synthase ( Russian patent application No. 2001112869), E. coli 382 strain (VKPM B-7926) (EP1170358A1), arginine producing strain introduced with argA gene encoding N-acetylglutamate synthetase Examples include, but are not limited to, (EP1170361A1).
  • L-arginine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased.
  • genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoylphosphate synthetase gene (carAB).
  • argC N-acetylglutamylphosphate reductase gene
  • argJ ornithine acetyltransferase gene
  • argB N-
  • L-valine producing bacteria examples include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. It is preferable to remove the region of the ilvGMEDA operon necessary for attenuation so that the expression of the operon is not attenuated by the produced L-valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.
  • L-valine-producing bacteria or parent strains for deriving them also include mutants having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766).
  • E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used.
  • E. coli VL1970 was assigned to GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation on June 24, 1988, on the Lucian National Collection of Industrial Microorganisms (VKPM). Deposited at B-4411.
  • a mutant strain (WO96 / 06926) that requires lipoic acid for growth and / or lacks H + -ATPase can be used as a parent strain.
  • L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing them include mutants resistant to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as the above, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882), but are not limited thereto.
  • a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).
  • L-Aspartic acid producing bacterium L-Asparagine is produced by adding an amino group to aspartic acid (Boehlein, SK, Richards, NGJ, & Schuster, SM (1994a) J. Biol. Chem. 269, 7450- 7457.)
  • Escherichia coli L-asparagine-producing bacteria include Escherichia coli strains in which the asparagine synthetase of L-aspartic acid-producing bacteria is enhanced.
  • the bacterium of the present invention may be a strain modified to increase the activity of pyruvate synthase or pyruvate: NADP + oxidoreductase.
  • Pyruvate synthase or pyruvate can be modified as NADP + oxide activity reductase is increased, the pyruvate synthase or pyruvate: NADP + oxidoreductase activity, the parent strain, for example, a wild strain or unmodified It is preferable to modify so as to increase compared to the strain.
  • the microorganism modified to have these enzyme activities is pyruvate synthase or pyruvate: NADP + oxidoreductase activity is increased compared to unmodified strains.
  • the “pyruvate synthase” in the present invention is an enzyme (EC 1.2) that catalyzes the following reaction for producing pyruvate from acetyl-CoA and CO 2 in the presence of an electron donor, for example, in the presence of ferredoxin or flavodoxin. .7.1).
  • Pyruvate synthase is sometimes abbreviated as PS and is sometimes named pyruvate oxidoreductase, pyruvate ferredoxin oxidoreductase, pyruvate flavodoxin oxidoreductase, or pyruvate oxidoreductase.
  • As the electron donor ferredoxin or flavodoxin can be used.
  • Confirmation that the activity of pyruvate synthase is enhanced is achieved by preparing a crude enzyme solution from the microorganism before enhancement and the microorganism after enhancement and comparing the activity of pyruvate synthase.
  • the activity of pyruvate synthase can be measured, for example, according to the method of Yoon et al. (Yoon, K. S. et al. 1997. Arch. Microbiol. 167: 275-279).
  • the amount of reduced methyl viologen that increases due to decarboxylation of pyruvic acid is measured spectroscopically. It can be measured by measuring.
  • One unit (U) of enzyme activity is expressed as a reduction amount of 1 ⁇ mol of methyl viologen per minute.
  • the enzyme activity is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more that of the parent strain.
  • pyruvate synthase is produced by introducing the pyruvate synthase gene, but the enzyme activity is enhanced to such an extent that it can be measured. Is preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more. Pyruvate synthase is sensitive to oxygen and is generally difficult to express and measure (Buckel, W.and Golding, B. T. 2006. Ann. Rev. of Microbiol. 60: 27-49). Therefore, when measuring enzyme activity, it is preferable to carry out the enzyme reaction by reducing the oxygen concentration in the reaction vessel.
  • pyruvate synthase gene As a gene encoding pyruvate synthase, it is possible to use a pyruvate synthase gene of a bacterium having a reductive TCA cycle, such as Chlorobium tepidum, Hydrogenobacter thermophilus, etc. is there. It is also possible to use a pyruvate synthase gene derived from bacteria belonging to the group of enterobacteria such as Escherichia coli.
  • genes encoding pyruvate synthase are autotrophic methane producers such as Methanococcus maripaludis, Methanococcus janasti, Methanothermobacter thermautotrophicus, and other methanothermobacter thermautotrophicus (Autotrophic (methanogens) pyruvate synthase gene can be used.
  • a gene having a base sequence located at base numbers 1534432 to 1537989 of the genome sequence of chlorobium tepidum (GenBank Accession No. NC_002932) Can do.
  • the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. AAC76906.
  • Hydrogenobacter thermophilus pyruvate synthase is composed of ⁇ subunit (GenBank Accession No. BAA95604), ⁇ subunit (GenBank Accession No. BAA95605), ⁇ subunit (GenBank Accession No. BAA95606), ⁇ subunit.
  • pyruvate synthase gene consisting of four genes, SSO1208, SSO7412, SSO1207, and SSO1206, represented by nucleotide numbers 1047593 to 1044711 in the genome sequence of Sulfolobus solfataricus (GenBank Accession No. NC 002754) it can. Furthermore, the pyruvate synthase gene is based on the homology with the genes exemplified above, based on the genus Chlorobium, the genus Desulfobacter, the genus Aquifex, the genus Hydrogenobacter, It may be cloned from bacteria of the genus Thermoproteus, Pyrobaculum, or the like.
  • the ydbK gene (b1378) having a nucleotide sequence located at nucleotide numbers 1435284 to 1438808 in the genome sequence of the K-12 strain (GenBank Accession No. U00096) is It is expected to encode a synoxide reductase, pyruvate synthase.
  • the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. AAC76906.
  • the pyruvate synthase gene is highly homologous to the Escherichia coli pyruvate synthase gene (ydbK), and the genera Escherichia, Salmonella, Serratia, Enterobacter, Shigella It may be a pyruvate synthase gene belonging to the group of enterobacteria such as (Shigella) and Citrobacter.
  • the pyruvate synthase from Methanococcus maripaludis is the genome sequence of Methanococcus maripardis (GenBank Accession No. NC_005791) (Hendrickson, E. L. et al. 2004. J. Bacteriol. 186: 6956-6969) Is encoded by the porCDABEF operon located at base numbers 1462535 to 1466397 (Lin, W. C. et al. 2003. Arch. Microbiol. 179: 444-456). This pyruvate synthase contains four subunits, ⁇ , ⁇ , ⁇ , and ⁇ .
  • PorE and PorF are also known to be important for the activity of pyruvate synthase.
  • the ⁇ subunit is encoded by the porA gene of base numbers 1465867 to 1466397 (complementary chain) of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988626.
  • the ⁇ subunit is encoded by the base number 1465595 to 1465852 (complementary chain) porB gene of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988627.
  • the ⁇ subunit is encoded by the porC gene of nucleotide numbers 1464410 to 1455773 (complementary chain) of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988625.
  • the ⁇ subunit is encoded by the porD gene of nucleotide numbers 1463497 to 14439393 (complementary strand) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_988624.
  • PorE is encoded by the porE gene of base numbers 1462970 to 1463473 (complementary chain) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_988623.
  • PorF is encoded by the porF gene of base numbers 1462535 to 1462951 (complementary chain) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_9886
  • pyruvate: NADP + oxidoreductase means reversibly catalyzing the following reaction for producing pyruvic acid from acetyl-CoA and CO 2 in the presence of an electron donor, for example, in the presence of NADPH or NADH. Means enzyme (EC 1.2.1.15).
  • Pyruvate: NADP + oxidoreductase is sometimes abbreviated as PNO and sometimes as pyruvate dehydrogenase.
  • pyruvate dehydrogenase activity is an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA, as described later.
  • Acid dehydrogenase is a separate enzyme from pyruvate: NADP + oxidoreductase.
  • the amount of reduced methyl viologen that increases due to the decarboxylation of pyruvate is measured spectroscopically. It can be measured by measuring.
  • One unit (U) of enzyme activity is expressed as a reduction amount of 1 ⁇ mol of methyl viologen per minute.
  • the enzyme activity is preferably increased 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to the parent strain. Is desirable.
  • pyruvate: NADP + oxidoreductase activity it is sufficient that pyruvate: NADP + oxidoreductase is generated by introducing the pyruvate synthase gene, but the enzyme activity is measured. It is preferably strengthened to the extent possible, preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more.
  • Pyruvate: NADP + oxidoreductase is sensitive to oxygen and is generally difficult to express and measure activity (Inui, H. et al. 1987. J. Biol. Chem. 262: 9130). -9135; Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710-720). Therefore, when measuring enzyme activity, it is preferable to carry out the enzyme reaction by reducing the oxygen concentration in the reaction vessel.
  • NADP + oxidoreductase is a photosynthetic eukaryotic microorganism and is also classified as a protozoan.
  • NADP + oxidoreductase gene of Euglena gracilis there is a gene having a base sequence shown in GenBank Accession No. AB021127. The amino acid sequence encoded by this gene is disclosed in GenBank Accession No. BAB12024.
  • the microorganism of the present invention is modified by increasing the activity of recycling the oxidized form of the electron donor necessary for the activity of pyruvate synthase to the reduced form as compared with the parent strain, for example, a wild strain or an unmodified strain,
  • the microorganism may be modified so that the activity of pyruvate synthase is increased.
  • Examples of the activity of recycling the oxidized form of the electron donor to the reduced form include ferredoxin-NADP + reductase activity.
  • the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the activity to increase pyruvate synthase activity.
  • the parent strain may have a gene encoding a protein inherently responsible for the electron donor recycling activity, or originally has no electron donor recycling activity,
  • the activity may be imparted by introducing a gene encoding a protein responsible for the activity, and the L-amino acid producing ability may be improved.
  • “Ferredoxin-NADP + reductase” refers to an enzyme (EC 1.18.1.2) that reversibly catalyzes the following reaction.
  • This reaction is a reversible reaction, and reduced ferredoxin can be produced in the presence of NADPH and oxidized ferredoxin.
  • Ferredoxin can be substituted for flavodoxin, and what is named flavodoxin-NADP + reductase also has an equivalent function.
  • Ferredoxin-NADP + reductase has been confirmed to exist widely from microorganisms to higher organisms (Carrillo, N. and Ceccarelli, EA 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, EA et al. 2004. Biochim Biophys. Acta. 1698: 155-165), some have been named ferredoxin-NADP + oxidoreductase, NADPH-ferredoxin oxidoreductase.
  • Confirmation that the activity of ferredoxin-NADP + reductase was enhanced is achieved by preparing a crude enzyme solution from the microorganism before modification and the microorganism after modification, and comparing the activity of ferredoxin-NADP + reductase.
  • the activity of ferredoxin-NADP + reductase can be measured, for example, according to the method of Blaschkowski et al. (Blaschkowski, H. P. et al. 1982. Eur. J. Biochem. 123: 563-569). For example, it can be measured by spectroscopically measuring the decreasing amount of NADPH using ferredoxin as a substrate.
  • One unit (U) of enzyme activity is expressed as an oxidation amount of 1 ⁇ mol NADPH per minute.
  • the parent strain has ferredoxin-NADP + reductase activity, it is not necessary to enhance the enzyme activity if the activity of the parent strain is sufficiently high, but it is preferably 1.5 times or more, more preferably 2 times that of the parent strain. As described above, it is more preferable that the enzyme activity is increased by 3 times or more.
  • a gene encoding ferredoxin-NADP + reductase has been found in many biological species, and any gene encoding an enzyme having activity in the target L-amino acid producing strain can be used.
  • the fpr gene has been identified as flavodoxin-NADP + reductase (Bianchi, V. et al. 1993. J. Bacteriol. 175: 1590-1595). It is also known that Pseedomonas putida has NADPH-Putidaredoxin reductase gene and Putidaredoxin gene as operons (Koga, H. et al. 1989). J. Biochem. (Tokyo) 106: 831-836).
  • Escherichia coli flavodoxin-NADP + reductase examples include the fpr gene having a nucleotide sequence located at nucleotide numbers 4111749 to 4112495 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096) can do.
  • the amino acid sequence of Fpr is disclosed in GenBank Accession No. AAC76906.
  • a ferredoxin-NADP + reductase gene has been found at the base numbers 25526234 to 2527211 of the genome sequence of Corynebacterium glutamicum (GenBank Accession No. BA00036) (GenBank Accession No. BAB99777).
  • the activity of pyruvate synthase requires that ferredoxin or flavodoxin be present as an electron donor. Therefore, the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the ferredoxin or flavodoxin so as to improve the production ability. Further, in addition to modification so that pyruvate synthase activity, or flavodoxin-NADP + reductase and pyruvate synthase activities are enhanced, modification may be made so that ferredoxin or flavodoxin production ability is improved.
  • the “ferredoxin” in the present invention is a protein that contains a non-heme iron atom (Fe) and a sulfur atom and binds an iron-sulfur cluster called a 4Fe-4S, 3Fe-4S, or 2Fe-2S cluster.
  • “Flavodoxin” refers to a protein that functions as a one- or two-electron carrier containing FMN (Flavin-mononucleotide) as a prosthetic genus.
  • FMN Fevin-mononucleotide
  • the parent strain used for the modification may have a gene that inherently encodes ferredoxin or flavodoxin, or originally has no ferredoxin or flavodoxin gene, but introduces a ferredoxin or flavodoxin gene. By doing so, the ability to produce these proteins may be imparted, and the ability to produce L-amino acids may be improved.
  • Confirmation that ferredoxin or flavodoxin production ability is improved compared to the parent strain, for example, wild strain or non-modified strain, can be confirmed by comparing the amount of ferredoxin or flavodoxin mRNA with the wild-type or non-modified strain.
  • Examples of the expression level confirmation method include Northern hybridization and RT-PCR (Sambrook, J. et al. 1989. Molecular CloningA Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York).
  • the expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.
  • ferredoxin or flavodoxin production is improved compared to the parent strain, for example, wild strain or unmodified strain, should be detected by SDS-PAGE, two-dimensional electrophoresis, or Western blot using an antibody.
  • the production amount may be any as long as it is improved as compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.
  • the activity of ferredoxin and flavodoxin can be measured by adding to an appropriate redox reaction system.
  • Boyer et al. Discloses a method of reducing the produced ferredoxin with ferredoxin-NADP + reductase and quantifying the reduction of cytochrome C by the resulting reduced ferredoxin (Boyer, ME et al. 2006. Biotechnol. Bioeng. 94: 128-138).
  • the activity of flavodoxin can be measured by the same method using flavodoxin-NADP + reductase.
  • the gene encoding ferredoxin or flavodoxin is widely distributed, and any encoded ferredoxin or flavodoxin can be used as long as pyruvate synthase and an electron donor regeneration system are available.
  • the fdx gene exists as a gene encoding ferredoxin having a 2Fe-2S cluster (Ta, D. T. and Vickery, L. E. 1992. J. Biol. Chem. 267: 11120 -11125), the yfhL gene is predicted as a ferredoxin gene having a 4Fe-4S cluster.
  • the flavodoxin gene includes fldA gene (Osborne, C. et al. 1991. J. Bacteriol.
  • ferredoxin I and ferredoxin II have been identified as 4Fe-4S type ferredoxin genes that serve as electron acceptors for pyruvate synthase (Yoon, K. S Et al. 2001. J. Biol. Chem. 276: 44027-44036).
  • Ferredoxin genes or flavodoxin genes derived from bacteria having a reductive TCA cycle such as Hydrogenobacter thermophilus can also be used.
  • the ferredoxin gene of Escherichia coli As the ferredoxin gene of Escherichia coli, the fdx gene located at base numbers 2654770-2655105 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096), and base numbers 2676885-2697945 The located yfhL gene can be exemplified. The amino acid sequences of Fdx and YfhL are disclosed in GenBank Accession No. AAC75578 and AAC75615, respectively.
  • Escherichia coli flavodoxin gene examples include the fldA gene located at nucleotide numbers 710688 to 710158 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096), and nucleotide numbers 3037877 to 3038398
  • the fldB gene located in can be exemplified.
  • the amino acid sequences encoded by the fldA gene and fldB gene are disclosed in GenBank Accession No. AAC73778 and AAC75933, respectively.
  • the ferredoxin gene of Chlorobium tepidum (Chelorobium tepidum) is located in the ferredoxin I gene located at nucleotide numbers 1184078 to 1184266 and nucleotide numbers 1184476 to 1184664 in the genome sequence of Chlorobium tepidum (GenBank Accession No. NC_002932)
  • a ferredoxin II gene can be exemplified.
  • the amino acid sequences of ferredoxin I and ferredoxin II are disclosed in GenBank Accession No. AAM72491 and AAM72490, respectively.
  • the ferredoxin gene (GenBank Accession No.
  • the genera Chlorobium, Desulfobacter, Aquifex, Hydrogenobacter, Thermoproteus, Thermoproteus May be cloned from bacteria belonging to the genus Pyrobaculum, and also ⁇ -proteobacteria such as Enterobacter, Klebsiella, Serratia, Erbinia, Yersinia, Corynebacterium glutamicum, etc.
  • coryneform bacteria such as Brevibacterium lactofermentum, Pseudomonas bacteria such as Pseudomonas aeruginosa, and Mycobacterium bacteria such as Mycobacterium tuberculosis.
  • the microorganism of the present invention may have reduced activity of malic enzyme.
  • the microorganism of the present invention is a bacterium belonging to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia, the activity of malic enzyme may be particularly reduced.
  • the activity of malic enzyme means the activity of reversibly catalyzing the following reaction that oxidatively decarboxylates malic acid to produce pyruvic acid.
  • These reactions are NADP-type malic enzyme using NADP as an electron acceptor (also expressed as malate dehydrogenase (oxaloacetate-decarboxylating) (NADP + )) (EC: 1.1.1.40 b2463 gene (also referred to as maeB gene)) 2 of NAD type malic enzyme (also referred to as malate dehydrogenase (oxaloacetate-decarboxylating) (NAD + )) (EC: 1.1.1.38 sfcA gene (also referred to as maeA gene)) using NAD as an electron acceptor Catalyzed by seed enzymes.
  • the confirmation of malic enzyme activity can be measured according to the method of Bologna et al. (Bologna, F. P. et al. 2007. J. Bacterio
  • NADP-dependent malic enzyme NADP + + malate ⁇ NADPH + CO 2 + pyruvate
  • NAD-dependent malic enzyme NAD + + malate ⁇ NADH + CO 2 + pyruvate
  • the microorganism of the present invention belongs to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia.
  • the bacterium belongs, it is preferable to reduce the activity of both types of malic enzyme.
  • the microorganism of the present invention preferably has reduced pyruvate dehydrogenase activity.
  • pyruvate dehydrogenase activity means an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA (acetyl-CoA). To do.
  • This reaction is PDH (E1p: pyruvate dehydrogenase, EC: 1.2.4.1, encoded by aceE gene), dihydrolipoyltransacetylase (E2p: dihydrolipoyltransacetylase, EC: 2.3.1.12, encoded by aceF gene), It is catalyzed by three enzymes: dihydrolipoamide dehydrogenase (E3: dihydrolipoamide dehydrogenase; EC: 1.8.1.4, encoded by the lpdA gene). That is, these three types of subunits each catalyze the following reaction, and the activity of catalyzing the combined reaction of these three reactions is called PDH activity. Confirmation of PDH activity can be measured according to the method of Visser and Strating (Visser, J. and Strating, M. 1982. Methods Enzymol. 89: 391-399).
  • E1p pyruvate + [dihydrolipoyllysine-residue succinyltransferase] lipoyllysine ⁇ [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO 2
  • E2p CoA + enzyme N6- (S-acetyldihydrolipoyl) lysine ⁇ acetyl-CoA + enzyme N6- (dihydrolipoyl) lysine
  • E3 protein N6- (dihydrolipoyl) lysine + NAD + ⁇ protein N6- (lipoyl) lysine + NADH + H +
  • the bacterium of the present invention is modified so that malate synthase, isocitrate lyase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) is constitutively expressed, or expression of the operon is enhanced. Strains may be used.
  • the constitutive expression of malate synthase, isocitrate triase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) means that the ace operon promoter is not repressed by the repressor protein iclR. It means that it has been released.
  • the constitutive expression of the ace operon and the enhanced expression of the operon are the proteins encoded by the ace operon, malate synthase (aceB), isocitrate triase (aceA), and isocyto This can be confirmed by the fact that the enzyme activity of rate dehydrogenase kinase / phosphatase (aceK) is increased compared to the unmodified strain or the wild strain.
  • Enzyme activity was determined by measuring glyoxylic acid-dependent degradation of the thioester bond of acetyl-CoA by reducing A232 for malate synthase (Dixon, GH, Kornberg, HL, 1960, Biochem. J, 1; 41: p217-233), for isocitrate lyase, a method for measuring glyoxylic acid generated from isocitrate as a 2,4-dinitrophenylhydrazone derivative (Roche, TE. Williams JO, 1970, Biochim. Biophys.
  • the binding site of the repressor (iclR) on the ace operon may be modified so that iclR cannot bind.
  • the suppression can be released by replacing the promoter of the operon with a strong promoter (such as the lac promoter) that is not subject to expression suppression by iclR.
  • the ace operon expression can be made constitutive by modifying the bacterium so that the expression of the iclR gene is reduced or deleted. Specifically, the expression control sequence of the gene encoding iclR is modified so that the gene does not express, or the coding region is modified so that the function of the repressor is lost, thereby suppressing the expression of the ace operon. Can be released.
  • the bacterium of the present invention can be obtained by modifying the enterobacteria having L-amino acid-producing ability as described above so that the activity of AldB protein is reduced. .
  • the bacterium of the present invention can also be obtained by imparting L-amino acid-producing ability to a bacterium modified so that the activity of the AldB protein is reduced.
  • “AldB protein” refers to a protein having a conservative mutation, such as a protein encoded by the aldb gene of Escherichia coli K-12 strain, and homologs and artificially modified forms thereof. A protein having such a conservative mutation is described as a conservative variant. Conservative variants will be described later.
  • the “activity of AldB protein” means an acetaldehyde dehydrogenase activity of AldB protein, that is, an activity that catalyzes a reaction of converting acetaldehyde into acetic acid, and particularly an NADP-dependent or NAD-dependent acetaldehyde dehydrogenase activity.
  • the protein having such activity is included in the AldB protein regardless of its name.
  • a protein encoded by the aldA gene of Pantoea ananatis has NAD-dependent acetaldehyde dehydrogenase activity and is included in the “AldB protein”.
  • AldB protein is also called Co-A independent aldehyde dehydrogenase (Co-A independent aldehyde dehydrogenase). Confirmation that the activity of the AldB protein has decreased can be confirmed by the method of K Ho et al. (Journal of Bacteriology Feb 2005. Vol 187 No. 3 p1067-1073).
  • AldB protein activity is reduced means that the activity of AldB protein is relatively reduced compared to the non-modified strain such as the parent strain or wild strain, and the activity of AldB protein is completely lost. If both are included.
  • the expression of the aldB gene encoding the AldB protein may be reduced.
  • aldB gene examples include the aldB gene of the Escherichia coli K-12 strain (GenBank Accession NC_000913.2 GI: 49175990 complement (3752996..3754534)).
  • the base sequence of this gene is shown in SEQ ID NO: 1.
  • SEQ ID NO: 2 shows the amino acid sequence of the AldB protein encoded by the same gene.
  • Pantoea Ananatis LMG 20103 strain (GenBank Accession NC_013956.1 GI: 291617493 complement (2166733..2168205)), Pectobacterium atrosepticum (formerly Erwinia carotovora) SCRI1043
  • the amino acid sequence of the AldB protein encoded by the aldB gene of the strain (GenBank Accession NC_004547.2 GI: 50119055 111626..113161)
  • Salmonella enterica CT18 strain NC_003198.1 GI: 16762629 3978586..3980124
  • the gene encoding the AldB protein homolog is called the aldA gene. This gene is described as the aldB gene, and the protein encoded by the gene is described as the AldB protein.
  • the alignment of these AldB proteins is shown in FIGS.
  • the AldB of Pantoea ananatis, Pectobacterium atrocepticam, and Salmonella enterica have 64.7%, 81.4%, and 95.8% homology with those of Escherichia coli, respectively.
  • AldB protein has acetaldehyde dehydrogenase activity, it may be a protein having a conservative mutation such as a homologue or artificially modified product thereof.
  • a protein having such a conservative mutation is described as a conservative variant.
  • AldB protein examples include, for example, a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of SEQ ID NO: 2 or 9-11 It may be a protein having
  • “One or several” amino acid residues vary depending on the position of the protein in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1-20, more preferably 1-10, Preferably 1 to 5 are meant.
  • a typical conservative mutation is a conservative substitution.
  • Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr.
  • substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
  • amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the microorganism from which the gene is derived. Also included by Such a protein is modified by, for example, site-directed mutagenesis so that the amino acid residue at a specific site of the encoded protein contains a substitution, deletion, insertion, or addition, so that the nucleotide sequence of the wild-type aldB gene is modified. Can be obtained by
  • the protein having the conservative mutation as described above is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more with respect to the entire amino acid sequence. It may be a protein having a homology of at least%, particularly preferably at least 99%, and having a function equivalent to that of a wild-type protein. In the present specification, “homology” may refer to “identity”.
  • the wild-type aldB gene is not limited to genes such as Escherichia coli, Pantoea ananatis, and Enterobacter aerogenes, as long as it encodes the amino acid sequence as described above. It may be what you did.
  • the wild-type aldB gene is a protein that hybridizes under stringent conditions with a probe complementary to the nucleotide sequence of SEQ ID NO: 1, or a probe that can be prepared from the complementary sequence, and that has acetaldehyde dehydrogenase activity. It may be DNA encoding.
  • stringent conditions refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, particularly preferably 99% or more.
  • the conditions include washing once, more preferably 2 to 3 times at a salt concentration and temperature corresponding to 0.1 ⁇ SSC, 0.1% SDS, more preferably 68 ° C., 0.1 ⁇ SSC, 0.1% SDS.
  • a part of the complementary sequence of the aldB gene can also be used.
  • Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
  • hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
  • a gene encoding a conservative variant of a protein as described above is a “conservative variant” of a wild-type gene.
  • Modifications that reduce the activity of the AldB protein are achieved, for example, by reducing the expression of the aldB gene.
  • the intracellular activity of the AldB protein can be reduced by deleting part or all of the coding region of the aldB gene on the chromosome.
  • the reduction in AldB protein activity can also be achieved by reducing the expression of the gene by modifying the expression regulatory sequence such as the promoter of aldB gene and Shine-Dalgarno (SD) sequence.
  • the expression level of a gene can also be reduced by modifying an untranslated region other than the expression regulatory sequence.
  • the entire gene including the sequence before and after the aldB gene on the chromosome may be deleted.
  • the gene expression can also be reduced (J. Biol. Chem. 272: 8611-8617 (1997), Proc. Natl. Acad. Sci. USA, 95: 5511-5515 (1998), J. Biol. Chem. 266: 20833-20839 (1991)).
  • the modification may be performed by X-ray or ultraviolet irradiation, or a normal mutation treatment with a mutation agent such as N-methyl-N′-nitro-N-nitrosoguanidine. Also good.
  • the modification of the expression regulatory sequence is preferably 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more.
  • the region to be deleted may be any of the N-terminal region, the internal region, and the C-terminal region as long as the activity of the AldB protein is reduced. It may be the whole. Usually, the longer region to be deleted can surely inactivate the gene. Further, it is preferable that the reading frames upstream and downstream of the region to be deleted do not match.
  • the insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated.
  • the sequences before and after the insertion site preferably do not match the reading frame.
  • Other sequences are not particularly limited as long as they reduce the function of the encoded AldB protein, and examples thereof include antibiotic resistance genes and transposons carrying genes useful for L-amino acid production.
  • a partial gene of the aldB gene is modified to produce a mutant gene that does not produce a normally functioning AldB protein, transformed into a microorganism belonging to the family Enterobacteriaceae with a DNA containing the gene, By causing recombination with a gene on the genome, the aldB gene on the genome can be replaced with a mutant.
  • the gene replacement using such homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A.
  • the above-described method for reducing the activity of the AldB protein can be similarly applied to the reduction of the enzyme activity in imparting or enhancing the L-amino acid producing ability.
  • the bacterium of the present invention preferably has enhanced activity of the AdhE protein.
  • AdhE protein the presence of AdhE having acetaldehyde dehydrogenase activity and alcohol dehydrogenase activity, which reversibly catalyzes the following reaction, is known as an enzyme that produces ethanol under anaerobic conditions.
  • the activity of AdhE protein means the activity of catalyzing these reactions, and “the activity of AdhE protein is enhanced” means that the activity of catalyzing at least the reaction of directly producing acetyl-CoA from acetaldehyde. It means being strengthened.
  • AdhE protein is also called Co-A dependent aldehyde dehydrogenase.
  • the sequence of the adhE gene encoding AdhE of Escherichia coli is disclosed in WO2009 / 031565, US Patent Application Publication No. 2009068712.
  • the base sequence of the adhE gene (NC_000913.2 GI: 49175990 1294669..1297344) of Escherichia coli K-12 strain is shown in SEQ ID NO: 3
  • amino acid sequence is shown in SEQ ID NO: 4.
  • Pantoea Ananatis LMG 20103 strain (GenBank Accession NC_013956.1 GI: 291617642 complement (4631008..4632396)), Pectobacterium atrosepticum (former name: Erwinia carotovora) SCRI1043 Strain (GenBank Accession NC_004547.2 GI: 50121254 2634501..2637176) and AdhE protein encoded by the adhE gene of Salmonella enterica CT18 strain (NC_003198.1 GI: 16760134 complement (1259893..1262571)) are shown in SEQ ID NOs: 12 to 14, respectively. The alignment of these AdhE proteins is shown in FIGS.
  • the activity of the AdhE protein can be enhanced, for example, by enhancing the expression of the adhE gene.
  • enhancing the expression of the adhE gene will be described. These methods can also be applied to the genes described above for L-amino acid producing bacteria.
  • the first method is to increase the copy number of the target gene.
  • the copy number of the gene can be increased by cloning the target gene on an appropriate vector and transforming the host bacterium with the obtained vector.
  • the vector used for transformation include a plasmid capable of autonomous replication with the microorganism used.
  • pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV are available from Takara Bio Inc.) , PMW119, pMW118, pMW219, pMW218 (pMW is available from Nippon Gene).
  • phage DNA may be used as a vector instead of a plasmid.
  • a transformation method for example, a method in which a recipient cell is treated with calcium chloride to increase DNA permeability as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), and electric pulse method (Japanese Patent Laid-Open No. 2-207791).
  • Increase in gene copy number can also be achieved by introducing multiple copies of the target gene onto the chromosomal DNA of the microorganism.
  • a homologous recombination method (MillerI, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory).
  • a sequence present in multiple copies on chromosomal DNA repetitive DNA and inverted repeats present at the end of a transposable element can be used.
  • a target gene can be mounted on a transposon, transferred, and introduced in multiple copies on chromosomal DNA.
  • the target gene can also be incorporated into the host chromosome by a method using Mu phage (Japanese Patent Laid-Open No. 2-109985). Confirmation that the target gene has been transferred onto the chromosome can be confirmed by Southern hybridization using a part of the gene as a probe.
  • the copy number of the gene is not particularly limited as long as the activity of the target gene product can be enhanced.
  • the microorganism originally has the target gene it is preferably 2 or more.
  • the number of copies of the introduced gene may be 1 or 2 or more.
  • the second method is a method for enhancing expression of a target gene by substituting an expression regulatory sequence such as a promoter of the target gene with an appropriate strength on a chromosomal DNA or a plasmid.
  • an expression regulatory sequence such as a promoter of the target gene
  • thr promoter, lac promoter, trp promoter, trc promoter, pL promoter, tac promoter and the like are known as frequently used promoters.
  • Methods for assessing promoter strength and examples of strong promoters are described in Goldstein and Doi (Goldstein, M. A. and Doi R. H. 1995. Prokaryotic promoters in biotechnology. Biotechnol.techAnnu. Rev., 1, 105- 128).
  • the expression regulatory sequence can also be replaced by a method using linear DNA, such as the other methods (see WO2005 / 010175).
  • the modification of the expression regulatory sequence may be combined with the method for increasing the gene copy number as described above.
  • the expression of the adhE gene is improved compared to the parent strain, for example, a wild strain or an unmodified strain, can be confirmed by comparing the amount of mRNA of the same gene with a wild type or an unmodified strain.
  • the expression level confirmation method include Northern hybridization and RT-PCR (Molecularolecloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)).
  • the expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more compared to the wild strain or the non-modified strain, It is desirable that it rises 3 times or more.
  • the bacterium of the present invention can assimilate ethanol aerobically.
  • Escherichia coli cannot assimilate ethanol under aerobic conditions
  • a strain modified so as to assimilate ethanol aerobically may be used.
  • expression under the control of a non-native promoter that functions under aerobic conditions examples include retaining a modified adh gene or retaining a mutant adhE gene having a mutation in the coding region that enables aerobic assimilation of ethanol (Clark, D. P. , And Cronan, J. E. Jr. 1980. J. Bacteriol.
  • this mutant adhE gene may be expressed under the control of a non-natural promoter that functions under aerobic conditions.
  • alcohol dehydrogenase can be expressed under aerobic conditions and ethanol can be assimilated aerobically by replacing the promoter upstream of the gene encoding alcohol dehydrogenase with a promoter that functions aerobically.
  • WO2008 / 010565 pamphlet As a non-natural promoter that functions under aerobic conditions, any promoter that can express the adhE gene beyond a certain level under aerobic conditions can be used. Aerobic conditions can be those normally used for culturing bacteria that are supplied with oxygen by methods such as shaking, aeration and agitation. Specifically, any promoter known to express a gene under aerobic conditions can be used.
  • promoters of genes involved in glycolysis, pentose phosphate pathway, TCA cycle, amino acid biosynthesis pathway, etc. can be used.
  • the Ptac promoter, lac promoter, trp promoter, trc promoter, PR promoter, or PL promoter of lambda phage are all strong promoters that function under aerobic conditions, and these are preferably used.
  • AdhE mutant having the mutation as described above, a mutant in which the glutamic acid residue at position 568 of AdhE of Escherichia coli is substituted with an amino acid residue other than glutamic acid and aspartic acid, for example, lysine (Glu568Lys, E568K) (International Publication Pamphlet WO2008 / 010565).
  • glutamic acid residue at position 568 of AdhE of Escherichia coli is substituted with an amino acid residue other than glutamic acid and aspartic acid, for example, lysine (Glu568Lys, E568K) (International Publication Pamphlet WO2008 / 010565).
  • the AdhE mutant may contain the following additional mutations.
  • AdhE of other microorganisms mutants having the same mutations as described above can be used.
  • Pantoea ananatis, Pectobacterium atrocepticam, and Salmonella enterica AdhE have 89.0%, 89.1%, and 97.2% homology, respectively, with Escherichia coli AdhE.
  • the amino acid residue corresponding to the position of the mutation can be identified according to the alignment shown in FIG.
  • the position where the mutation is introduced can be specified by creating an alignment between the encoded amino acid sequence of AdhE and the known amino acid sequence of AdhE.
  • Alcohol dehydrogenase activity in is 1.5 units or more, preferably 5 units or more, and more preferably 10 units or more per mg of protein.
  • the bacterium of the present invention may be modified so that the activity of ribonuclease G is reduced.
  • L-amino acid can be produced by culturing the bacterium of the present invention in a medium containing ethanol as a carbon source and collecting L-amino acid from the medium.
  • the amount of ethanol contained in the medium used in the method of the present invention may be any amount as long as the bacterium used in the method of the present invention can assimilate as a carbon source. / v% or less, preferably 5 w / v% or less, more preferably 2 w / v% or less. In addition, when added as a single carbon source in the medium, it is desirable that it be contained at 0.2 w / v% or more, preferably 0.5 w / v% or more, more preferably 1.0 w / v% or more.
  • carbon sources may be added to the medium used in the method of the present invention in addition to ethanol.
  • Preferred are sugars such as glucose, fructose, sucrose, lactose, galactose, molasses, starch hydrolyzate and molasses obtained by hydrolysis of biomass, and organic acids such as fumaric acid, citric acid and succinic acid.
  • the ethanol ratio in the carbon source is preferably 10% by weight or more, preferably 30% by weight or more, more preferably 50% by weight or more.
  • the concentration in the medium after fed-batch is 5 w / v% or less, preferably 2 w / v% or less, more preferably It is preferably contained at 1 w / v% or less.
  • it should be controlled in an amount of 0.01 w / v% or more, preferably 0.02 w / v% or more, more preferably 0.05 w / v% or more. Is preferred.
  • ethanol may be contained at a constant concentration in all the steps of the culture, may be added only to the fed-batch medium or only to the initial medium, and if other carbon sources are satisfied, There may be a period where the etarule is insufficient for a certain period of time.
  • ethanol may be deficient in the time of 10%, 20%, and 30% at the maximum of the entire fermentation time.
  • the ethanol concentration temporarily becomes 0, if there is a culture period in a medium containing ethanol, it is included in the phrase “cultivate in a medium containing ethanol” of the present invention. It is.
  • a nitrogen source As a component other than the carbon source to be added to the medium, a nitrogen source, inorganic ions, and other organic components as required can be used.
  • the nitrogen source contained in the culture medium of the present invention ammonium salts such as ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate, urea and the like can be used, and used for pH adjustment. Ammonia gas and aqueous ammonia can also be used as a nitrogen source.
  • peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean hydrolyzate and the like can also be used. Only one of these nitrogen sources may be included in the medium, or two or more thereof may be included.
  • nitrogen sources can be used for both the initial medium and the fed-batch medium. Further, the same nitrogen source may be used for both the initial culture medium and the fed-batch medium, or the nitrogen source of the fed-feed medium may be changed to the nitrogen source of the initial culture medium.
  • the medium of the present invention preferably contains a phosphate source and a sulfur source in addition to a carbon source and a nitrogen source.
  • phosphoric acid source phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and pyrophosphoric acid can be used.
  • the sulfur source may be any one containing sulfur atoms, but sulfates such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione are desirable. However, ammonium sulfate is desirable.
  • the medium may contain a growth promoting factor (a nutrient having a growth promoting effect) in addition to the above components.
  • a growth promoting factor a nutrient having a growth promoting effect
  • trace metals include iron, manganese, magnesium, calcium and the like
  • vitamins include vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12.
  • the initial medium and fed-batch medium may have the same or different medium composition.
  • the initial culture medium and the fed-batch medium may have the same or different sulfur concentration.
  • the composition of each feeding medium may be the same or different.
  • the culture is preferably carried out by aeration culture at a fermentation temperature of 20 to 45 ° C, particularly preferably 33 to 42 ° C.
  • the oxygen concentration is adjusted to 5 to 50%, preferably about 10%.
  • calcium carbonate can be added or neutralized with an alkali such as ammonia gas or ammonia water.
  • bacterial culture in order to keep L-amino acid accumulation at a certain level or more, bacterial culture may be divided into seed culture and main culture.
  • the main culture may be performed by fed-batch culture or continuous culture, and both seed culture and main culture may be performed by batch culture.
  • the feed medium when fed-batch culture or continuous culture is performed, the feed medium may be intermittently fed so that ethanol or other carbon sources are temporarily stopped. In addition, it is preferable to stop the feeding of the fed-batch medium at a maximum of 30% or less, desirably 20% or less, particularly desirably 10% or less of the feeding time.
  • the fed-batch culture is fed intermittently, the fed-batch medium is added for a certain period of time, and the second and subsequent additions are performed when the carbon source in the fermentation medium is depleted in the addition stop period preceding the addition stage. Control to start when a rise in pH or an increase in dissolved oxygen concentration is detected by the computer, so that the substrate concentration in the culture tank may always be automatically maintained at a low level (US Pat. No. 5,912,113). book).
  • the fed-batch medium used for fed-batch culture is preferably a medium containing ethanol and other carbon sources and nutrients that have a growth-promoting effect (growth-promoting factor), and the ethanol concentration in the fermentation medium is controlled to be below a certain level. May be.
  • growth-promoting factor growth-promoting factor
  • other carbon sources to be added to the feed medium glucose, sucrose, and fructose are preferable, and as a growth promoting factor, a nitrogen source, phosphoric acid, amino acid and the like are preferable.
  • a nitrogen source ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate, and urea, nitrates, and the like can be used.
  • the feed medium may be one kind or a mixture of two or more kinds.
  • the media may be mixed and fed with a single feed can, or fed with a plurality of feed cans.
  • the withdrawal may be performed simultaneously with the feeding, or the feeding may be performed after a part of the withdrawal is performed.
  • a continuous culture method may be used in which the culture medium is drawn out while containing L-amino acid and cells, and the cells are returned to the fermenter and reused (see French Patent No. 2669935).
  • a method for feeding the nutrient source continuously or intermittently the same method as the fed-batch culture is used.
  • the continuous culture method that recycles bacterial cells means that when a predetermined amino acid concentration is reached, the fermentation medium is withdrawn intermittently or continuously, only L-amino acids are extracted, and the filtration residue containing the bacterial cells is fermented. This is a method of recirculation in a tank, and can be carried out with reference to, for example, French Patent No. 2669935.
  • the culture solution when the culture solution is withdrawn intermittently, when a predetermined L-amino acid concentration is reached, a part of the L-amino acid is withdrawn, and a culture medium is newly fed to carry out the culture.
  • the amount of the medium to be added is set so as to be the same as the amount of the culture solution before the final withdrawal.
  • the same amount means an amount of about 93 to 107% of the amount of the culture solution before drawing.
  • the withdrawal start time is within 5 hours from the start of feeding. Preferably it is within 3 hours, more preferably within 1 hour.
  • the amount of the culture solution to be withdrawn is preferably with the same amount as that to be fed.
  • the pH during the cultivation is controlled to 6.5 to 9.0, and the pH of the medium at the end of the cultivation is controlled to 7.2 to 9.0.
  • the basic amino acid may be recovered by a method (JP 2002-65287 A, US 2002-0025564A, EP 1813677A).
  • the pH of the medium during the culture is 6.5 to 9.0, preferably 6.5 to 9.0.
  • the pH of the medium at the end of the culture is controlled to be 7.2 to 9.0, and further the pressure in the fermenter during the fermentation is controlled to be positive, or carbon dioxide or It is known to supply a mixed gas containing carbon dioxide gas to a culture medium (Japanese Patent Laid-Open No. 2002-65287, US Patent Application Publication No. 20020025564, EP1813677A).
  • both control so that the pressure in the fermenter during fermentation may be positive and carbon dioxide or a mixed gas containing carbon dioxide may be supplied to the culture medium.
  • the pressure in the fermenter, the supply amount of carbon dioxide or a mixed gas containing carbon dioxide, or the limited supply amount is measured, for example, by measuring bicarbonate ions or carbonate ions in the medium, or by measuring pH or ammonia concentration. Can be determined.
  • the pH of the medium during the culture is controlled to 6.0 to 9.0, preferably 6.5 to 8.0, and the pH of the medium at the end of the culture is controlled to 7.2 to 9.0.
  • the pH of the culture medium for making the quantity of bicarbonate ion and / or carbonate ion which are required as a counter ion exist in a culture medium.
  • ammonia is supplied to increase the pH, which can be an N source of basic amino acids.
  • Examples of cations other than basic amino acids contained in the medium include K, Na, Mg, Ca and the like derived from medium components. These are preferably 50% or less of the total cations.
  • the supply air pressure may be set higher than the exhaust pressure.
  • the carbon dioxide gas generated by fermentation dissolves in the culture solution to produce bicarbonate ions or carbonate ions, which can be counter ions of basic amino acids.
  • the pressure in the fermenter is 0.03 to 0.2 MPa, preferably 0.05 to 0.15 MPa, more preferably 0.1 to 0.3 MPa in terms of gauge pressure (differential pressure with respect to atmospheric pressure).
  • gauge pressure Differential pressure with respect to atmospheric pressure.
  • carbon dioxide gas may be dissolved in the culture solution by supplying carbon dioxide gas or a mixed gas containing carbon dioxide gas to the culture solution.
  • the supply air pressure may be set to be higher than the exhaust pressure.
  • carbon dioxide when carbon dioxide is supplied to the culture solution, for example, pure carbon dioxide or a mixed gas containing 5% by volume or more of carbon dioxide may be blown.
  • the above-mentioned method for dissolving bicarbonate ions and / or carbonate ions in the medium may be used alone or in combination.
  • a sufficient amount of ammonium sulfate or ammonium chloride is usually added to the medium as a counter anion of the basic amino acid to be produced, and a sulfate or hydrolyzate of protein or the like as a nutrient component is added to the medium.
  • the culture medium contains sulfate ions and chloride ions. Therefore, the concentration of carbonate ion, which is weakly acidic, is extremely low during the culture, and is in ppm.
  • the above aspect is characterized in that the sulfate ions and chloride ions are reduced, and carbon dioxide released by the microorganisms during fermentation is dissolved in the medium in the fermentation environment to form counter ions.
  • sulfate ions or chloride ions it is not necessary to add sulfate ions or chloride ions to the culture medium in an amount necessary for growth.
  • an appropriate amount of ammonium sulfate or the like is fed to the medium at the beginning of the culture, and the feed is stopped during the culture. Or you may feed ammonium sulfate etc., maintaining the balance with the dissolved amount of the carbonate ion or bicarbonate ion in a culture medium.
  • ammonia may be fed to the medium as a nitrogen source for basic amino acids. Ammonia can be supplied to the medium alone or with other gases.
  • the concentration of bicarbonate ions and / or other anions other than carbonate ions contained in the medium is preferably low as long as it is an amount necessary for the growth of microorganisms.
  • Such anions include chloride ions, sulfate ions, phosphate ions, ionized organic acids, hydroxide ions, and the like.
  • the total molar concentration of these other ions is preferably usually 900 mM or less, more preferably 700 mM or less, particularly preferably 500 mM or less, still more preferably 300 mM or less, and particularly preferably 200 mM or less.
  • one of the purposes is to reduce the amount of sulfate ion and / or chloride ion used, and the sulfate ion or chloride ion contained in the medium, or the total of these, is usually 700 mM.
  • it is preferably 500 mM or less, more preferably 300 mM or less, still more preferably 200 mM or less, and particularly preferably 100 mM or less.
  • the total ammonia concentration in the medium is controlled to such an extent that “the production of basic amino acids is not inhibited”.
  • Such conditions include, for example, preferably 50% or more, more preferably 70% or more, particularly preferably 90%, as compared to the yield and / or productivity in the case of producing a basic amino acid under optimum conditions.
  • Conditions for obtaining the above yield and / or productivity are included.
  • the total ammonia concentration in the medium is preferably 300 mM or less, more preferably 250 mM, and particularly preferably 200 mM or less.
  • the degree of ammonia dissociation decreases with increasing pH. Undissociated ammonia is more toxic to bacteria than ammonium ions.
  • the upper limit of the total ammonia concentration also depends on the pH of the culture solution. That is, the higher the pH of the culture solution, the lower the allowable total ammonia concentration. Therefore, the total ammonia concentration that “does not inhibit the production of basic amino acids” is preferably set for each pH. However, the total ammonia concentration range allowed at the highest pH during the culture may be the upper limit range of the total ammonia concentration throughout the culture period.
  • the total ammonia concentration as a nitrogen source necessary for the growth of microorganisms and the production of basic substances decreases the productivity of target substances by microorganisms due to the lack of a nitrogen source that does not continuously deplete ammonia during culture.
  • the ammonia concentration may be measured over time during the culture, and a small amount of ammonia may be added to the medium when the ammonia in the medium is depleted.
  • the concentration when ammonia is added is not particularly limited.
  • the total ammonia concentration is preferably 1 mM or more, more preferably 10 mM or more, and particularly preferably 20 mM or more.
  • L-amino acids from fermentation broth is usually performed by ion exchange resin method (Nagai, H. et al., Separation, Science and Technology, 39 (16), 3691-3710), precipitation method, membrane separation method No. 164323, JP-A-9-173792), crystallization methods (WO2008 / 078448, WO2008 / 078646), and other known methods can be combined.
  • ion exchange resin method Naagai, H. et al., Separation, Science and Technology, 39 (16), 3691-3710
  • precipitation method precipitation method
  • membrane separation method No. 164323 JP-A-9-173792
  • crystallization methods WO2008 / 078448, WO2008 / 078646
  • other known methods can be combined.
  • L-amino acid accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves, etc., and the microbial cells are removed by centrifugation. Can be recovered.
  • the recovered L-amino acid may contain bacterial cells, medium components, water, and bacterial metabolic byproducts in addition to the L-amino acid.
  • the purity of the collected L-amino acid is 50% or more, preferably 85% or more, particularly preferably 95% or more (JP1214636B, USP 5,431,933, 4,956,471, 4,777,051, 4946654, 45,840,358, 6,238,714, US2005 / 0025878)).
  • L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration.
  • the L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.
  • Example 1 Construction of L-lysine-producing bacterium with reduced AldB activity ⁇ 1-1> Construction of L-lysine-producing bacterium with ethanol-assimilating ability L-lysine-producing bacterium is imparted with ethanol-assimilating ability Therefore, a mutant alcohol dehydrogenase gene (adhE *) was introduced. As the mutant alcohol dehydrogenase gene, a gene derived from MG1655 :: PL-tacadhE * (WO2008 / 010565) was used.
  • a DNA fragment in which a chloramphenicol resistance gene (cat) and a mutant adhE gene controlled by the PL-tac promoter are linked is inserted into the genome of Escherichia coli MG1655 strain. This is the stock obtained.
  • the mutant adhE gene encodes a mutant in which the glutamic acid residue at position 568 is replaced with lysine. Escherichia coli that retains this mutant alcohol dehydrogenase can assimilate ethanol under aerobic conditions.
  • the cat gene was replaced with a DNA fragment (att-tet) linking the attachment site of lambda phage and the tetracycline resistance gene.
  • Red-driven integration originally developed by Datsenko and Wanner described in WO2005 / 010175 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645).
  • a synthetic oligonucleotide designed with a part of the target gene on the 5 ′ side of the synthetic oligonucleotide and a part of the antibiotic resistance gene on the 3 ′ side is used as a primer.
  • a gene-disrupted strain can be constructed in one step.
  • the antibiotic resistance gene incorporated into the gene-disrupted strain is removed by combining the excision system derived from ⁇ phage (Cho, EH et al., J. Bacteriol. 2002 Sep; 184 (18): 5200-5203) I can do it.
  • the primers of SEQ ID NOs: 5 and 6 were used as primers for replacing the cat gene with the att-tet gene.
  • the MG1655-att-tet-PL-tacadhE * strain in which the cat gene of MG1655 :: PL-tacadhE * was replaced with the att-tet gene was obtained.
  • L-lysine-producing bacteria WC196 ⁇ cadA ⁇ ldcC also referred to as “WC196LC”
  • MG1655-att-tet-PL-tacadhE * as a donor
  • WC196LC-att-tet-PL-tacadhE * strain was obtained.
  • pMW-intxis-ts US Patent Application Publication 200601415866
  • pMW-intxis-ts is a plasmid carrying a gene encoding ⁇ phage integrase (Int) and gene encoding excisionase (Xis) and having temperature-sensitive replication ability.
  • Competent cells of the WC196LC-att-tet-PL-tacadhE * strain obtained above were prepared according to a conventional method, transformed with the helper plasmid pMW-intxis-ts, and 50 mg / L ampicillin at 30 ° C. Plated on an LB agar medium containing ampicillin resistant strains. In order to remove the pMW-intxis-ts plasmid, it was cultured on LB agar medium at 42 ° C, and the resulting colonies were tested for ampicillin resistance and tetracycline resistance. Att-tet and pMW-intxis-ts were removed. We obtained tetracycline and ampicillin sensitive strains, which are PL-tacadhE * introduced strains. This strain was named WC196LC PL-tacadhE * strain.
  • the WC196LC PL-tacadhE * strain was transformed with the Lys production plasmid pCABD2 (International Publication No. WO01 / 53459 pamphlet) carrying the dapA, dapB and lysC genes according to a conventional method to obtain the WC196LC PL-tacadhE * / pCABD2 strain. It was.
  • pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from E. coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine.
  • DDPS dihydrodipicolinate synthase
  • a mutant lysC gene encoding aspartokinase III derived from E. coli, dapB gene encoding dihydrodipicolinate reductase derived from E. coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.
  • MG1655 ⁇ aldB Tet strain was used as a donor, P1-transduction was performed on L-lysine-producing WC196LC PL-tacadhE * strain, and AldB non-producing strain WC196LC PL-tacadhE * ⁇ aldB :: Tet / pCABD2 strain was obtained. After culturing these strains in L medium containing 20 mg / L streptomycin at 37 ° C. so that the final OD600 ⁇ 0.6, an equal amount of 40% glycerol solution and the same amount as the culture solution were added and stirred. Aliquots were stored at -80 ° C. This is called glycerol stock.
  • Example 2 Evaluation of L-lysine-producing ability of non-AldB-producing strain Melt the glycerol stock of the above-mentioned strain, apply 100 ⁇ L of each uniformly onto an L plate containing 20 mg / L of streptomycin, at 37 ° C. Cultured for 15 hours. Approximately 1/8 of the cells on the obtained plate was inoculated into 5 mL of a fermentation medium containing 20 mg / L of streptomycin in a large test tube (inner diameter: 18 mm) and stirred at 120 rpm with a reciprocating shaker. Cultured at 37 ° C under conditions. The same culture was performed for the AldB non-modified strain WC196LC PL-tacadhE * / pCABD2 strain. The composition of the fermentation medium using ethanol as a carbon source is shown below.
  • the AldB non-producing strain (WC196LC PL-tacadhE * ⁇ aldB :: Tet / pCABD2 strain) showed significantly higher L-lysine production than the control strain (WC196LC PL-tacadhE * / pCABD2 strain).
  • SEQ ID NO: 1 E. coli aldb gene base sequence
  • SEQ ID NO: 2 E. coli AldB amino acid sequence
  • SEQ ID NO: 3 E. coli adhE gene base sequence
  • SEQ ID NO: 4 E. coli AdhE amino acid sequence
  • SEQ ID NO: 5 Primer for replacing cat gene with att-tet gene
  • SEQ ID NO: 6 Primer for replacing cat gene with att-tet gene
  • SEQ ID NO: 7 primer for deletion of aldB gene
  • SEQ ID NO: 8 of aldB gene Primer for deletion
  • SEQ ID NO: 9 AldB amino acid sequence of P. ananatis
  • SEQ ID NO: 10 AldB amino acid sequence of P.
  • SEQ ID NO: 11 AldB amino acid sequence of S. enterica
  • SEQ ID NO: 12 AdhE amino acid of P. ananatis
  • SEQ ID NO: 13 AdhE amino acid sequence of P. atrosepticum
  • SEQ ID NO: 14 AdhE amino acid sequence of S. enterica
  • L-amino acid can be efficiently fermented and produced using ethanol as a carbon source.

Abstract

Provided is a method for efficiently producing an L-amino acid from ethanol as the starting material using a bacterium belonging to the family Enterobacteriaceae, and also provided is a bacterium used in this method. A bacterium modified such that the activity of Aldb protein is reduced, preferably a bacterium further modified such that the activity of AdhE protein is enhanced, is used in the method for producing an L-amino acid in which a bacterium belonging to the family Enterobacteriaceae capable of producing an L-amino acid is cultured in a medium containing ethanol and the L-amino acid is recovered from the medium.

Description

Ｌ－アミノ酸の製造法Method for producing L-amino acid
　本発明は、細菌を用いたＬ－アミノ酸の製造法に関し、特にエタノールを原料とするＬ－アミノ酸の製造法、及び該方法に用いる細菌に関する。Ｌ－アミノ酸は、動物飼料用の添加物、健康食品の成分、又はアミノ酸輸液等として、産業上有用である。 The present invention relates to a method for producing L-amino acids using bacteria, and more particularly, to a method for producing L-amino acids using ethanol as a raw material, and bacteria used in the method. L-amino acids are industrially useful as additives for animal feeds, health food ingredients, amino acid infusions, and the like.
　Ｌ－アミノ酸は、ブレビバクテリウム属、コリネバクテリウム属、エシェリヒア属等に属する微生物を用いた発酵法により工業生産されている。これらの製造法においては、自然界から分離された菌株または該菌株の人工変異株、さらには、組換えDNA技術により塩基性Ｌ－アミノ酸生合成酵素の活性が増大するように改変された微生物などが用いられている。(特許文献１～９） L-amino acids are industrially produced by fermentation using microorganisms belonging to the genera Brevibacterium, Corynebacterium, Escherichia and the like. In these production methods, strains isolated from nature, artificial mutants of the strains, and microorganisms modified so as to increase the activity of basic L-amino acid biosynthetic enzymes by recombinant DNA technology are used. It is used. (Patent Documents 1 to 9)
　一般的に、微生物を用いてアミノ酸生産を行う際には、炭素源に糖質を主成分として用いているが、エタノールも糖質と同様に炭素源として用いることが可能である（特許文献１０）。細胞に取り込まれたエタノールはアルコールデヒドロゲナーゼにより、まずアセトアルデヒドに変換される。生じたアセトアルデヒドは、アセトアルデヒドデヒドロゲナーゼにより酢酸に、または、アセトアルデヒドCoAデヒドロゲナーゼによりアセチル-CoAへと変換されると考えられる。 Generally, when amino acids are produced using microorganisms, a saccharide is used as a carbon source as a main component, but ethanol can also be used as a carbon source in the same manner as a saccharide (Patent Document 10). ). Ethanol taken up by cells is first converted to acetaldehyde by alcohol dehydrogenase. The resulting acetaldehyde is thought to be converted to acetic acid by acetaldehyde dehydrogenase or acetyl-CoA by acetaldehyde CoA dehydrogenase.
　エシェリヒア・コリにおいて見いだされたAldBタンパク質は、NADP依存型のアセトアルデヒドデヒドロゲナーゼ活性を有することが知られており、MgCl2存在時にはさまざまな基質に対する活性が向上するなど、ヒトミトコンドリアのアルデヒドデヒドロゲナーゼと似た性質を持つ（非特許文献１）。 AldB protein found in Escherichia coli is known to have NADP-dependent acetaldehyde dehydrogenase activity, and has similar properties to human mitochondrial aldehyde dehydrogenase, such as increased activity against various substrates in the presence of MgCl 2. (Non-Patent Document 1).
　また、エタノールが存在すると培養定常期にAldBの発現が上昇することから、アルコールやアルデヒドによるストレスを低減する機能を持つと考えられている（非特許文献２）。
　しかしながら、実際にエタノールを資化する際にAldBによりアセトアルデヒドが酢酸に変換されるかは不明であった。また、AldBの活性と、エタノールからのＬ－アミノ酸生産との関係は、全く知られていなかった。 Moreover, since the expression of AldB increases in the stationary culture phase in the presence of ethanol, it is considered to have a function of reducing stress caused by alcohol or aldehyde (Non-patent Document 2).
However, it was not clear whether AldB converted acetaldehyde into acetic acid when actually assimilating ethanol. Furthermore, the relationship between AldB activity and L-amino acid production from ethanol was not known at all.
欧州特許公開EP0643135BEuropean Patent Publication EP0643135B 欧州特許公開EP0733712BEuropean Patent Publication EP0733712B 欧州特許公開EP1477565AEuropean Patent Publication EP1477565A 欧州特許公開EP0796912AEuropean Patent Publication EP0796912A 欧州特許公開EP0837134AEuropean Patent Publication EP0837134A 国際公開WO01/53459International Publication WO01 / 53459 欧州特許公開EP1170376AEuropean Patent Publication EP1170376A 国際公開WO2005/010175International Publication WO2005 / 010175 国際公開WO96/17930International Publication WO96 / 17930 WO2008/010565WO2008 / 010565
　本発明は、エタノールを原料として、腸内細菌科に属する細菌を用いて効率よくＬ－アミノ酸を製造する方法、及び該方法に用いる細菌を提供することを課題とする。 An object of the present invention is to provide a method for efficiently producing an L-amino acid using ethanol as a raw material and a bacterium belonging to the family Enterobacteriaceae, and a bacterium used in the method.
　エタノールの資化にAldBが関与しているとすると、AldBのアセトアルデヒドデヒドロゲナーゼ活性によってアセトアルデヒドから酢酸が生じると考えられる。次いで、酢酸はアセチルCoA合成酵素（ACS）、又は、ホスホトランスアセチラーゼ（PTA）及びアセテートキナーゼ（ACK）により触媒される反応によってアセチル-CoAに変換されるが、いずれもATPからAMP又はADPへの変換を伴う。それに対し、アセトアルデヒドデヒドロゲナーゼ活性を持つ他のタンパク質であるAdhEは、アルコールデヒドロゲナーゼ活性も有しており、アセトアルデヒドを直接アセチル-CoAに変換する反応を触媒するため、ATPの損失がなく、エネルギー的には有利と考えた。一方、Xu, J. et al., J. Bacteriol., 177 (1995) 3166-3175には、エタノールが存在すると培養定常期にAldBの発現が上昇することが示されており、AldBがエタノール代謝に関わるとすると、aldB欠損株ではエタノールの資化性が低下する可能性も考えられた。ところが、予想に反し、Ｌ－アミノ酸生産菌のAldBの活性を低下させたところ、エタノールを炭素源としたときにＬ－アミノ酸生産能が向上することを見出し、本発明を完成するに至った。
　すなわち本発明は、以下のとおりである。 If AldB is involved in the assimilation of ethanol, it is considered that acetic acid is produced from acetaldehyde by the acetaldehyde dehydrogenase activity of AldB. Acetic acid is then converted to acetyl-CoA by a reaction catalyzed by acetyl-CoA synthase (ACS) or phosphotransacetylase (PTA) and acetate kinase (ACK), both from ATP to AMP or ADP. With conversion. In contrast, AdhE, another protein with acetaldehyde dehydrogenase activity, also has alcohol dehydrogenase activity and catalyzes the reaction of directly converting acetaldehyde to acetyl-CoA, so there is no loss of ATP and it is energetically I thought it was advantageous. On the other hand, Xu, J. et al., J. Bacteriol., 177 (1995) 3166-3175 show that the presence of ethanol increases AldB expression in the stationary phase of culture. As a result, ethanol utilization may be reduced in aldb-deficient strains. However, contrary to expectation, when the activity of AldB of L-amino acid producing bacteria was reduced, it was found that L-amino acid producing ability was improved when ethanol was used as a carbon source, and the present invention was completed.
That is, the present invention is as follows.
（１）Ｌ－アミノ酸生産能を有する腸内細菌科に属する細菌を、エタノールを含む培地で培養し、該培地からＬ－アミノ酸を採取する、Ｌ－アミノ酸の製造法であって、
　前記細菌は、ＡｌｄＢタンパク質の活性が低下するように改変されていることを特徴とする方法。
（２）ａｌｄＢ遺伝子のコード領域及び／又は同遺伝子の発現制御領域に変異が導入されたことにより、前記ＡｌｄＢタンパク質の活性が低下した、前記方法。
（３）前記細菌は染色体上のａｌｄＢ遺伝子が破壊されたことを特徴とする、前記方法。
（４）前記ＡｌｄＢタンパク質は、下記（Ａ）又は（Ｂ）のいずれかのタンパク質である、前記方法。
　（Ａ）配列番号２に示すアミノ酸配列を有するタンパク質、又は、
　（Ｂ）配列番号２に示すアミノ酸配列において、１又は数個のアミノ酸の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、アセトアルデヒドデヒドロゲナーゼ活性を有するタンパク質。
（５）前記ａｌｄＢ遺伝子は、下記（ａ）又は（ｂ）のＤＮＡである前記方法。
　（ａ）配列番号１の塩基配列を有するＤＮＡ、又は、
　（ｂ）配列番号１の塩基配列に相補的な配列、又は同配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、アセトアルデヒドデヒドロゲナーゼ活性を有するタンパク質をコードするＤＮＡ。
（６）前記細菌がエシェリヒア属、エンテロバクター属、又はパントエア属に属する細菌である、前記方法。
（７）前記細菌がエシェリヒア・コリである、前記方法。
（８）前記細菌は、さらにＡｄｈＥタンパク質の活性が増強された、前記方法。
（９）前記細菌は、好気的にエタノールを資化できるように改変された、前記方法。
（１０）前記Ｌ－アミノ酸がＬ－リジンである、前記方法。
（１１）前記細菌は、ジヒドロジピコリン酸レダクターゼ、ジアミノピメリン酸デカルボキシラーゼ、ジアミノピメリン酸デヒドロゲナーゼ、フォスフォエノールピルビン酸カルボキシラーゼ、アスパルテートアミノトランスフェラーゼ、ジアミノピメリン酸エピメラーゼ、アスパルテートセミアルデヒドデヒドロゲナーゼ、テトラヒドロジピコリン酸スクシニラーゼ、及び、スクシニルジアミノピメリン酸デアシラーゼからなる群より選択される１種または２種以上の酵素の活性が増強されている、及び／または、リジンデカルボキシラーゼの活性が弱化されている、前記方法。 (1) A method for producing an L-amino acid, wherein a bacterium belonging to the family Enterobacteriaceae having L-amino acid producing ability is cultured in a medium containing ethanol, and the L-amino acid is collected from the medium,
The method wherein the bacterium is modified so that the activity of the AldB protein is reduced.
(2) The method, wherein the activity of the AldB protein is reduced by introducing a mutation into the coding region of the aldB gene and / or the expression control region of the gene.
(3) The method as described above, wherein the bacterium has a chromosomal aldB gene disrupted.
(4) The method as described above, wherein the AldB protein is any one of the following proteins (A) and (B).
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2, or
(B) A protein having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids and having acetaldehyde dehydrogenase activity in the amino acid sequence shown in SEQ ID NO: 2.
(5) The said method whose said aldB gene is DNA of the following (a) or (b).
(A) DNA having the base sequence of SEQ ID NO: 1, or
(B) A DNA that hybridizes under stringent conditions with a sequence that is complementary to the base sequence of SEQ ID NO: 1 or that can be prepared from the same sequence, and that encodes a protein having acetaldehyde dehydrogenase activity.
(6) The method as described above, wherein the bacterium belongs to the genus Escherichia, Enterobacter, or Pantoea.
(7) The method as described above, wherein the bacterium is Escherichia coli.
(8) The method as described above, wherein the bacterium further has enhanced activity of AdhE protein.
(9) The method as described above, wherein the bacterium is modified so as to assimilate ethanol aerobically.
(10) The method as described above, wherein the L-amino acid is L-lysine.
(11) The bacterium includes dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and The method, wherein the activity of one or more enzymes selected from the group consisting of succinyl diaminopimelate deacylase is enhanced and / or the activity of lysine decarboxylase is attenuated.
各種AldBのアミノ酸配列のアラインメントを示す図。The figure which shows the alignment of the amino acid sequence of various AldB. 各種AldBのアミノ酸配列のアラインメントを示す図（続き）。The figure which shows the alignment of the amino acid sequence of various AldB (continuation). 各種AdhEのアミノ酸配列のアラインメントを示す図。The figure which shows the alignment of the amino acid sequence of various AdhE. 各種AdhEのアミノ酸配列のアラインメントを示す図（続き）。The figure which shows the alignment of the amino acid sequence of various AdhE (continuation). 各種AdhEのアミノ酸配列のアラインメントを示す図（続き）。The figure which shows the alignment of the amino acid sequence of various AdhE (continuation).
　以下、本発明を詳細に説明する。
＜１＞本発明の細菌
　本発明に用いる細菌は、Ｌ－アミノ酸生産能を有する腸内細菌科に属する細菌であって、かつ、AldBタンパク質の活性が低下するように改変されている細菌である。 Hereinafter, the present invention will be described in detail.
<1> Bacteria of the Present Invention The bacterium used in the present invention is a bacterium belonging to the family Enterobacteriaceae having L-amino acid-producing ability and modified so that the activity of the AldB protein is reduced. .
　Ｌ－アミノ酸生産能とは、本発明に用いる細菌（以下、「本発明の細菌」ともいう）を培地中で培養したときに、Ｌ－アミノ酸を生成し、培地中または菌体内に蓄積する能力をいう。好ましくは、目的とするＬ－アミノ酸を好ましくは0.5g/L以上、より好ましくは1.0g/L以上の量で培地に蓄積させることができる能力をいう。Ｌ－アミノ酸の生産能を有する細菌としては、本来的にＬ－アミノ酸の生産能を有するものであってもよいが、後述の細菌を、変異法や組換えDNA技術を利用して、Ｌ－アミノ酸の生産能を有するように改変したものであってもよい。 The ability to produce L-amino acid refers to the ability to produce L-amino acid and accumulate it in the medium or in the cells when the bacterium used in the present invention (hereinafter also referred to as “the bacterium of the present invention”) is cultured in the medium. Say. Preferably, it refers to the ability to accumulate the target L-amino acid in the medium in an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more. The bacterium having L-amino acid-producing ability may be inherently L-amino acid-producing bacterium, but a bacterium described later can be transformed into L-amino acid using a mutation method or recombinant DNA technology. It may be modified so as to have an amino acid-producing ability.
　Ｌ－アミノ酸の種類は特に制限されないが、Ｌ－リジン、Ｌ－オルニチン、Ｌ－アルギニン、Ｌ－ヒスチジン、Ｌ－シトルリン等の塩基性アミノ酸、Ｌ－イソロイシン、Ｌ－アラニン、Ｌ－バリン、Ｌ－ロイシン、グリシン等の脂肪族アミノ酸、Ｌ－スレオニン、Ｌ－セリン等のヒドロキシモノアミノカルボン酸であるアミノ酸、Ｌ－プロリン等の環式アミノ酸、Ｌ－フェニルアラニン、Ｌ－チロシン、Ｌ－トリプトファン等の芳香族アミノ酸、Ｌ－システイン、Ｌ－シスチン、Ｌ－メチオニン等の含硫アミノ酸、Ｌ－グルタミン酸、Ｌ－アスパラギン酸等の酸性アミノ酸、Ｌ－グルタミン、Ｌ－アスパラギン等の側鎖にアミド基を持つアミノ酸が挙げられる。本発明の細菌は２種類以上のＬ－アミノ酸の生産能を有するものであってもよい。 The type of L-amino acid is not particularly limited, but basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L- Aliphatic amino acids such as leucine and glycine, amino acids that are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, and fragrances such as L-phenylalanine, L-tyrosine and L-tryptophan Amino acids, sulfur-containing amino acids such as L-cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, amino acids having an amide group in the side chain such as L-glutamine and L-asparagine Is mentioned. The bacterium of the present invention may be capable of producing two or more types of L-amino acids.
　本発明においてＬ－アミノ酸とは、フリー体のＬ－アミノ酸とＬ－アミノ酸塩、たとえば硫酸塩、塩酸塩、及び炭酸塩を含む。 In the present invention, L-amino acids include free L-amino acids and L-amino acid salts such as sulfates, hydrochlorides, and carbonates.
　本発明の細菌を得るために用いる腸内細菌科に属する細菌としては、特に限定されないが、エシェリヒア、エンテロバクター、エルビニア、クレブシエラ、パントエア、ペクトバクテリウム、フォトルハブドゥス、プロビデンシア、サルモネラ、セラチア、シゲラ、モルガネラ、イェルシニア等の属に属する細菌を含む。特に、NCBI (National Center for Biotechnology Information)のデータベース(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347)で用いられている分類法により腸内細菌科に分類されている細菌が好ましい。 Bacteria belonging to the family Enterobacteriaceae used to obtain the bacterium of the present invention are not particularly limited, but Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Pectobacterium, Photorubadus, Providencia, Salmonella, Serratia, Includes bacteria belonging to genera such as Shigella, Morganella, and Yersinia. In particular, enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) Bacteria classified in the family are preferred.
　エシェリヒア属に属する細菌とは、特に制限されないが、当該細菌が微生物学の専門家に知られている分類により、エシェリヒア属に分類されていることを意味する。例えば、ナイトハルトらの著書（Neidhardt, F. C. Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology/Second Edition pp. 2477-2483. Table 1. American Society for Microbiology Press, Washington, D.C.）に記述されている系統のものが含まれる。具体的には、プロトタイプの野生株Ｋ１２株由来のエシェリヒア・コリ W3110 （ATCC 27325）、エシェリヒア・コリ MG1655 (ATCC 47076)等が挙げられる。 The bacterium belonging to the genus Escherichia is not particularly limited, but means that the bacterium is classified into the genus Escherichia according to the classification known to experts in microbiology. For example, written by Knighthard et al. (Neidhardt, F. C. Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology / Second Edition pp. 2477-2483. Table 1. American Society for Microbiology Press, Washington The ones that are being used are included. Specific examples include Escherichia coli W3110 (ATCC 32525) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.
　これらの菌株は、例えばアメリカン・タイプ・カルチャー・コレクション（住所 P.O. Box 1549 Manassas, VA 20108, United States of America）より分譲を受けることが出来る。すなわち各菌株に対応する登録番号が付与されており、この登録番号を利用して分譲を受けることが出来る。各菌株に対応する登録番号は、アメリカン・タイプ・カルチャー・コレクションのカタログに記載されている。以下に記載する他のATCC菌株も同様である。 These strains can be sold, for example, from the American Type Culture Collection (address P.O. Box 1549 Manassas, VA 20108, United States of America). That is, the registration number corresponding to each strain is given, and it can receive distribution using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. The same applies to other ATCC strains described below.
　パントエア属に属する細菌とは、当該細菌が微生物学の専門家に知られている分類により、パントエア属に分類されていることを意味する。エンテロバクター・アグロメランス（Enterobacter agglomerans）のある種のものは、最近、16S rRNAの塩基配列分析等に基づき、パントエア・アグロメランス（Pantoea agglomerans）、パントエア・アナナティス（Pantoea ananatis）、パントエア・ステワルティイ（Pantoea stewartii）、又はその他に再分類された(Int. J. Syst. Bacteriol., 43, 162-173 (1993))。本発明において、パントエア属に属する細菌には、このようにパントエア属に再分類された細菌も含まれる。 The bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to microbiologists. Certain types of Enterobacter agglomerans, recently based on 16S rRNA sequence analysis, etc., are based on Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii Or others (Int.tJ. Syst. Bacteriol., 43, 162-173 (1993)). In the present invention, the bacteria belonging to the genus Pantoea include bacteria that have been reclassified to the genus Pantoea in this way.
　パントエア・アナナティスとしては、パントエア・アナナティスAJ13355株（FERM BP-6614）、AJ13356株（FERM BP-6615）、AJ13601株（FERM BP-7207）及びそれらの誘導体等を用いることができる。これらの株は、分離された当時はエンテロバクター・アグロメランスと同定され、エンテロバクター・アグロメランスとして寄託されたが、上記のとおり、16S rRNAの塩基配列解析などにより、パントエア・アナナティスに再分類されている。 As Pantoea ananatis, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans at the time of isolation, and deposited as Enterobacter agglomerans, but as described above, they were reclassified as Pantoea ananatis by 16S rRNA sequence analysis, etc. .
　エンテロバクター属細菌とは、特に制限されないが、当該細菌が微生物学の専門家に知られている分類により、エンテロバクター属に分類されていることを意味する。例えば、エンテロバクター・アグロメランス、エンテロバクター・アエロゲネス（Enterobacter aerogenes）等が挙げられる。具体的には欧州特許出願公開EP952221号に例示された菌株を使用することが出来る。エンテロバクター属の代表的な株としては、エンテロバクター・アグロメランスATCC12287株、エンテロバクター・アエロゲネスATCC13048株、エンテロバクター・アエロゲネスNBRC12010株（Biotechonol Bioeng.2007 Mar 27; 98(2) 340-348）、及びエンテロバクター・アエロゲネスAJ110637(FERM BP-10955)株等が挙げられる。 The Enterobacter bacterium is not particularly limited, but means that the bacterium is classified into the genus Enterobacter according to a classification known to microbiologists. Examples thereof include Enterobacter agglomerans and Enterobacter aerogenes. Specifically, strains exemplified in European Patent Application Publication No. EP952221 can be used. Representative strains of the genus Enterobacter include Enterobacter agglomerans ATCC 12287, Enterobacter aerogenes ATCC 13048, Enterobacter aerogenes NBRC 12010 (Biotechonol Bioeng.2007 Mar 27; 98 (2) 340-348), and Entero Examples include Bacter Aerogenes AJ110637 (FERM-10BP-10955).
　エルビニア属細菌としては、エルビニア・アミロボーラ（Erwinia amylovora）等が挙げられ、クレブシエラ属細菌としては、クレブシエラ・プランティコーラ（Klebsiella planticola）等が挙げられ、ペクトバクテリウム属細菌としては、ペクトバクテリウム・アトロセプティカム（Pectobacterium atrosepticum）（旧名、エルビニア・カロトボーラ(Erwinia carotovora)）等が挙げられる。 Examples of the genus Erwinia include Erwinia amylovora, examples of the genus Klebsiella include Klebsiella planticola, and examples of the bacterium belonging to the genus Pectobacterium include: Atrocepticum (Pectobacterium atrosepticum) (former name Erwinia carotovora) and the like.
＜１－１＞Ｌ－アミノ酸生産菌、及びＬ－アミノ酸生産能の付与又は増強
　以下、腸内細菌科に属するＬ－アミノ酸生菌、並びに細菌にＬ－アミノ酸生産能を付与する方法、又は細菌のＬ－アミノ酸生産能を増強する方法について述べる。
　Ｌ－アミノ酸生産能を付与するには、栄養要求性変異株、Ｌ－アミノ酸のアナログ耐性株又は代謝制御変異株の取得や、Ｌ－アミノ酸の生合成系酵素の発現が増強された組換え株の創製等、従来、エシェリヒア属細菌等のアミノ酸生産菌の育種に採用されてきた方法を適用することができる（アミノ酸発酵、（株）学会出版センター、1986年５月30日初版発行、第７７～１００頁参照）。ここで、Ｌ－アミノ酸生産菌の育種において、付与される栄養要求性、アナログ耐性、代謝制御変異等の性質は、単独でもよく、２種又は３種以上であってもよい。また、発現が増強されるＬ－アミノ酸生合成系酵素も、単独であっても、２種又は３種以上であってもよい。さらに、栄養要求性、アナログ耐性、代謝制御変異等の性質の付与と、生合成系酵素の増強が組み合わされてもよい。 <1-1> L-amino acid producing bacterium, and imparting or enhancing L-amino acid producing ability Hereinafter, L-amino acid live bacteria belonging to the family Enterobacteriaceae, and a method for imparting L-amino acid producing ability to bacteria, or bacteria A method for enhancing the L-amino acid-producing ability is described.
In order to confer L-amino acid-producing ability, acquisition of auxotrophic mutants, L-amino acid analog resistant strains or metabolic control mutants, and recombinant strains with enhanced expression of L-amino acid biosynthetic enzymes The method that has been conventionally used for breeding amino acid-producing bacteria such as Escherichia bacteria can be applied (Amino Acid Fermentation, Japan Society for Publishing Press, May 30, 1986, first edition published, 77th) See page 100). Here, in the breeding of L-amino acid-producing bacteria, the auxotrophy, analog resistance, metabolic control mutation and other properties imparted may be singly or may be two or more. Further, the L-amino acid biosynthetic enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.
　Ｌ－アミノ酸生産能を有する栄養要求性変異株、アナログ耐性株、又は代謝制御変異株を取得するには、親株又は野生株を通常の変異処理、すなわちＸ線や紫外線の照射、またはＮ－メチル－Ｎ’－ニトロ－Ｎ－ニトロソグアニジン等の変異剤処理などによって処理し、得られた変異株の中から、栄養要求性、アナログ耐性、又は代謝制御変異を示し、かつＬ－アミノ酸生産能を有するものを選択することによって得ることができる。 In order to obtain an auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having L-amino acid-producing ability, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl. -Treated with a treatment with a mutant such as -N'-nitro-N-nitrosoguanidine, among the obtained mutant strains, shows auxotrophy, analog resistance, or metabolic control mutation, and has an ability to produce L-amino acid It can be obtained by selecting what it has.
　また、Ｌ－アミノ酸生産能の付与又は増強は、遺伝子組換えによって、酵素活性を増強することによっても行うことが出来る。酵素活性の増強は、例えば、Ｌ－アミノ酸の生合成に関与する酵素をコードする遺伝子の発現が増強するように細菌を改変する方法を挙げることができる。遺伝子の発現を増強するための方法としては、目的遺伝子を含むＤＮＡ断片を、適当なプラスミド、例えば微生物内でプラスミドの複製増殖機能を司る遺伝子を少なくとも含むプラスミドベクターに導入した組換えプラスミドを導入すること、または、目的遺伝子を含むＤＮＡ断片を染色体上で接合、転移等により多コピー化すること、及び、目的遺伝子のプロモーター領域に変異を導入すること等が挙げられる（国際公開パンフレットWO95/34672号参照）。 Also, the imparting or enhancing of the ability to produce L-amino acid can be performed by enhancing the enzyme activity by gene recombination. The enzyme activity can be enhanced by, for example, a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, a recombinant plasmid in which a DNA fragment containing a target gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication and replication function of the plasmid in a microorganism is introduced. Or making multiple copies of DNA fragments containing the gene of interest by joining, transferring, etc. on the chromosome, and introducing mutations into the promoter region of the gene of interest (International Publication Pamphlet WO95 / 34672). reference).
　上記増幅プラスミドまたは染色体上に目的遺伝子を導入する場合、これらの遺伝子を発現させるためのプロモーターは腸内細菌科において機能するものであればいかなるプロモーターであっても良く、用いる遺伝子自身のプロモーターであってもよいし、改変したものでもよい。腸内細菌科で強力に機能するプロモーターを適宜選択することや、プロモーターの－35、－10領域をコンセンサス配列に近づけることによっても遺伝子の発現量の調節が可能である。以上のような、酵素遺伝子の発現を増強する方法は、WO00/18935号パンフレット、欧州特許出願公開1010755号明細書等に記載されている。 When the target genes are introduced into the amplification plasmid or chromosome, the promoter for expressing these genes may be any promoter that functions in the family Enterobacteriaceae, and is the promoter of the gene itself used. It may be modified or modified. The expression level of the gene can also be controlled by appropriately selecting a promoter that functions strongly in the family Enterobacteriaceae, or by bringing the −35 and −10 regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in WO00 / 18935 pamphlet, European Patent Application Publication No. 1010755, and the like.
　以下、細菌にＬ－アミノ酸生産能を付与する方法、及びＬ－アミノ酸生産能が付与された細菌について例示する。 Hereinafter, a method for imparting L-amino acid-producing ability to bacteria and a bacterium imparted with L-amino acid-producing ability will be exemplified.
Ｌ－リジン生産菌
　エシェリヒア・コリのＬ－リジン生産菌又はそれを誘導するための親株の例としては、Ｌ－リジンアナログに耐性を有する変異株が挙げられる。Ｌ－リジンアナログはエシェリヒア・コリの生育を阻害するが、この阻害は、Ｌ－リジンが培地に共存するときには完全にまたは部分的に解除される。Ｌ－リジンアナログの例としては、オキサリジン、リジンヒドロキサメート、Ｓ－（２－アミノエチル）－Ｌ－システイン(AEC)、γ－メチルリジン、α－クロロカプロラクタムなどが挙げられるが、これらに限定されない。これらのリジンアナログに対して耐性を有する変異株は、エシェリヒア・コリを通常の人工変異処理に付すことによって得ることができる。Ｌ－リジンの生産に有用な細菌株の具体例としては、E. coli AJ11442 (FERM BP-1543, NRRL B-12185; 米国特許第4,346,170号参照)及びE. coli VL611等が挙げられる。これらの菌株では、アスパルトキナーゼのＬ－リジンによるフィードバック阻害が解除されている。 Examples of L-lysine-producing bacteria such as Escherichia coli L-lysine-producing bacteria or parent strains for inducing the same include mutants having resistance to L-lysine analogs. L-lysine analogs inhibit the growth of Escherichia coli, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and the like. . Mutants having resistance to these lysine analogs can be obtained by subjecting Escherichia coli to normal artificial mutation treatment. Specific examples of bacterial strains useful for the production of L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and E. coli VL611. In these strains, feedback inhibition of aspartokinase by L-lysine is released.
　WC196株は、E. coliのＬ－リジン生産菌として使用できる。この菌株は、E. coli K-12に由来するW3110株から取得された株で、352位のスレオニンをイソロイシンに置換することによりＬ－リジンによるフィードバック阻害が解除されたアスパルトキナーゼIIIをコードする変異型lysC遺伝子（米国特許第5,661,012号）でW3110株の染色体上の野生型lysC遺伝子を置き換えた後、AEC耐性を付与することにより育種された（米国特許第5,827,698号）。同株は、Escherichia coli AJ13069と命名され、1994年12月6日、工業技術院生命工学工業技術研究所（現 独立行政法人 産業技術総合研究所 特許生物寄託センター、〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託番号FERM P-14690として寄託され、1995年9月29日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-5252が付与されている（米国特許第5,827,698号）。 WC196 strain can be used as an L-lysine producing bacterium of E.coli. This strain was obtained from the W3110 strain derived from E. coli K-12 and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine. After the wild type lysC gene on the chromosome of the W3110 strain was replaced with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance (US Pat. No. 5,827,698). The stock was named Escherichia coli AJ13069. On December 6, 1994, Biotechnology Institute of Industrial Technology (currently National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, 305-8566 茨 Ibaraki, Japan Deposited as FERM P-14690 at Tsukuba City Higashi 1-chome 1-1 1 Chuo 6), transferred to international deposit based on the Budapest Treaty on September 29, 1995, and assigned FERM BP-5252 (US Pat. No. 5,827,698).
　Ｌ－リジン生産菌又はそれを誘導するための親株の例としては、Ｌ－リジン生合成系酵素の１種又は２種以上の活性が増強されている株も挙げられる。かかる酵素の例としては、ジヒドロジピコリン酸シンターゼ(dapA)、アスパルトキナーゼ(lysC)、ジヒドロジピコリン酸レダクターゼ(dapB)、ジアミノピメリン酸デカルボキシラーゼ(lysA)、ジアミノピメリン酸デヒドロゲナーゼ(ddh) (米国特許第6,040,160号)、フォスフォエノールピルビン酸カルボキシラーゼ(ppc)、アスパルテートアミノトランスフェラーゼ(aspC)、アスパルテートセミアルデヒドデヒドロゲナーゼ（asd）、ジアミノピメリン酸エピメラーゼ(dapF)、テトラヒドロジピコリン酸スクシニラーゼ(dapD)、スクシニルジアミノピメリン酸デアシラーゼ(dapE)及びアスパルターゼ(aspA) (EP 1253195 A)が挙げられるが、これらに限定されない。これらの酵素の中では、ジヒドロジピコリン酸レダクターゼ、ジアミノピメリン酸デカルボキシラーゼ、ジアミノピメリン酸デヒドロゲナーゼ、フォスフォエノールピルビン酸カルボキシラーゼ、アスパルテートアミノトランスフェラーゼ、ジアミノピメリン酸エピメラーゼ、アスパルテートセミアルデヒドデヒドロゲナーゼ、テトラヒドロジピコリン酸スクシニラーゼ、及び、スクシニルジアミノピメリン酸デアシラーゼが特に好ましい。また、親株は、エネルギー効率に関与する遺伝子(cyo) (EP 1170376 A)、ニコチンアミドヌクレオチドトランスヒドロゲナーゼをコードする遺伝子(pntAB) (米国特許第5,830,716号)、ybjE遺伝子(WO2005/073390)、または、これらの組み合わせの発現レベルが増大していてもよい。なお、カッコ内は、それらの遺伝子の略記号である。 Examples of L-lysine-producing bacteria or parent strains for inducing them include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced. Examples of such enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160). ), Phosphoenolpyruvate carboxylase (ppc), aspartate aminotransferase (aspC), aspartate semialdehyde dehydrogenase (asd), diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (dapE) and aspartase (aspA) (EP 1253195 A), but are not limited to these. Among these enzymes, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and Succinyl diaminopimelate deacylase is particularly preferred. The parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Patent No. 5,830,716), ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased. The parentheses are abbreviations for these genes.
　エシェリヒア・コリ由来の野生型ジヒドロジピコリン酸合成酵素はＬ－リジンによるフィードバック阻害を受けることが知られており、エシェリヒア・コリ由来の野生型アスパルトキナーゼはＬ－リジンによる抑制及びフィードバック阻害を受けることが知られている。したがって、dapA遺伝子及びlysC遺伝子を用いる場合、これらの遺伝子は、Ｌ－リジンによるフィードバック阻害を受けない変異型酵素をコードする変異型遺伝子、又はＬ－リジンによる抑制を受けない変異型遺伝子であることが好ましい。 Wild-type dihydrodipicolinate synthase derived from Escherichia coli is known to undergo feedback inhibition by L-lysine, and wild-type aspartokinase derived from Escherichia coli is subject to inhibition and feedback inhibition by L-lysine. It has been known. Therefore, when using the dapA gene and the lysC gene, these genes must be mutant genes that encode mutant enzymes that are not subject to feedback inhibition by L-lysine, or mutant genes that are not subject to suppression by L-lysine. Is preferred.
　Ｌ－リジンによるフィードバック阻害を受けない変異型ジヒドロジピコリン酸合成酵素をコードするＤＮＡとしては、118位のヒスチジン残基がチロシン残基に置換された配列を有するタンパク質をコードするＤＮＡが挙げられる。また、Ｌ－リジンによるフィードバック阻害を受けない変異型アスパルトキナーゼをコードするＤＮＡとしては、352位のスレオニン残基がイソロイシン残基に置換、323位のグリシン残基がアスパラギン残基に置換、318位のメチオニンがイソロイシンに置換された配列を有するAKIIIをコードするＤＮＡが挙げられる（これらの変異体については米国特許第5661012号及び第6040160号明細書参照）。変異型ＤＮＡはＰＣＲなどによる部位特異的変異法により取得することができる。 Examples of DNA encoding a mutant dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine include DNA encoding a protein having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue. As a DNA encoding a mutant aspartokinase that is not subject to feedback inhibition by L-lysine, the threonine residue at position 352 is replaced with an isoleucine residue, the glycine residue at position 323 is replaced with an asparagine residue, 318 Examples include DNA encoding AKIII having a sequence in which the methionine at the position is replaced with isoleucine (see US Pat. Nos. 5,610,010 and 6,040,160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.
　なお、変異型変異型ジヒドロジピコリン酸合成酵素をコードする変異型dapA及び変異型アスパルトキナーゼをコードする変異型lysCを含むプラスミドとして、広宿主域プラスミドRSFD80、pCAB1、pCABD2が知られている（米国特許第6040160号明細書）。RSFD80で形質転換されたエシェリヒア・コリ JM109株（米国特許第6040160号明細書）は、AJ12396と命名され、同株は1993年10月28日に通産省工業技術院生命工学工業技術研究所（現 独立行政法人 産業技術総合研究所 特許生物寄託センター）に受託番号FERM P-13936として寄託され、1994年11月1日にブダペスト条約に基づく国際寄託に移管され、FERM BP-4859の受託番号のもとで寄託されている。RSFD80は、AJ12396株から、公知の方法によって取得することができる。 Wide host range plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant mutant dihydrodipicolinate synthase and mutant lysC encoding mutant aspartokinase (USA) Patent No. 6040160). Escherichia coli strain JM109 transformed with RSFD80 (US Pat. No. 6,040,160) was named AJ12396, and this strain was established on October 28, 1993 at the Institute of Biotechnology, Ministry of International Trade and Industry Deposited under the accession number FERM P-13936 at the administrative body (National Institute of Advanced Industrial Science and Technology Patent Biological Depositary), transferred to an international deposit under the Budapest Treaty on November 1, 1994, under the FERM BP-4859 accession number Deposited at RSFD80 can be obtained from AJ12396 strain by a known method.
　Ｌ－リジン生産菌又はそれを誘導するための親株の例としては、Ｌ－リジンの生合成経路から分岐してＬ－リジン以外の化合物を生成する反応を触媒する酵素の活性が低下または欠損している株も挙げられる。Ｌ－リジンの生合成経路から分岐してＬ－リジン以外の化合物を生成する反応を触媒する酵素の例としては、ホモセリンデヒドロゲナーゼ、リジンデカルボキシラーゼ(米国特許第5,827,698号)、及び、リンゴ酸酵素(WO2005/010175)が挙げられる。ここで、リジンデカルボキシラーゼ活性を低下または欠損させるためには、リジンデカルボキシラーゼをコードするcadA遺伝子とldcC遺伝子の両方の発現を低下させることが好ましい（国際公開第WO2006/038695号パンフレット）。 Examples of L-lysine-producing bacteria or parent strains for deriving the same include reduction or loss of the activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthetic pathway to produce compounds other than L-lysine. There are also stocks. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175). Here, in order to reduce or eliminate the lysine decarboxylase activity, it is preferable to reduce the expression of both the cadA gene and the ldcC gene encoding lysine decarboxylase (International Publication No. WO2006 / 038695).
　cadA遺伝子とldcC遺伝子が破壊された菌株としては、エシェリヒア・コリWC196LC（WC196ΔcadAΔldcC）（US5,827,698、US20060160191）が挙げられる。WC196LC株は、AJ110692と命名され、2008年10月7日に独立行政法人 産業技術総合研究所 特許生物寄託センター（〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に国際寄託され、受託番号FERM BP-11027が付与されている。 Examples of strains in which the cadA gene and the ldcC gene are disrupted include Escherichia coli WC196LC (WC196ΔcadAΔldcC) (US 5,827,698, US20060160191). The WC196LC strain was named AJ110692 and was internationally established on October 7, 2008 by the National Institute of Advanced Industrial Science and Technology (AIST), the Patent Biological Deposit Center (1-6 Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, 305-8566). Deposited and given accession number FERM BP-11027.
Ｌ－スレオニン生産菌
　Ｌ－スレオニン生産菌又はそれを誘導するための親株の例としては、E. coli TDH-6/pVIC40 (VKPM B-3996) (米国特許第5,175,107号、米国特許第5,705,371号)、E. coli 472T23/pYN7 (ATCC 98081) (米国特許第5,631,157号)、E. coli NRRL-21593 (米国特許第5,939,307号)、E. coli FERM BP-3756 (米国特許第5,474,918号)、E. coli FERM BP-3519及びFERM BP-3520 (米国特許第5,376,538号)、E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978))、E. coli VL643及びVL2055 (EP 1149911 A)などが挙げられるが、これらに限定されない。 Examples of L-threonine-producing bacteria L-threonine-producing bacteria or parent strains for inducing them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371) E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli. coli FERM BP-3519 and FERM BP-3520 (US Pat.No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A) and the like, but are not limited thereto.
　TDH-6株はthrC遺伝子を欠損し、スクロース資化性であり、また、そのilvA遺伝子がリーキー(leaky)変異を有する。この株はまた、rhtA遺伝子に、高濃度のスレオニンまたはホモセリンに対する耐性を付与する変異を有する。B-3996株は、RSF1010由来ベクターに、変異thrA遺伝子を含むthrA*BCオペロンを挿入したプラスミドpVIC40を保持する。この変異thrA遺伝子は、スレオニンによるフィードバック阻害が実質的に解除されたアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする。B-3996株は、1987年11月19日、オールユニオン・サイエンティフィック・センター・オブ・アンチビオティクス(Nagatinskaya Street 3-A, 117105 Moscow, Russia)に、受託番号RIA 1867で寄託されている。この株は、また、1987年4月7日、ルシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオルガニズムズ(VKPM)(GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation)に、受託番号B-3996で寄託されている。 The TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 was deposited on 19 November 1987 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) under the deposit number RIA 1867. . The strain was also assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation) on April 7, 1987. Deposited at -3996.
　E. coli VKPM B-5318 (EP 0593792B)も、Ｌ－スレオニン生産菌又はそれを誘導するための親株として使用できる。B-5318株は、イソロイシン非要求性であり、プラスミドpVIC40中のスレオニンオペロンの制御領域が、温度感受性ラムダファージC1リプレッサー及びPRプロモーターにより置換されている。VKPM B-5318は、1990年5月3日、ルシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオルガニズムズ(VKPM) (GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation)に、受託番号VKPM B-5318で寄託されている。 E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned on May 3, 1990 to Lucian National Collection of Industrial Microorganisms (VKPM) (GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation). Deposited at B-5318.
　好ましくは、本発明に用いる細菌は、さらに、下記の遺伝子の１種以上の発現が増大するように改変されたものである。
－スレオニンによるフィードバック阻害に耐性のアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする変異thrA遺伝子
－ホモセリンキナーゼをコードするthrB遺伝子
－スレオニンシンターゼをコードするthrC遺伝子
－推定トランスメンブランタンパク質をコードするrhtA遺伝子
－アスパルテート－β－セミアルデヒドデヒドロゲナーゼをコードするasd遺伝子
－アスパルテートアミノトランスフェラーゼ（アスパルテートトランスアミナーゼ）をコードするaspC遺伝子 Preferably, the bacterium used in the present invention is further modified so that expression of one or more of the following genes is increased.
-Aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine-Mutant thrA gene encoding homoserine kinase-ThrC gene encoding threonine synthase-rhtA gene encoding putative transmembrane protein-Aspartate- asd gene encoding β-semialdehyde dehydrogenase-aspC gene encoding aspartate aminotransferase (aspartate transaminase)
　E. coliのアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードするthrA遺伝子は明らかにされている(ヌクレオチド番号337～2799, GenBank accession NC_000913.2, gi: 49175990)。thrA遺伝子は、E. coli K-12の染色体において、thrL遺伝子とthrB遺伝子との間に位置する。E. coliのホモセリンキナーゼをコードするthrB遺伝子は明らかにされている(ヌクレオチド番号2801～3733, GenBank accession NC_000913.2, gi: 49175990)。thrB遺伝子は、E. coli K-12の染色体において、thrA遺伝子とthrC遺伝子との間に位置する。E. coliのスレオニンシンターゼをコードするthrC遺伝子は明らかにされている(ヌクレオチド番号3734～5020, GenBank accession NC_000913.2, gi: 49175990)。thrC遺伝子は、E. coli K-12の染色体において、thrB遺伝子とyaaXオープンリーディングフレームとの間に位置する。これら三つの遺伝子は、全て、単一のスレオニンオペロンとして機能する。スレオニンオペロンの発現を増大させるには、転写に影響するアテニュエーター領域を、好ましくは、オペロンから除去する(WO2005/049808, WO2003/097839)。 The thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding E. セ リ ン coli homoserine kinase has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 99049175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon. To increase the expression of the threonine operon, the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).
　スレオニンによるフィードバック阻害に耐性のアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする変異thrA遺伝子、ならびに、thrB遺伝子及びthrC遺伝子は、スレオニン生産株E. coli VKPM B-3996に存在する周知のプラスミドpVIC40から一つのオペロンとして取得できる。プラスミドpVIC40の詳細は、米国特許第5,705,371号に記載されている。 The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.
　rhtA遺伝子は、グルタミン輸送系の要素をコードするglnHPQ オペロンに近いE. coli染色体の18分に存在する。rhtA遺伝子は、ORF1 (ybiF遺伝子, ヌクレオチド番号764～1651, GenBank accession number AAA218541, gi:440181)と同一であり、pexB遺伝子とompX遺伝子との間に位置する。ORF1によりコードされるタンパク質を発現するユニットは、rhtA遺伝子と呼ばれている(rht: ホモセリン及びスレオニンに耐性)。また、高濃度のスレオニンまたはホモセリンに対する耐性を付与するrhtA23変異は、ATG開始コドンに対して-1位のG→A置換であることが判明している(ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A)。 The rhtA gene is present on the 18th minute of the E. 染色体 coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant to homoserine and threonine). The rhtA23 mutation conferring resistance to high concentrations of threonine or homoserine has been found to be a G → A substitution at position -1 relative to the ATG initiation codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).
　E. coliのasd遺伝子は既に明らかにされており(ヌクレオチド番号3572511～3571408, GenBank accession NC_000913.1, gi:16131307)、その遺伝子の塩基配列に基づいて作製されたプライマーを用いるPCRにより得ることができる(White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189参照)。他の微生物のasd遺伝子も同様に得ることができる。 The E. coli asd gene has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene. (See White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189). The asd gene of other microorganisms can be obtained similarly.
　また、E. coliのaspC遺伝子も既に明らかにされており(ヌクレオチド番号983742～984932, GenBank accession NC_000913.1, gi:16128895)、PCRにより得ることができる。他の微生物のaspC遺伝子も同様に得ることができる。 In addition, the aspC gene of E.coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895) and can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.
Ｌ－システイン生産菌
　Ｌ－システイン生産菌又はそれを誘導するための親株の例としては、フィードバック阻害耐性のセリンアセチルトランスフェラーゼをコードする複数種のcysEアレルで各々形質転換されたE. coli JM15(米国特許第6,218,168号、ロシア特許出願第2003121601号)、細胞に毒性の物質を排出するのに適したタンパク質をコードする過剰発現遺伝子を有するE. coli W3110 (米国特許第5,972,663号)、システインデスルフォヒドラーゼ活性が低下したE. coli株 (JP11155571A2)、cysB遺伝子によりコードされる正のシステインレギュロンの転写制御因子の活性が上昇したE. coli W3110 (WO0127307A1)などの菌株が挙げられるが、これらに限定されない。 Examples of L-cysteine-producing bacteria L-cysteine-producing bacteria or parent strains for deriving L-cysteine-producing bacteria include E. coli JM15 (US) each transformed with a plurality of cysE alleles encoding a feedback inhibition resistant serine acetyltransferase. (Patent No. 6,218,168, Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfide Examples include, but are not limited to, strains such as E. coli strains (JP11155571A2) that have reduced enzyme activity and E. coli W3110 (WO0127307A1) that have increased activity of the transcriptional regulator of the positive cysteine regulon encoded by the cysB gene. Not.
Ｌ－ロイシン生産菌
　Ｌ－ロイシン生産菌又はそれを誘導するための親株の例としては、ロイシン耐性のE. coli株 (例えば、57株 (VKPM B-7386, 米国特許第6,124,121号))またはβ－２－チエニルアラニン、３－ヒドロキシロイシン、４－アザロイシン、5,5,5-トリフルオロロイシンなどのロイシンアナログ耐性のE. coli株(特公昭62-34397号及び特開平8-70879号)、WO96/06926に記載された遺伝子工学的方法で得られたE. coli株、E. coli H-9068 (特開平8-70879号)などの株が挙げられるが、これらに限定されない。 Examples of L-leucine-producing bacteria L-leucine-producing bacteria or parent strains for deriving them include leucine-resistant E. coli strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or β E. coli strains resistant to leucine analogs such as -2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP-B-62-34397 and JP-A-8-70879), Examples include, but are not limited to, E. coli strains obtained by the genetic engineering method described in WO96 / 06926, E. coli H-9068 (JP-A-8-70879), and the like.
　本発明に用いる細菌は、Ｌ－ロイシン生合成に関与する遺伝子の１種以上の発現が増大されることにより改良されていてもよい。このような遺伝子の例としては、好ましくはＬ－ロイシンによるフィードバック阻害が解除されたイソプロピルマレートシンターゼをコードする変異leuA遺伝子(米国特許第6,403,342号)に代表される、leuABCDオペロンの遺伝子が挙げられる。さらに、本発明に用いる細菌は、細菌の細胞からＬ－アミノ酸を排出するタンパク質をコードする遺伝子の１種以上の発現が増大されることにより改良されていてもよい。このような遺伝子の例としては、b2682遺伝子及びb2683遺伝子(ygaZH遺伝子) (EP 1239041 A2)が挙げられる。 The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis. As an example of such a gene, a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase which is preferably desensitized to feedback inhibition by L-leucine can be mentioned. . Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (EP 1239041 A2).
Ｌ－ヒスチジン生産菌
　Ｌ－ヒスチジン生産菌又はそれを誘導するための親株の例としては、E. coli 24株 (VKPM B-5945, RU2003677)、E. coli 80株 (VKPM B-7270, RU2119536)、E. coli NRRL B-12116 - B12121 (米国特許第4,388,405号)、E. coli H-9342 (FERM BP-6675)及びH-9343 (FERM BP-6676) (米国特許第6,344,347号)、E. coli H-9341 (FERM BP-6674) (EP1085087)、E. coli AI80/pFM201 (米国特許第6,258,554号)などの株が挙げられるが、これらに限定されない。 Examples of L-histidine-producing bacteria L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, RU2003677), E. coli 80 strain (VKPM B-7270, RU2119536) E. coli NRRL B-12116-B12121 (U.S. Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat.No. 6,344,347), E. coli. Examples include, but are not limited to, E. coli H-9341 (FERM BP-6674) (EP1085087) and E. coli AI80 / pFM201 (US Pat. No. 6,258,554).
　Ｌ－ヒスチジン生産菌又はそれを誘導するための親株の例としては、Ｌ－ヒスチジン生合成系酵素をコードする遺伝子の１種以上の発現が増大した株も挙げられる。かかる遺伝子の例としては、ATPフォスフォリボシルトランスフェラーゼ遺伝子(hisG)、フォスフォリボシルAMPサイクロヒドロラーゼ遺伝子(hisI)、フォスフォリボシル-ATPピロフォスフォヒドロラーゼ遺伝子(hisI)、フォスフォリボシルフォルミミノ-5-アミノイミダゾールカルボキサミドリボタイドイソメラーゼ遺伝子(hisA)、アミドトランスフェラーゼ遺伝子(hisH)、ヒスチジノールフォスフェイトアミノトランスフェラーゼ遺伝子(hisC)、ヒスチジノールフォスファターゼ遺伝子(hisB)、ヒスチジノールデヒドロゲナーゼ遺伝子(hisD)などが挙げられる。 Examples of L-histidine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.
　hisG及びhisBHAFIにコードされるＬ－ヒスチジン生合成系酵素はＬ－ヒスチジンにより阻害されることが知られており、従って、Ｌ－ヒスチジン生産能は、ATPフォスフォリボシルトランスフェラーゼ遺伝子(hisG)にフィードバック阻害への耐性を付与する変異を導入することにより効率的に増大させることができる(ロシア特許第2003677号及び第2119536号)。 L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).
　Ｌ－ヒスチジン生産能を有する株の具体例としては、Ｌ－ヒスチジン生合成系酵素をコードするDNAを保持するベクターを導入したE. coli FERM P-5038及びFERM P-5048 (特開昭56-005099号)、アミノ酸輸送の遺伝子を導入したE. coli株(EP1016710A)、スルファグアニジン、DL-1,2,4-トリアゾール-3-アラニン及びストレプトマイシンに対する耐性を付与したE. coli 80株(VKPM B-7270, ロシア特許第2119536号)などの菌株が挙げられる。 Specific examples of strains capable of producing L-histidine include E. coli FERM P-5038 and FERM P-5048 introduced with a vector carrying a DNA encoding an L-histidine biosynthetic enzyme (JP-A-56-56). No. 005099), E.licoli strain (EP1016710A) introduced with a gene for amino acid transport, E. coli 80 strain (VKPM) with resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin B-7270, (Russian Patent No. 2119536) and the like.
Ｌ－グルタミン酸生産菌
　Ｌ－グルタミン酸生産菌又はそれを誘導するための親株の例としては、E. coli VL334thrC+ (EP 1172433)などの菌株が挙げられるが、これらに限定されない。E. coli VL334 (VKPM B-1641)は、thrC遺伝子及びilvA遺伝子に変異を有するＬ－イソロイシン及びＬ－スレオニン要求性株である(米国特許第4,278,765号)。thrC遺伝子の野生型アレルは、野生型E. coli K12株 (VKPM B-7)の細胞で増殖したバクテリオファージP1を用いる一般的形質導入法により導入された。この結果、Ｌ－イソロイシン要求性のＬ－グルタミン酸生産菌VL334thrC+ (VKPM B-8961) が得られた。 Examples of L-glutamic acid-producing bacteria L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, E. coli VL334thrC + (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7). As a result, L-isoleucine-requiring L-glutamic acid-producing bacterium VL334thrC + (VKPM B-8961) was obtained.
　Ｌ－グルタミン酸生産菌又はそれを誘導するための親株の例としては、Ｌ－グルタミン酸生合成系酵素をコードする遺伝子の１種以上の発現が増大した株が挙げられるが、これらに限定されない。かかる遺伝子の例としては、グルタメートデヒドロゲナーゼ（gdhA）、グルタミンシンテターゼ(glnA)、グルタメートシンテターゼ(gltAB)、イソシトレートデヒドロゲナーゼ(icdA)、アコニテートヒドラターゼ(acnA, acnB)、シトレートシンターゼ(gltA)、フォスフォエノールピルベートカルボシラーゼ(ppc)、ピルベートデヒドロゲナーゼ(aceEF, lpdA)、ピルベートキナーゼ(pykA, pykF)、フォスフォエノールピルベートシンターゼ(ppsA)、エノラーゼ(eno)、フォスフォグリセロムターゼ(pgmA, pgmI)、フォスフォグリセレートキナーゼ(pgk)、グリセルアルデヒド-3-フォスフェートデヒドロゲナーゼ(gapA)、トリオースフォスフェートイソメラーゼ(tpiA)、フルクトースビスフォスフェートアルドラーゼ(fbp)、フォスフォフルクトキナーゼ（pfkA, pfkB）、グルコースフォスフェートイソメラーゼ(pgi)などが挙げられる。 Examples of L-glutamic acid-producing bacteria or parent strains for deriving the same include, but are not limited to, strains in which expression of one or more genes encoding L-glutamic acid biosynthetic enzymes are increased. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Phosphoenol pyruvate carbosylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenol pyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase ( pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (PfkA, pfkB), glucose phosphine Toisomeraze (pgi), and so forth.
　シトレートシンテターゼ遺伝子、フォスフォエノールピルベートカルボキシラーゼ遺伝子、及び／またはグルタメートデヒドロゲナーゼ遺伝子の発現が増大するように改変された株の例としては、EP1078989A、EP955368A及びEP952221Aに開示されたものが挙げられる。 Examples of strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A and EP952221A.
　Ｌ－グルタミン酸生産菌又はそれを誘導するための親株の例としては、Ｌ－グルタミン酸の生合成経路から分岐してＬ－グルタミン酸以外の化合物の合成を触媒する酵素の活性が低下または欠損している株も挙げられる。このような酵素の例としては、イソシトレートリアーゼ(aceA)、α-ケトグルタレートデヒドロゲナーゼ(sucA)、フォスフォトランスアセチラーゼ(pta)、アセテートキナーゼ(ack)、アセトヒドロキシ酸シンターゼ(ilvG)、アセトラクテートシンターゼ(ilvI)、フォルメートアセチルトランスフェラーゼ(pfl)、ラクテートデヒドロゲナーゼ(ldh)、グルタメートデカルボキシラーゼ(gadAB)、γ-グルタミル転移酵素(ggt)、γ-グルタミルシステイン合成酵素(gshA)、γ-グルタミン酸プトレシン合成酵素(ycjK)などが挙げられる。α-ケトグルタレートデヒドロゲナーゼ活性が欠損した、または、α-ケトグルタレートデヒドロゲナーゼ活性が低下したエシェリヒア・コリ、及び、それらの取得方法は米国特許第5,378,616 号及び第5,573,945号に記載されている。 Examples of L-glutamic acid-producing bacteria or parent strains for deriving the same are those in which the activity of an enzyme that catalyzes the synthesis of compounds other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or absent Stocks are also mentioned. Examples of such enzymes include isocitrate triase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), γ-glutamyltransferase (ggt), γ-glutamylcysteine synthetase (gshA), γ-glutamic acid And putrescine synthase (ycjK). Escherichia coli lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945.
　具体例としては下記のものが挙げられる。
E. coli W3110sucA::Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881) Specific examples include the following.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)
　E. coli W3110sucA::Kmr は、E. coli W3110のα-ケトグルタレートデヒドロゲナーゼ遺伝子（以下、「sucA遺伝子」ともいう）を破壊することにより得られた株である。この株は、α-ケトグルタレートデヒドロゲナーゼを完全に欠損している。 E. coli W3110sucA :: Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter also referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.
　Ｌ－グルタミン酸生産菌の他の例としては、アスパラギン酸代謝拮抗物質に耐性を有するエシェリヒア・コリが挙げられる。このような株は、α-ケトグルタレートデヒドロゲナーゼを欠損していてもよく、例えば、E. coli AJ13199 (FERM BP-5807) (米国特許第5.908,768号)、さらにＬ－グルタミン酸分解能が低下したFFRM P-12379(米国特許第5,393,671号); AJ13138 (FERM BP-5565) (米国特許第6,110,714号)などの菌株が挙げられる。 Other examples of L-glutamic acid-producing bacteria include Escherichia coli that has resistance to an aspartic acid antimetabolite. Such a strain may be deficient in α-ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5.908,768) and further reduced L-glutamate resolution. Examples include strains such as FFRM P-12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714).
　パントアエ・アナナティスのＬ－グルタミン酸生産菌の例としては、パントエア・アナナティスAJ13355株が挙げられる。同株は、静岡県磐田市の土壌から、低pHでＬ－グルタミン酸及び炭素源を含む培地で増殖できる株として分離された株である。パントエア・アナナティスAJ13355は、1998年2月19日に、独立行政法人 産業技術総合研究所 特許生物寄託センター（住所　〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に、受託番号FERM P-16644として寄託され、1999年1月11日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-6614が付与されている。尚、同株は、分離された当時はエンテロバクター・アグロメランス（Enterobacter agglomerans）と同定され、エンテロバクター・アグロメランスAJ13355として寄託されたが、近年16S rRNAの塩基配列解析などにより、パントエア・アナナティス（Pantoea ananatis）に再分類されている。 An example of an L-glutamic acid-producing bacterium of Pantoae ananatis is Pantoea ananatis AJ13355 strain. This strain was isolated from the soil of Iwata City, Shizuoka Prefecture as a strain that can grow on a medium containing L-glutamic acid and a carbon source at a low pH. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center (address: 1st, 1st, 1st, 1-chome, Tsukuba, Ibaraki, Japan, 305-8566). Deposited under the number FERM644P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999 and given the accession number FERM BP-6614. The strain was identified as Enterobacter agglomerans at the time of its isolation and deposited as Enterobacter グ ロ agglomerans AJ13355, but recently, Pantoea ananatis (Pantoea ananatis) was analyzed by 16S rRNA sequence analysis. ).
　また、パントアエ・アナナティスのＬ－グルタミン酸生産菌として、α-ケトグルタレートデヒドロゲナーゼ（αKGDH）活性が欠損した、または、αKGDH活性が低下したパントエア属に属する細菌が挙げられる。このような株としては、AJ13355株のαKGDH-E1サブユニット遺伝子（sucA）を欠損させたAJ13356(米国特許第6,331,419号)、及びAJ13355株から粘液質低生産変異株として選択されたSC17株由来のsucA遺伝子欠損株であるSC17sucA(米国特許第6,596,517号)がある。AJ13356は、1998年2月19日、工業技術院生命工学工業技術研究所（現 独立行政法人 産業技術総合研究所 特許生物寄託センター、〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託番号FERM P-16645として寄託され、1999年1月11日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-6616が付与されている。AJ13355及びAJ13356は、上記寄託機関にEnterobacter agglomeransとして寄託されているが、本明細書では、Pantoea ananatisとして記載する。また、SC17sucA株は、ブライベートナンバーAJ417株が付与され、2004年2月26日に産業技術総合研究所特許生物寄託センターに受託番号FERM BP-08646として寄託されている。 Further, examples of L-glutamic acid-producing bacteria of Pantoae ananatis include bacteria belonging to the genus Pantoea in which α-ketoglutarate dehydrogenase (αKGDH) activity is deficient or αKGDH activity is reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the αKGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected from AJ13355 strain as a low mucus production mutant. There is SC17sucA (US Pat. No. 6,596,517) which is a gene-deficient strain. AJ13356 was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain has been assigned the private number AJ417, and was deposited at the National Institute of Advanced Industrial Science and Technology as the accession number FERM BP-08646 on February 26, 2004.
　さらに、パントエア・アナナティスのＬ－グルタミン酸生産菌として、SC17sucA/RSFCPG+pSTVCB株、AJ13601株、NP106株、及びNA1株が挙げられる。SC17sucA/RSFCPG+pSTVCB株は、SC17sucA株に、エシェリヒア・コリ由来のクエン酸シンターゼ遺伝子（gltA）、ホスホエノールピルビン酸カルボキシラーゼ遺伝子（ppsA）、およびグルタメートデヒドロゲナーゼ遺伝子（gdhA）を含むプラスミドRSFCPG、並びに、ブレビバクテリウム・ラクトファーメンタム由来のクエン酸シンターゼ遺伝子（gltA）を含むプラスミドpSTVCBを導入して得た株である。AJ13601株は、このSC17sucA/RSFCPG+pSTVCB株から低pH下で高濃度のＬ－グルタミン酸に耐性を示す株として選択された株である。また、NP106株は、AJ13601株からプラスミドRSFCPG+pSTVCBを脱落させた株である。AJ13601株は、1999年8月18日に、独立行政法人 産業技術総合研究所 特許生物寄託センター（〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託番号FERM P-17516として寄託され、2000年7月6日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-7207が付与されている。 Further, as L-glutamic acid-producing bacteria of Pantoea ananatis, SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain can be mentioned. The SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppsA), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (305-1856, Ibaraki, Japan, 1st-chome, 1st-chome, 1st-chome, 1st-centre, 6th). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.
Ｌ－フェニルアラニン生産菌
　Ｌ－フェニルアラニン生産菌又はそれを誘導するための親株の例としては、E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197)、変異型pheA34遺伝子を保持するE. coli HW1089 (ATCC 55371) (米国特許第 5,354,672号)、E. coli MWEC101-b (KR8903681)、E.coli NRRL B-12141, NRRL B-12145, NRRL B-12146及びNRRL B-12147 (米国特許第4,407,952号)などの菌株が挙げられるが、これらに限定されない。また、親株として、E. coli K-12 [W3110 (tyrA)/pPHAB] (FERM BP-3566)、E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659)、E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662)、及びAJ 12604と命名されたE. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] (FERM BP-3579)も使用できる(EP 488424 B1)。さらに、yedA遺伝子またはyddG遺伝子にコードされるタンパク質の活性が増大したエシェリヒア・コリのＬ－フェニルアラニン生産菌も使用できる(米国特許出願公開2003/0148473 A1及び2003/0157667 A1)。 Examples of L-phenylalanine producing bacteria L-phenylalanine producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197), E carrying a mutant pheA34 gene coli HW1089 (ATCC 55371) (US Pat.No. 5,354,672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patents) No. 4,407,952) and the like, but is not limited thereto. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) designated AJ 12604 Can be used (EP 488424 B1). Furthermore, Escherichia coli L-phenylalanine-producing bacteria having increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).
Ｌ－トリプトファン生産菌
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、trpS遺伝子によりコードされるトリプトファニル-tRNAシンテターゼの機能が欠損したE. coli JP4735/pMU3028 (DSM10122)及びJP6015/pMU91 (DSM10123) (米国特許第5,756,345号)、セリンによるフィードバック阻害を受けないフォスフォグリセリレートデヒドロゲナーゼをコードするserAアレル及びトリプトファンによるフィードバック阻害を受けないアントラニレートシンターゼをコードするtrpEアレルを有するE. coli SV164 (pGH5) (米国特許第6,180,373号)、トリプトファナーゼが欠損したE. coli AGX17 (pGX44) (NRRL B-12263)及びAGX6(pGX50)aroP (NRRL B-12264) (米国特許第4,371,614号)、フォスフォエノールピルビン酸生産能が増大したE. coli AGX17/pGX50,pACKG4-pps (WO9708333, 米国特許第6,319,696号)などの菌株が挙げられるが、これらに限定されない。yedA遺伝子またはyddG遺伝子にコードされるタンパク質の活性が増大したエシェリヒア・コリのＬ－トリプトファン生産菌も使用できる(米国特許出願公開2003/0148473 A1及び2003/0157667 A1)。 Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / which lack the function of tryptophanyl-tRNA synthetase encoded by the trpS gene. pMU91 (DSM10123) (U.S. Pat.No. 5,756,345), E having a serA allele encoding phosphoglycerate dehydrogenase that is not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase that is not subject to feedback inhibition by tryptophan. coli SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) ), E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased phosphoenolpyruvate production capacity Strains including but not limited to. Escherichia coli L-tryptophan-producing bacteria having increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、アントラニレートシンターゼ(trpE)、フォスフォグリセレートデヒドロゲナーゼ(serA)、及び、トリプトファンシンターゼ(trpAB)から選ばれる酵素の活性の一種以上が増大した株も挙げられる。アントラニレートシンターゼ及びフォスフォグリセレートデヒドロゲナーゼは共にＬ－トリプトファン及びＬ－セリンによるフィードバック阻害を受けるので、フィードバック阻害を解除する変異をこれらの酵素に導入してもよい。このような変異を有する株の具体例としては、脱感作型アントラニレートシンターゼを保持するE. coli SV164、及び、フィードバック阻害が解除されたフォスフォグリセレートデヒドロゲナーゼをコードする変異serA遺伝子を含むプラスミドpGH5 (WO 94/08031)をE. coli SV164に導入することにより得られた形質転換株が挙げられる。 Examples of L-tryptophan-producing bacteria or parent strains for inducing the same include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and a kind of activity of an enzyme selected from tryptophan synthase (trpAB) Also included are strains with increased above. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase with desensitized feedback inhibition Examples include a transformant obtained by introducing the plasmid pGH5 (WO 94/08031) into E.coli SV164.
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、阻害解除型アントラニレートシンターゼをコードする遺伝子を含むトリプトファンオペロンが導入された株(特開昭57-71397号, 特開昭62-244382号, 米国特許第4,371,614号)も挙げられる。さらに、トリプトファンオペロン(trpBA)中のトリプトファンシンターゼをコードする遺伝子の発現を増大させることによりＬ－トリプトファン生産能を付与してもよい。トリプトファンシンターゼは、それぞれtrpA及びtrpB遺伝子によりコードされるα及びβサブユニットからなる。さらに、イソシトレートリアーゼ－マレートシンターゼオペロンの発現を増大させることによりＬ－トリプトファン生産能を改良してもよい(WO2005/103275)。 Examples of L-tryptophan-producing bacteria or parent strains for deriving the same include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open Nos. 57-71397 and 1994 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO2005 / 103275).
Ｌ－プロリン生産菌
　Ｌ－プロリン生産菌又はそれを誘導するための親株の例としては、ilvA遺伝子が欠損し、Ｌ－プロリンを生産できるE. coli 702ilvA (VKPM B-8012) (EP 1172433)などの菌株が挙げられるが、これらに限定されない。 Examples of L-proline-producing bacteria L-proline-producing bacteria or parent strains for deriving them include E. coli 702ilvA (VKPM B-8012) (EP 1172433) that lacks the ilvA gene and can produce L-proline However, it is not limited to these.
　本発明に用いる細菌は、Ｌ－プロリン生合成に関与する遺伝子の一種以上の発現を増大することにより改良してもよい。Ｌ－プロリン生産菌に好ましい遺伝子の例としては、Ｌ－プロリンによるフィードバック阻害が解除されたグルタメートキナーゼをコードするproB遺伝子(ドイツ特許第3127361号)が挙げられる。さらに、本発明に用いる細菌は、細菌の細胞からＬ－アミノ酸を排出するタンパク質をコードする遺伝子の一種以上の発現が増大することにより改良してもよい。このような遺伝子としては、b2682 遺伝子及びb2683遺伝子(ygaZH遺伝子) (EP1239041 A2)が挙げられる。 The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) gene (EP1239041 gene A2).
　Ｌ－プロリン生産能を有するエシェリヒア・コリの例としては、NRRL B-12403及びNRRL B-12404 (英国特許第2075056号)、VKPM B-8012 (ロシア特許出願2000124295)、及び、ドイツ特許第3127361号に記載のプラスミド変異体、又は、Bloom F.R. et al (The 15th Miami winter symposium, 1983, p.34)に記載のプラスミド変異体を持つE. coli 株等が挙げられる。 Examples of Escherichia coli having L-proline-producing ability include NRRL B-12403 and NRRL B-12404 英国 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), and German Patent No. 3127361 Or the E. coli strain having the plasmid variant described in Bloom FR et al (The 15th Miami winter symposium, 1983, p.34).
Ｌ－アルギニン生産菌
　Ｌ－アルギニン生産菌又はそれを誘導するための親株の例としては、E. coli 237株 (VKPM B-7925) (米国特許出願公開2002/058315 A1)、及び、変異N-アセチルグルタメートシンターゼを保持するその誘導株(ロシア特許出願第2001112869号)、E. coli 382株 (VKPM B-7926) (EP1170358A1)、N-アセチルグルタメートシンテターゼをコードするargA遺伝子が導入されたアルギニン生産株(EP1170361A1)などの菌株が挙げられるが、これらに限定されない。 Examples of L-arginine producing bacteria L-arginine producing bacteria or parent strains for inducing them include E. coli strain 237 (VKPM B-7925) (US Patent Application Publication 2002/058315 A1) and mutant N- Derivatives carrying acetylglutamate synthase (Russian patent application No. 2001112869), E. coli 382 strain (VKPM B-7926) (EP1170358A1), arginine producing strain introduced with argA gene encoding N-acetylglutamate synthetase Examples include, but are not limited to, (EP1170361A1).
　Ｌ－アルギニン生産菌又はそれを誘導するための親株の例としては、Ｌ－アルギニン生合成系酵素をコードする遺伝子の１種以上の発現が増大した株も挙げられる。かかる遺伝子の例としては、N-アセチルグルタミルフォスフェートレダクターゼ遺伝子(argC)、オルニチンアセチルトランスフェラーゼ遺伝子(argJ)、N-アセチルグルタメートキナーゼ遺伝子(argB)、アセチルオルニチントランスアミナーゼ遺伝子(argD)、オルニチンカルバモイルトランスフェラーゼ遺伝子(argF)、アルギノコハク酸シンテターゼ遺伝子(argG)、アルギノコハク酸リアーゼ遺伝子(argH)、カルバモイルフォスフェートシンテターゼ遺伝子(carAB)が挙げられる。 Examples of L-arginine-producing bacteria or parent strains for inducing them include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoylphosphate synthetase gene (carAB).
Ｌ－バリン生産菌
　Ｌ－バリン生産菌又はそれを誘導するための親株の例としては、ilvGMEDAオペロンを過剰発現するように改変された株(米国特許第5,998,178号)が挙げられるが、これらに限定されない。アテニュエーションに必要なilvGMEDAオペロンの領域を除去し、生産されるＬ－バリンによりオペロンの発現が減衰しないようにすることが好ましい。さらに、オペロンのilvA遺伝子が破壊され、スレオニンデアミナーゼ活性が減少することが好ましい。 Examples of L-valine producing bacteria L-valine producing bacteria or parent strains for inducing them include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. It is preferable to remove the region of the ilvGMEDA operon necessary for attenuation so that the expression of the operon is not attenuated by the produced L-valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.
　Ｌ－バリン生産菌又はそれを誘導するための親株の例としては、アミノアシルt-RNAシンテターゼの変異を有する変異株(米国特許第5,658,766号)も挙げられる。例えば、イソロイシンtRNAシンテターゼをコードするileS 遺伝子に変異を有するE. coli VL1970が使用できる。E. coli VL1970は、1988年6月24日、ルシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオルガニズムズ(VKPM) (GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation)に、受託番号VKPM B-4411で寄託されている。
　さらに、生育にリポ酸を要求する、及び／または、H+-ATPaseを欠失している変異株(WO96/06926)を親株として用いることができる。 Examples of L-valine-producing bacteria or parent strains for deriving them also include mutants having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was assigned to GNII genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russian Federation on June 24, 1988, on the Lucian National Collection of Industrial Microorganisms (VKPM). Deposited at B-4411.
Furthermore, a mutant strain (WO96 / 06926) that requires lipoic acid for growth and / or lacks H + -ATPase can be used as a parent strain.
Ｌ－イソロイシン生産菌
　Ｌ－イソロイシン生産菌又はそれを誘導するための親株の例としては、６－ジメチルアミノプリンに耐性を有する変異株(特開平5-304969号)、チアイソロイシン、イソロイシンヒドロキサメートなどのイソロイシンアナログに耐性を有する変異株、さらにDL-エチオニン及び／またはアルギニンヒドロキサメートに耐性を有する変異株(特開平5-130882号).が挙げられるが、これらに限定されない。さらに、スレオニンデアミナーゼ、アセトヒドロキシ酸シンターゼなどのＬ－イソロイシン生合成に関与するタンパク質をコードする遺伝子で形質転換された組換え株もまた親株として使用できる(特開平2-458号, FR 0356739, 及び米国特許第5,998,178号)。 Examples of L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing them include mutants resistant to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as the above, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882), but are not limited thereto. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).
Ｌ－アスパラギン酸生産菌
　Ｌ－アスパラギンは、アスパラギン酸へアミノ基を付与することにより生産される（Boehlein, S. K., Richards, N. G. J., & Schuster, S. M. (1994a) J. Biol. Chem. 269, 7450-7457.）。したがって、エシェリヒア・コリのＬ－アスパラギン生産菌として、Ｌ－アスパラギン酸生産菌のアスパラギンシンテターゼが増強されたエシェリヒア・コリ菌株が挙げられる。 L-Aspartic acid producing bacterium L-Asparagine is produced by adding an amino group to aspartic acid (Boehlein, SK, Richards, NGJ, & Schuster, SM (1994a) J. Biol. Chem. 269, 7450- 7457.) Thus, Escherichia coli L-asparagine-producing bacteria include Escherichia coli strains in which the asparagine synthetase of L-aspartic acid-producing bacteria is enhanced.
　本発明の細菌は、ピルビン酸シンターゼ、または、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性が増大するように改変された菌株であってもよい。ピルビン酸シンターゼの、あるいは、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性が増大するように改変するには、ピルビン酸シンターゼ、または、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性が、親株、例えば野生株や非改変株と比べて増大するように改変することが好ましい。尚、微生物が元来ピルビン酸シンターゼ活性、又はピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性を有していない場合、これらの酵素活性を有するように改変された微生物は、ピルビン酸シンターゼ、または、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性が、非改変株に比べて増大している。 The bacterium of the present invention may be a strain modified to increase the activity of pyruvate synthase or pyruvate: NADP + oxidoreductase. Pyruvate synthase or pyruvate: can be modified as NADP + oxide activity reductase is increased, the pyruvate synthase or pyruvate: NADP + oxidoreductase activity, the parent strain, for example, a wild strain or unmodified It is preferable to modify so as to increase compared to the strain. In addition, when the microorganism originally does not have pyruvate synthase activity or pyruvate: NADP + oxidoreductase activity, the microorganism modified to have these enzyme activities is pyruvate synthase or pyruvate: NADP + oxidoreductase activity is increased compared to unmodified strains.
　本発明における「ピルビン酸シンターゼ」とは、アセチル-CoAとCO2からピルビン酸を生成する下記の反応を、電子供与体存在下、例えばフェレドキシンあるいはフラボドキシン存在下で可逆的に触媒する酵素（EC 1.2.7.1）を意味する。ピルビン酸シンターゼは、ＰＳと略称されることもあり、ピルビン酸オキシドレダクターゼ、ピルビン酸フェレドキシンオキシドレダクターゼ、ピルビン酸フラボドキシンオキシドレダクターゼ、または、ピルビン酸オキシドレダクターゼと命名されている場合もある。電子供与体としては、フェレドキシンまたはフラボドキシンを用いることが出来る。 The “pyruvate synthase” in the present invention is an enzyme (EC 1.2) that catalyzes the following reaction for producing pyruvate from acetyl-CoA and CO 2 in the presence of an electron donor, for example, in the presence of ferredoxin or flavodoxin. .7.1). Pyruvate synthase is sometimes abbreviated as PS and is sometimes named pyruvate oxidoreductase, pyruvate ferredoxin oxidoreductase, pyruvate flavodoxin oxidoreductase, or pyruvate oxidoreductase. As the electron donor, ferredoxin or flavodoxin can be used.
還元型フェレドキシン + アセチル-CoA + CO2 → 酸化型フェレドキシン + ピルビン酸 + ＣｏＡ Reduced ferredoxin + acetyl-CoA + CO 2 → oxidized ferredoxin + pyruvate + CoA
　ピルビン酸シンターゼの活性が増強されたことの確認は、増強前の微生物と、増強後の微生物より粗酵素液を調製し、そのピルビン酸シンターゼ活性を比較することにより達成される。ピルビン酸シンターゼの活性は、例えば、Yoonらの方法（Yoon, K. S. et al. 1997. Arch. Microbiol. 167: 275-279）に従って測定できる。例えば、電子受容体としての酸化型メチルビオロゲンとＣｏＡと粗酵素液を含む反応液にピルビン酸を添加した際に、ピルビン酸の脱炭酸反応によって増大する還元型メチルビオロゲンの量を分光学的に測定することによって、測定可能である。酵素活性１ユニット（U）は１分間あたり１μmolのメチルビオロゲンの還元量で表される。親株がピルビン酸シンターゼ活性を有している場合、親株と比較して、好ましくは1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上酵素活性が上昇していることが望ましい。また親株がピルビン酸シンターゼ活性を有していない場合には、ピルビン酸シンターゼ遺伝子を導入することにより、ピルビン酸シンターゼが生成されていればよいが、酵素活性が測定できる程度に強化されていることが好ましく、好ましくは0.001U/mg（菌体タンパク質）以上、より好ましくは0.005U/mg以上、さらに好ましくは0.01U/mg以上が望ましい。ピルビン酸シンターゼは、酸素に対して感受性であり、一般的に活性発現や測定は困難であることも多い（Buckel, W.and Golding, B. T. 2006. Ann. Rev. of Microbiol. 60: 27-49）。したがって、酵素活性の測定に際しては、反応容器中の酸素濃度を低下させて酵素反応を行うことが好ましい。 Confirmation that the activity of pyruvate synthase is enhanced is achieved by preparing a crude enzyme solution from the microorganism before enhancement and the microorganism after enhancement and comparing the activity of pyruvate synthase. The activity of pyruvate synthase can be measured, for example, according to the method of Yoon et al. (Yoon, K. S. et al. 1997. Arch. Microbiol. 167: 275-279). For example, when pyruvic acid is added to a reaction solution containing oxidized methyl viologen as an electron acceptor, CoA, and a crude enzyme solution, the amount of reduced methyl viologen that increases due to decarboxylation of pyruvic acid is measured spectroscopically. It can be measured by measuring. One unit (U) of enzyme activity is expressed as a reduction amount of 1 μmol of methyl viologen per minute. When the parent strain has pyruvate synthase activity, the enzyme activity is preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more that of the parent strain. If the parent strain does not have pyruvate synthase activity, it is sufficient that pyruvate synthase is produced by introducing the pyruvate synthase gene, but the enzyme activity is enhanced to such an extent that it can be measured. Is preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more. Pyruvate synthase is sensitive to oxygen and is generally difficult to express and measure (Buckel, W.and Golding, B. T. 2006. Ann. Rev. of Microbiol. 60: 27-49). Therefore, when measuring enzyme activity, it is preferable to carry out the enzyme reaction by reducing the oxygen concentration in the reaction vessel.
　ピルビン酸シンターゼをコードする遺伝子としては、クロロビウム・テピダム(Chlorobium tepidum)、ハイドロジェノバクター・サーモファイラス（Hydrogenobacter thermophilus）等、還元的ＴＣＡサイクルを持つ細菌のピルビン酸シンターゼ遺伝子を利用することが可能である。また、エシェリヒア・コリ（Escherichia coli）をはじめとする、腸内細菌群に属する細菌由来のピルビン酸シンターゼ遺伝子を利用することも可能である。さらに、ピルビン酸シンターゼをコードする遺伝子は、メタノコッカス・マリパルディス（Methanococcus maripaludis）、メタノカルドコッカス・ジャナスチ（Methanocaldococcus jannaschii）、メタノサーモバクター・サーマトトロフィカス（Methanothermobacter thermautotrophicus）などの独立栄養性メタン生成古細菌（autotrophic methanogens）のピルビン酸シンターゼ遺伝子を利用することが可能である。 As a gene encoding pyruvate synthase, it is possible to use a pyruvate synthase gene of a bacterium having a reductive TCA cycle, such as Chlorobium tepidum, Hydrogenobacter thermophilus, etc. is there. It is also possible to use a pyruvate synthase gene derived from bacteria belonging to the group of enterobacteria such as Escherichia coli. In addition, genes encoding pyruvate synthase are autotrophic methane producers such as Methanococcus maripaludis, Methanococcus janasti, Methanothermobacter thermautotrophicus, and other methanothermobacter thermautotrophicus (Autotrophic （methanogens) pyruvate synthase gene can be used.
　具体的には、クロロビウム・テピダム（Chlorobium tepidum）のピルビン酸シンターゼ遺伝子として、クロロビウム・テピダムのゲノム配列（GenBank Accession No. NC_002932）の塩基番号1534432～1537989に位置する塩基配列を有する遺伝子を例示することができる。同遺伝子がコードするアミノ酸配列はGenBank Accession No. AAC76906に開示されている。また、ハイドロジェノバクター・サーモファイラスのピルビン酸シンターゼは、δサブユニット（GenBank Accession No. BAA95604）、αサブユニット（GenBank Accession No. BAA95605）、βサブユニット（GenBank Accession No. BAA95606）、γサブユニット（GenBank Accession No. BAA95607）の４つのサブユニットによる複合体を形成していることが知られている（Ikeda, T. et al. 2006. Biochem. Biophys. Res. Commun. 340: 76-82）。さらに、ヘリコバクター・ピロリ（Helicobacter pylori）のゲノム配列（GenBank Accession No. NC 000915）の塩基番号1170138～1173296番に位置するHP1108、HP1109、HP1110、HP1111の4つの遺伝子からなるピルビン酸シンターゼ遺伝子、スルフォロバス・ソルファタリカス（Sulfolobus solfataricus）のゲノム配列（GenBank Accession No. NC 002754）の塩基番号1047593～1044711番で示されるSSO1208、SSO7412、SSO1207、SSO1206の4つの遺伝子からなるピルビン酸シンターゼ遺伝子を例示することができる。さらに、ピルビン酸シンターゼ遺伝子は、上記で例示された遺伝子との相同性に基づいて、クロロビウム（Chlorobium）属、デスルホバクター（Desulfobacter）属、アクイフェクス（Aquifex）属、ハイドロジェノバクター（Hydrogenobacter）属、サーモプロテウス（Thermoproteus）属、パイロバキュラム（Pyrobaculum）属細菌等からクローニングされるものであってもよい。 Specifically, as a pyruvate synthase gene of Chlorobium tepidum (Chlorobium tepidum), a gene having a base sequence located at base numbers 1534432 to 1537989 of the genome sequence of chlorobium tepidum (GenBank Accession No. NC_002932) Can do. The amino acid sequence encoded by this gene is disclosed in GenBank Accession No. AAC76906. Hydrogenobacter thermophilus pyruvate synthase is composed of δ subunit (GenBank Accession No. BAA95604), α subunit (GenBank Accession No. BAA95605), β subunit (GenBank Accession No. BAA95606), γ subunit. It is known to form a complex with four subunits of the unit (GenBank Accession No. BAA95607) (Ikeda, T. et al. 2006. Biochem. Biophys. Res. Commun. 340: 76-82 ). Furthermore, the pyruvate synthase gene comprising 4 genes of HP1108, HP1109, HP1110, and HP1111 located at base numbers 1170138 to 1173296 of the genome sequence of Helicobacter pylori (GenBank Accession No. An example is a pyruvate synthase gene consisting of four genes, SSO1208, SSO7412, SSO1207, and SSO1206, represented by nucleotide numbers 1047593 to 1044711 in the genome sequence of Sulfolobus solfataricus (GenBank Accession No. NC 002754) it can. Furthermore, the pyruvate synthase gene is based on the homology with the genes exemplified above, based on the genus Chlorobium, the genus Desulfobacter, the genus Aquifex, the genus Hydrogenobacter, It may be cloned from bacteria of the genus Thermoproteus, Pyrobaculum, or the like.
　エシェリヒア・コリにおいては、K-12株のゲノム配列（GenBank Accession No. U00096）の塩基番号1435284～1438808に位置する塩基配列を有するydbK遺伝子（b1378）が、配列上の相同性からピルビン酸フラボドキシンオキシドレダクターゼ、すなわちピルビン酸シンターゼをコードしていると予想されている。同遺伝子がコードするアミノ酸配列はGenBank Accession No. AAC76906に開示されている。さらに、ピルビン酸シンターゼ遺伝子は、エシェリヒア・コリのピルビン酸シンターゼ遺伝子（ydbK）と高い相同性を有する、エシェリヒア属、サルモネラ属（Salmonella）、セラチア属（Serratia）、エンテロバクター属（Enterobacter）、シゲラ属（Shigella）、サイトロバクター属（Citrobacter）などの腸内細菌群に属するピルビン酸シンターゼ遺伝子であってもよい。 In Escherichia coli, the ydbK gene (b1378) having a nucleotide sequence located at nucleotide numbers 1435284 to 1438808 in the genome sequence of the K-12 strain (GenBank Accession No. U00096) is It is expected to encode a synoxide reductase, pyruvate synthase. The amino acid sequence encoded by this gene is disclosed in GenBank Accession No. AAC76906. Furthermore, the pyruvate synthase gene is highly homologous to the Escherichia coli pyruvate synthase gene (ydbK), and the genera Escherichia, Salmonella, Serratia, Enterobacter, Shigella It may be a pyruvate synthase gene belonging to the group of enterobacteria such as (Shigella) and Citrobacter.
　メタノコッカス・マリパルディス（Methanococcus maripaludis）のピルビン酸シンターゼは、メタノコッカス・マリパルディスのゲノム配列（GenBank Accession No. NC_005791）（Hendrickson, E. L. et al. 2004. J. Bacteriol. 186: 6956-6969）の塩基番号1462535～1466397に位置するporCDABEFオペロンにコードされている（Lin, W. C. et al. 2003. Arch. Microbiol. 179: 444-456）。このピルビン酸シンターゼは、γ、α、β、及びδの４つのサブユニットを含んでおり、これらのサブユニットに加えて、PorE及びPorFもピルビン酸シンターゼの活性に重要であることが知られている（Lin, W. and Whitman, W. B. 2004. Arch. Microbiol. 181: 68-73）。γサブユニットは、前記ゲノム配列の塩基番号1465867～1466397（相補鎖）のporA遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988626に開示されている。δサブユニットは、前記ゲノム配列の塩基番号1465595～1465852（相補鎖）porB遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988627に開示されている。αサブユニットは、前記ゲノム配列の塩基番号1464410～1465573（相補鎖）のporC遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988625に開示されている。βサブユニットは、ゲノム配列の塩基番号1463497～1464393（相補鎖）のporD遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988624に開示されている。PorEは、ゲノム配列の塩基番号1462970～1463473（相補鎖）のporE遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988623に開示されている。PorFは、ゲノム配列の塩基番号1462535～1462951（相補鎖）のporF遺伝子にコードされており、同遺伝子がコードするアミノ酸配列はGenBank Accession No. NP_988622に開示されている。 The pyruvate synthase from Methanococcus maripaludis is the genome sequence of Methanococcus maripardis (GenBank Accession No. NC_005791) (Hendrickson, E. L. et al. 2004. J. Bacteriol. 186: 6956-6969) Is encoded by the porCDABEF operon located at base numbers 1462535 to 1466397 (Lin, W. C. et al. 2003. Arch. Microbiol. 179: 444-456). This pyruvate synthase contains four subunits, γ, α, β, and δ. In addition to these subunits, PorE and PorF are also known to be important for the activity of pyruvate synthase. (Lin, W. and Whitman, W. B. 2004. Arch. Microbiol. 181: 68-73). The γ subunit is encoded by the porA gene of base numbers 1465867 to 1466397 (complementary chain) of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988626. The δ subunit is encoded by the base number 1465595 to 1465852 (complementary chain) porB gene of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988627. The α subunit is encoded by the porC gene of nucleotide numbers 1464410 to 1455773 (complementary chain) of the genome sequence, and the amino acid sequence encoded by the gene is disclosed in GenBank Accession No. NP_988625. The β subunit is encoded by the porD gene of nucleotide numbers 1463497 to 14439393 (complementary strand) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_988624. PorE is encoded by the porE gene of base numbers 1462970 to 1463473 (complementary chain) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_988623. PorF is encoded by the porF gene of base numbers 1462535 to 1462951 (complementary chain) of the genome sequence, and the amino acid sequence encoded by this gene is disclosed in GenBank Accession No. NP_988622.
　独立栄養性のメタン生成古細菌のメタノカルドコッカス・ジャナスチ（Methanocaldococcus jannaschii）、メタノサーモバクター・サーマトトロフィカス（Methanothermobacter thermautotrophicus）なども同じ遺伝子構造のピルビン酸シンターゼ遺伝子を有していることが知られており、これらを利用することが可能である。 Autotrophic methanogenic archaea Methananocaldococcus jannaschii and Methanothermobacter thermautotrophicus are also known to have pyruvate synthase genes with the same gene structure. These can be used.
　本発明における「ピルビン酸：ＮＡＤＰ+オキシドレダクターゼ」とは、アセチル-CoAとCO2からピルビン酸を生成する下記の反応を、電子供与体存在下、例えばＮＡＤＰＨあるいはＮＡＤＨ存在下で可逆的に触媒する酵素（EC 1.2.1.15）を意味する。ピルビン酸：ＮＡＤＰ+オキシドレダクターゼは、ＰＮＯと略称されることもあり、ピルビン酸デヒドロゲナーゼと命名されている場合もある。しかしながら、本発明において「ピルビン酸デヒドロゲナーゼ活性」というときは、後述するように、ピルビン酸を酸化的に脱炭酸し、アセチル-CoAを生成する反応を触媒する活性であり、この反応を触媒するピルビン酸デヒドロゲナーゼ（PDH）は、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼとは別の酵素である。ピルビン酸：ＮＡＤＰ+オキシドレダクターゼは、電子供与体としては、ＮＡＤＰＨあるいはＮＡＤＨを用いることが出来る。 In the present invention, “pyruvate: NADP + oxidoreductase” means reversibly catalyzing the following reaction for producing pyruvic acid from acetyl-CoA and CO 2 in the presence of an electron donor, for example, in the presence of NADPH or NADH. Means enzyme (EC 1.2.1.15). Pyruvate: NADP + oxidoreductase is sometimes abbreviated as PNO and sometimes as pyruvate dehydrogenase. However, in the present invention, “pyruvate dehydrogenase activity” is an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA, as described later. Acid dehydrogenase (PDH) is a separate enzyme from pyruvate: NADP + oxidoreductase. Pyruvate: NADP + oxidoreductase can use NADPH or NADH as an electron donor.
ＮＡＤＰＨ + アセチル-CoA + CO2 → ＮＡＤＰ+ + ピルビン酸 + CoA NADPH + Acetyl-CoA + CO 2 → NADP + + Pyruvate + CoA
　ピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性が増強されたことの確認は、増強前の微生物と、増強後の微生物より粗酵素液を調製し、そのピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性を比較することにより達成される。ピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性は、例えば、Inuiらの方法（Inui, H. et al. 1987. J. Biol. Chem. 262: 9130-9135）に従って測定できる。例えば、電子受容体としての酸化型メチルビオロゲンとCoAと粗酵素液を含む反応液に、ピルビン酸を添加した際にピルビン酸の脱炭酸反応によって増大する還元型メチルビオロゲンの量を分光学的に測定することによって、測定可能である。酵素活性１ユニット（U）は１分間あたり１μmolのメチルビオロゲンの還元量で表される。親株がピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性を有している場合、親株と比較して、好ましくは1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上酵素活性が上昇していることが望ましい。また親株がピルビン酸：ＮＡＤＰ+オキシドレダクターゼ活性を有していない場合には、ピルビン酸シンターゼ遺伝子を導入することにより、ピルビン酸：ＮＡＤＰ+オキシドレダクターゼが生成されていればよいが、酵素活性が測定できる程度に強化されていることが好ましく、好ましくは0.001U/mg（菌体タンパク質）以上、より好ましくは0.005U/mg以上、さらに好ましくは0.01U/mg以上が望ましい。ピルビン酸：ＮＡＤＰ+オキシドレダクターゼは、酸素に対して感受性であり、一般的に活性発現や測定は困難であることも多い（Inui, H. et al. 1987. J. Biol. Chem. 262: 9130-9135; Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710-720）。したがって、酵素活性の測定に際しては、反応容器中の酸素濃度を低下させて酵素反応を行うことが好ましい。 Confirmation that the activity of pyruvate: NADP + oxidoreductase was enhanced was made by preparing a crude enzyme solution from the microorganism before enhancement and the microorganism after enhancement, and comparing the activity of pyruvate: NADP + oxidoreductase. Achieved. The activity of pyruvate: NADP + oxidoreductase can be measured, for example, according to the method of Inui et al. (Inui, H. et al. 1987. J. Biol. Chem. 262: 9130-9135). For example, when pyruvate is added to a reaction solution containing oxidized methyl viologen as an electron acceptor, CoA, and a crude enzyme solution, the amount of reduced methyl viologen that increases due to the decarboxylation of pyruvate is measured spectroscopically. It can be measured by measuring. One unit (U) of enzyme activity is expressed as a reduction amount of 1 μmol of methyl viologen per minute. When the parent strain has pyruvate: NADP + oxidoreductase activity, the enzyme activity is preferably increased 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to the parent strain. Is desirable. If the parent strain does not have pyruvate: NADP + oxidoreductase activity, it is sufficient that pyruvate: NADP + oxidoreductase is generated by introducing the pyruvate synthase gene, but the enzyme activity is measured. It is preferably strengthened to the extent possible, preferably 0.001 U / mg (bacterial protein) or more, more preferably 0.005 U / mg or more, and still more preferably 0.01 U / mg or more. Pyruvate: NADP + oxidoreductase is sensitive to oxygen and is generally difficult to express and measure activity (Inui, H. et al. 1987. J. Biol. Chem. 262: 9130). -9135; Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710-720). Therefore, when measuring enzyme activity, it is preferable to carry out the enzyme reaction by reducing the oxygen concentration in the reaction vessel.
　ピルビン酸：ＮＡＤＰ+オキシドレダクターゼをコードする遺伝子は、光合成真核微生物で原生動物にも分類されるユーグレナ・グラシリス（Euglena gracilis）のピルビン酸：ＮＡＤＰ+オキシドレダクターゼ遺伝子（Nakazawa, M. et al. 2000. FEBS Lett. 479: 155-156）、原生生物クリプトスポルジウム・パルバム（Cryptosporidium parvum）のピルビン酸：ＮＡＤＰ+オキシドレダクターゼ遺伝子（Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710-720）の他、珪藻タラシオシラ・スードナナ（Tharassiosira pseudonana）にも相同な遺伝子が存在することが知られている（Ctrnacta, V. et al. 2006. J. Eukaryot. Microbiol. 53: 225-231）。 The gene encoding pyruvate: NADP + oxidoreductase is a photosynthetic eukaryotic microorganism and is also classified as a protozoan. The pyruvate: NADP + oxidoreductase gene of Euglena gracilis (Nakazawa, M. et al. 2000) FEBS Lett. 479: 155-156), the protist Cryptosporidium parvum pyruvate: NADP + oxidoreductase gene (Rotte, C. et al. 2001. Mol. Biol. Evol. 18: 710 -720) and homologous genes are known to exist in the diatom Tharassiosira pseudonana (Ctrnacta, V. et al. 2006. J. Eukaryot. Microbiol. 53: 225-231) .
　具体的には、ユーグレナ・グラシリス（Euglena gracilis）のピルビン酸：ＮＡＤＰ+オキシドレダクターゼ遺伝子として、GenBank Accession No. AB021127に示す塩基配列を有する遺伝子が挙げられる。同遺伝子がコードするアミノ酸配列はGenBank Accession No. BAB12024に開示されている。 Specifically, as a pyruvate: NADP + oxidoreductase gene of Euglena gracilis, there is a gene having a base sequence shown in GenBank Accession No. AB021127. The amino acid sequence encoded by this gene is disclosed in GenBank Accession No. BAB12024.
　本発明の微生物は、ピルビン酸シンターゼの活性に必要な電子供与体の酸化型を還元型にリサイクルする活性が、親株、例えば野生株や非改変株と比べて増大するように改変することによって、ピルビン酸シンターゼの活性が増大するように改変された微生物でもよい。電子供与体の酸化型を還元型にリサイクルする活性としては、フェレドキシン-ＮＡＤＰ+レダクターゼ活性を挙げることができる。また、電子供与体のリサイクル活性の増強に加えて、ピルビン酸シンターゼ活性が増大するように改変することによって、ピルビン酸シンターゼの活性が増大するように改変された微生物でもよい。なお、上記親株は、本来内在的に電子供与体のリサイクル活性を担うタンパク質をコードする遺伝子を有しているものであってもよいし、本来は電子供与体のリサイクル活性を有さないが、当該活性を担うタンパク質をコードする遺伝子を導入することにより活性が付与され、Ｌ-アミノ酸生産能が向上するものであってもよい。 The microorganism of the present invention is modified by increasing the activity of recycling the oxidized form of the electron donor necessary for the activity of pyruvate synthase to the reduced form as compared with the parent strain, for example, a wild strain or an unmodified strain, The microorganism may be modified so that the activity of pyruvate synthase is increased. Examples of the activity of recycling the oxidized form of the electron donor to the reduced form include ferredoxin-NADP + reductase activity. In addition to enhancing the recycling activity of the electron donor, the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the activity to increase pyruvate synthase activity. The parent strain may have a gene encoding a protein inherently responsible for the electron donor recycling activity, or originally has no electron donor recycling activity, The activity may be imparted by introducing a gene encoding a protein responsible for the activity, and the L-amino acid producing ability may be improved.
　「フェレドキシン-ＮＡＤＰ+レダクターゼ」とは、以下の反応を可逆的に触媒する酵素（EC 1.18.1.2）をいう。 “Ferredoxin-NADP + reductase” refers to an enzyme (EC 1.18.1.2) that reversibly catalyzes the following reaction.
還元型フェレドキシン + NADP+ → 酸化型フェレドキシン + NADPH + H+ Reduced ferredoxin + NADP + → oxidized ferredoxin + NADPH + H +
　本反応は、可逆反応であり、ＮＡＤＰＨと酸化型フェレドキシン存在下で、還元型フェレドキシンを産生することが可能である。フェレドキシンはフラボドキシンと代替可能であり、フラボドキシン-ＮＡＤＰ+レダクターゼと命名されているものも同等の機能を有する。フェレドキシン-ＮＡＤＰ+レダクターゼは微生物から高等生物まで幅広く存在が確認されており（Carrillo, N. and Ceccarelli, E. A. 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, E. A. et al. 2004. Biochim. Biophys. Acta. 1698: 155-165参照）、フェレドキシン-ＮＡＤＰ+オキシドレダクターゼ、ＮＡＤＰＨ-フェレドキシンオキシドレダクターゼと命名されているものもある。 This reaction is a reversible reaction, and reduced ferredoxin can be produced in the presence of NADPH and oxidized ferredoxin. Ferredoxin can be substituted for flavodoxin, and what is named flavodoxin-NADP + reductase also has an equivalent function. Ferredoxin-NADP + reductase has been confirmed to exist widely from microorganisms to higher organisms (Carrillo, N. and Ceccarelli, EA 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, EA et al. 2004. Biochim Biophys. Acta. 1698: 155-165), some have been named ferredoxin-NADP + oxidoreductase, NADPH-ferredoxin oxidoreductase.
　フェレドキシン-ＮＡＤＰ+レダクターゼの活性が増強されたことの確認は、改変前の微生物と、改変後の微生物より粗酵素液を調製し、そのフェレドキシン-ＮＡＤＰ+レダクターゼ活性を比較することにより達成される。フェレドキシン-ＮＡＤＰ+レダクターゼの活性は、例えば、Blaschkowskiらの方法（Blaschkowski, H. P. et al. 1982. Eur. J. Biochem. 123: 563-569）に従って測定できる。例えば、基質としてフェレドキシンを用い、減少するNADPH量を分光学的に測定することによって測定可能である。酵素活性１ユニット（U）は１分間あたり１μmolのNADPHの酸化量で表される。親株がフェレドキシン-ＮＡＤＰ+レダクターゼ活性を有している場合、親株の活性が十分高ければ、酵素活性を増強する必要はないが、親株と比較して、好ましくは1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上酵素活性が上昇していることが望ましい。 Confirmation that the activity of ferredoxin-NADP + reductase was enhanced is achieved by preparing a crude enzyme solution from the microorganism before modification and the microorganism after modification, and comparing the activity of ferredoxin-NADP + reductase. The activity of ferredoxin-NADP + reductase can be measured, for example, according to the method of Blaschkowski et al. (Blaschkowski, H. P. et al. 1982. Eur. J. Biochem. 123: 563-569). For example, it can be measured by spectroscopically measuring the decreasing amount of NADPH using ferredoxin as a substrate. One unit (U) of enzyme activity is expressed as an oxidation amount of 1 μmol NADPH per minute. When the parent strain has ferredoxin-NADP + reductase activity, it is not necessary to enhance the enzyme activity if the activity of the parent strain is sufficiently high, but it is preferably 1.5 times or more, more preferably 2 times that of the parent strain. As described above, it is more preferable that the enzyme activity is increased by 3 times or more.
　フェレドキシン-ＮＡＤＰ+レダクターゼをコードする遺伝子は、多くの生物種で見出されており、目的のＬ-アミノ酸生産株中で活性を有する酵素をコードするものであれば使用することが可能である。エシェリヒア・コリではフラボドキシン-ＮＡＤＰ+レダクターゼとしてfpr遺伝子が同定されている（Bianchi, V. et al. 1993. J. Bacteriol. 175:1590-1595）。また、シュードモナス・プチダ(Psuedomonas putida)には、ＮＡＤＰＨ-プチダレドキシンレダクターゼ（Putidaredoxin reductase）遺伝子とプチダレドキシン（Putidaredoxin）遺伝子がオペロンとして存在することが知られている(Koga, H. et al. 1989. J. Biochem. (Tokyo) 106: 831-836）。 A gene encoding ferredoxin-NADP + reductase has been found in many biological species, and any gene encoding an enzyme having activity in the target L-amino acid producing strain can be used. In Escherichia coli, the fpr gene has been identified as flavodoxin-NADP + reductase (Bianchi, V. et al. 1993. J. Bacteriol. 175: 1590-1595). It is also known that Pseedomonas putida has NADPH-Putidaredoxin reductase gene and Putidaredoxin gene as operons (Koga, H. et al. 1989). J. Biochem. (Tokyo) 106: 831-836).
　エシェリヒア・コリのフラボドキシン-ＮＡＤＰ+レダクターゼとしては、エシェリヒア・コリK-12株のゲノム配列（GenBank Accession No. U00096）の塩基番号4111749～4112495（相補鎖）に位置する塩基配列を有するfpr遺伝子を例示することができる。Fprのアミノ酸配列は、GenBank Accession No. AAC76906に開示されている。また、コリネバクテリウム・グルタミカムのゲノム配列（GenBank Accession No. BA00036）の塩基番号2526234～2527211にフェレドキシン-ＮＡＤＰ+レダクターゼ遺伝子が見出されている（GenBank Accession No. BAB99777）。 Examples of Escherichia coli flavodoxin-NADP + reductase include the fpr gene having a nucleotide sequence located at nucleotide numbers 4111749 to 4112495 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096) can do. The amino acid sequence of Fpr is disclosed in GenBank Accession No. AAC76906. Further, a ferredoxin-NADP + reductase gene has been found at the base numbers 25526234 to 2527211 of the genome sequence of Corynebacterium glutamicum (GenBank Accession No. BA00036) (GenBank Accession No. BAB99777).
　ピルビン酸シンターゼの活性には、フェレドキシン又はフラボドキシンが電子供与体として存在することが必要である。従って、フェレドキシン又はフラボドキシンの産生能が向上するように改変することによって、ピルビン酸シンターゼの活性が増大するように改変された微生物であってもよい。
　また、ピルビン酸シンターゼ活性、又は、フラボドキシン-ＮＡＤＰ+レダクターゼ及びピルビン酸シンターゼ活性が増強するように改変することに加えて、フェレドキシン又はフラボドキシンの産生能が向上するように改変してもよい。 The activity of pyruvate synthase requires that ferredoxin or flavodoxin be present as an electron donor. Therefore, the microorganism may be modified so that the activity of pyruvate synthase is increased by modifying the ferredoxin or flavodoxin so as to improve the production ability.
Further, in addition to modification so that pyruvate synthase activity, or flavodoxin-NADP + reductase and pyruvate synthase activities are enhanced, modification may be made so that ferredoxin or flavodoxin production ability is improved.
　本発明における「フェレドキシン」とは、非ヘム鉄原子（Fe）と、硫黄原子を含み、4Fe-4S、3Fe-4S、あるいは、2Fe-2Sクラスターと呼ばれる鉄-硫黄クラスターを結合したタンパク質で1電子の伝達体として機能するものを指す。「フラボドキシン」とはFMN（Flavin-mononucleotide）を補欠分子属として含む1あるいは2電子の伝達体として機能するタンパク質を指す。フェレドキシンとフラボドキシンについては、McLeanらの文献に記載されている（McLean, K. J. et al. 2005. Biochem. Soc. Trans. 33: 796-801）。 The “ferredoxin” in the present invention is a protein that contains a non-heme iron atom (Fe) and a sulfur atom and binds an iron-sulfur cluster called a 4Fe-4S, 3Fe-4S, or 2Fe-2S cluster. The one that functions as a transmitter. “Flavodoxin” refers to a protein that functions as a one- or two-electron carrier containing FMN (Flavin-mononucleotide) as a prosthetic genus. Ferredoxin and flavodoxin are described in McLean et al. (McLean, K. J. et al. 2005. Biochem. Soc. Trans. 33: 796-801).
　なお、改変に用いる親株は、本来内在的にフェレドキシン又はフラボドキシンをコードする遺伝子を有しているものであってもよいし、本来はフェレドキシン又はフラボドキシン遺伝子を有さないが、フェレドキシン又はフラボドキシン遺伝子を導入することによりこれらのタンパク質の産生能が付与され、Ｌ-アミノ酸生産能が向上するものであってもよい。 The parent strain used for the modification may have a gene that inherently encodes ferredoxin or flavodoxin, or originally has no ferredoxin or flavodoxin gene, but introduces a ferredoxin or flavodoxin gene. By doing so, the ability to produce these proteins may be imparted, and the ability to produce L-amino acids may be improved.
　フェレドキシン又はフラボドキシンの産生能が親株、例えば野生株や非改変株と比べて向上していることの確認は、フェレドキシン又はフラボドキシンのmRNAの量を野生型、あるいは非改変株と比較することによって確認できる。発現量の確認方法としては、ノーザンハイブリダイゼーション、RT-PCRが挙げられる（Sambrook, J. et al. 1989. Molecular CloningA Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, New York）。発現量については、野生株あるいは非改変株と比較して、上昇していればいずれでもよいが、例えば野生株、非改変株と比べて1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上上昇していることが望ましい。 Confirmation that ferredoxin or flavodoxin production ability is improved compared to the parent strain, for example, wild strain or non-modified strain, can be confirmed by comparing the amount of ferredoxin or flavodoxin mRNA with the wild-type or non-modified strain. . Examples of the expression level confirmation method include Northern hybridization and RT-PCR (Sambrook, J. et al. 1989. Molecular CloningA Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). The expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.
　また、フェレドキシン又はフラボドキシンの産生能が親株、例えば野生株や非改変株と比べて向上していることの確認は、SDS-PAGEや二次元電気泳動あるいは、抗体を用いたウェスタンブロットによって検出することが出来る（Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, New York）。生産量については、野生株あるいは非改変株と比較して、向上していればいずれでもよいが、例えば野生株、非改変株と比べて1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上上昇していることが望ましい。 Confirmation that ferredoxin or flavodoxin production is improved compared to the parent strain, for example, wild strain or unmodified strain, should be detected by SDS-PAGE, two-dimensional electrophoresis, or Western blot using an antibody. (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). The production amount may be any as long as it is improved as compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.
　フェレドキシン及びフラボドキシンの活性は、適切な酸化還元反応系に加えることで測定することが可能である。例えば、Boyerらにより、産生されたフェレドキシンをフェレドキシン-ＮＡＤＰ+レダクターゼにより還元し、生じた還元型フェレドキシンによるチトクロームCの還元を定量する方法が開示されている（Boyer, M. E. et al. 2006. Biotechnol. Bioeng. 94: 128-138）。また、フラボドキシンの活性は、フラボドキシン-ＮＡＤＰ+レダクターゼを用いることで、同じ方法で測定が可能である。 The activity of ferredoxin and flavodoxin can be measured by adding to an appropriate redox reaction system. For example, Boyer et al. Discloses a method of reducing the produced ferredoxin with ferredoxin-NADP + reductase and quantifying the reduction of cytochrome C by the resulting reduced ferredoxin (Boyer, ME et al. 2006. Biotechnol. Bioeng. 94: 128-138). The activity of flavodoxin can be measured by the same method using flavodoxin-NADP + reductase.
　フェレドキシン、又はフラボドキシンをコードする遺伝子は、広く分布しており、コードされるフェレドキシン又はフラボドキシンがピルビン酸シンターゼと電子供与体再生系が利用可能なものであれば、どのようなものでも用いることができる。例えば、エシェリヒア・コリには、2Fe-2Sクラスターを有するフェレドキシンをコードする遺伝子としてfdx遺伝子が存在し（Ta, D. T. and Vickery, L. E. 1992. J. Biol. Chem. 267:11120-11125）、4Fe-4Sクラスターを有するフェレドキシン遺伝子としてyfhL遺伝子が予想されている。また、フラボドキシン遺伝子としては、fldA遺伝子（Osborne, C. et al. 1991. J. Bacteriol. 173: 1729-1737）とfldB遺伝子（Gaudu, P. and Weiss, B. 2000. J. Bacteriol. 182:1788-1793）の存在が知られている。コリネバクテリウム・グルタミカムのゲノム配列（GenBank Accession No. BA00036）においては、塩基番号562643～562963に複数のフェレドキシン遺伝子fdx（GenBank Accession No. BAB97942）及び塩基番号1148953～1149270にfer（GenBank Accession No. BAB98495）が見出されている。また、クロロビウム・テピダムにおいては、多くのフェレドキシン遺伝子が存在するが、ピルビン酸シンターゼの電子受容体となる4Fe-4S型のフェレドキシン遺伝子としてフェレドキシンＩ及びフェレドキシンIIが同定されている（Yoon, K. S. et al. 2001. J. Biol. Chem. 276: 44027-44036）。ハイドロジェノバクター・サーモファイラス等、還元的ＴＣＡサイクルを持つ細菌由来のフェレドキシン遺伝子あるいはフラボドキシン遺伝子を用いることもできる。 The gene encoding ferredoxin or flavodoxin is widely distributed, and any encoded ferredoxin or flavodoxin can be used as long as pyruvate synthase and an electron donor regeneration system are available. . For example, in Escherichia coli, the fdx gene exists as a gene encoding ferredoxin having a 2Fe-2S cluster (Ta, D. T. and Vickery, L. E. 1992. J. Biol. Chem. 267: 11120 -11125), the yfhL gene is predicted as a ferredoxin gene having a 4Fe-4S cluster. The flavodoxin gene includes fldA gene (Osborne, C. et al. 1991. J. Bacteriol. 173: 1729-1737) and fldB gene (Gaudu, P. and Weiss, B. 2000. J. Bacteriol. 182: 1788-1793) is known. In the genome sequence of Corynebacterium glutamicum (GenBank Accession No. BA00036), multiple ferredoxin genes fdx (GenBank Accession No. BAB97942) at base numbers 562643 to 562963 and fer (GenBank Accession No. BAB98495 at base numbers 1148953 to 1149270 ) Has been found. In Chlorobium tepidum, there are many ferredoxin genes, but ferredoxin I and ferredoxin II have been identified as 4Fe-4S type ferredoxin genes that serve as electron acceptors for pyruvate synthase (Yoon, K. S Et al. 2001. J. Biol. Chem. 276: 44027-44036). Ferredoxin genes or flavodoxin genes derived from bacteria having a reductive TCA cycle such as Hydrogenobacter thermophilus can also be used.
　エシェリヒア・コリのフェレドキシン遺伝子として、エシェリヒア・コリK-12株のゲノム配列（GenBank Accession No. U00096）の塩基番号2654770～2655105番（相補鎖）に位置するfdx遺伝子、及び塩基番号2697685～2697945番に位置するyfhL遺伝子を例示することができる。Fdx及びYfhLのアミノ酸配列は、それぞれ、GenBank Accession No. AAC75578及びAAC75615に開示されている。エシェリヒア・コリのフラボドキシン遺伝子としては、エシェリヒア・コリK-12株のゲノム配列（GenBank Accession No. U00096）の塩基番号710688～710158番（相補鎖）に位置するfldA遺伝子、及び塩基番号3037877～3038398 番に位置するfldB遺伝子を例示することができる。fldA遺伝子及びfldB遺伝子がコードするアミノ酸配列は、それぞれ、GenBank Accession No. AAC73778及びAAC75933に開示されている。 As the ferredoxin gene of Escherichia coli, the fdx gene located at base numbers 2654770-2655105 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096), and base numbers 2676885-2697945 The located yfhL gene can be exemplified. The amino acid sequences of Fdx and YfhL are disclosed in GenBank Accession No. AAC75578 and AAC75615, respectively. Examples of the Escherichia coli flavodoxin gene include the fldA gene located at nucleotide numbers 710688 to 710158 (complementary strand) of the genome sequence of Escherichia coli K-12 strain (GenBank Accession No. U00096), and nucleotide numbers 3037877 to 3038398 The fldB gene located in can be exemplified. The amino acid sequences encoded by the fldA gene and fldB gene are disclosed in GenBank Accession No. AAC73778 and AAC75933, respectively.
　クロロビウム・テピダム(Chlorobium tepidum)のフェレドキシン遺伝子としては、クロロビウム・テピダムのゲノム配列（GenBank Accession No. NC_002932）の塩基番号1184078～1184266番に位置するフェレドキシンＩ遺伝子、及び塩基番号1184476～1184664番に位置するフェレドキシンII遺伝子を例示することができる。フェレドキシンＩ及びフェレドキシンIIのアミノ酸配列は、それぞれ、GenBank Accession No. AAM72491及びAAM72490に開示されている。また、ハイドロジェノバクター・サーモファイラス（Hydrogenobacter thermophilus）のフェレドキシン遺伝子（GenBank Accession No. BAE02673）や、スルフォロバス・ソルファタリカス（Sulfolobus solfataricus）のゲノム配列中の塩基番号2345414～2345728番で示されるスルフォロバス・ソルファタリカスのフェレドキシン遺伝子を例示することができる。さらに、上記で例示された遺伝子との相同性に基づいて、クロロビウム（Chlorobium）属、デスルホバクター（Desulfobacter）属、アクイフェクス（Aquifex）属、ハイドロジェノバクター（Hydrogenobacter）属、サーモプロテウス（Thermoproteus）属、パイロバキュラム（Pyrobaculum）属細菌等からクローニングされるものであってもよく、さらにはエンテロバクター属、クレブシエラ属、セラチア属、エルビニア属、エルシニア属等のγ-プロテオバクテリア、コリネバクテリウム・グルタミカム、ブレビバクテリウム・ラクトファーメンタム等のコリネ型細菌、シュードモナス・アエルジノーサ等のシュードモナス属細菌、マイコバクテリウム・ツベルクロシス等のマイコバクテリウム属細菌等からクローニングされるものであってもよい。 The ferredoxin gene of Chlorobium tepidum (Chelorobium tepidum) is located in the ferredoxin I gene located at nucleotide numbers 1184078 to 1184266 and nucleotide numbers 1184476 to 1184664 in the genome sequence of Chlorobium tepidum (GenBank Accession No. NC_002932) A ferredoxin II gene can be exemplified. The amino acid sequences of ferredoxin I and ferredoxin II are disclosed in GenBank Accession No. AAM72491 and AAM72490, respectively. In addition, the ferredoxin gene (GenBank Accession No. BAE02673) of Hydrogenobacter thermophilus and the sulphobus bus sequence shown by nucleotide numbers 2345414 to 2345728 in the genome sequence of Sulfolobus solfataricus An example is the ferredoxin gene of Solfataricus. Furthermore, based on the homology with the genes exemplified above, the genera Chlorobium, Desulfobacter, Aquifex, Hydrogenobacter, Thermoproteus, Thermoproteus May be cloned from bacteria belonging to the genus Pyrobaculum, and also γ-proteobacteria such as Enterobacter, Klebsiella, Serratia, Erbinia, Yersinia, Corynebacterium glutamicum, etc. It may be cloned from coryneform bacteria such as Brevibacterium lactofermentum, Pseudomonas bacteria such as Pseudomonas aeruginosa, and Mycobacterium bacteria such as Mycobacterium tuberculosis.
　また、本発明の微生物は、ピルビン酸シンターゼ、又はピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性の増強に加えて、マリックエンザイムの活性が低下していてもよい。本発明の微生物がエシェリヒア属、エンテロバクター属、パントエア属、クレブシエラ属、又はセラチア属に属する細菌である場合は、特にマリックエンザイムの活性を低下させてもよい。 In addition to the enhancement of pyruvate synthase or pyruvate: NADP + oxidoreductase activity, the microorganism of the present invention may have reduced activity of malic enzyme. When the microorganism of the present invention is a bacterium belonging to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia, the activity of malic enzyme may be particularly reduced.
　本発明において、マリックエンザイムの活性とは、リンゴ酸を酸化的に脱炭酸し、ピルビン酸を生成する下記の反応を可逆的触媒する活性を意味する。これらの反応は、ＮＡＤＰを電子受容体とするＮＡＤＰ型マリックエンザイム（malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)とも表記される）（EC:1.1.1.40 b2463遺伝子（maeB遺伝子とも表記される））、あるいは、ＮＡＤを電子受容体とするＮＡＤ型マリックエンザイム（malate dehydrogenase (oxaloacetate-decarboxylating) (NAD+) とも表記される）（EC:1.1.1.38 sfcA遺伝子（maeA遺伝子とも表記される））の２種の酵素によって触媒される。マリックエンザイム活性の確認は、Bolognaらの方法（Bologna, F. P. et al. 2007. J. Bacteriol. 2007 189: 5937-5946）に従って測定することができる。 In the present invention, the activity of malic enzyme means the activity of reversibly catalyzing the following reaction that oxidatively decarboxylates malic acid to produce pyruvic acid. These reactions are NADP-type malic enzyme using NADP as an electron acceptor (also expressed as malate dehydrogenase (oxaloacetate-decarboxylating) (NADP + )) (EC: 1.1.1.40 b2463 gene (also referred to as maeB gene)) 2 of NAD type malic enzyme (also referred to as malate dehydrogenase (oxaloacetate-decarboxylating) (NAD + )) (EC: 1.1.1.38 sfcA gene (also referred to as maeA gene)) using NAD as an electron acceptor Catalyzed by seed enzymes. The confirmation of malic enzyme activity can be measured according to the method of Bologna et al. (Bologna, F. P. et al. 2007. J. Bacteriol. 2007 189: 5937-5946).
NADP-dependent malic enzyme : NADP+ + malate → NADPH + CO2 + pyruvate
NAD-dependent malic enzyme：NAD+ + malate → NADH + CO2 + pyruvate NADP-dependent malic enzyme: NADP + + malate → NADPH + CO 2 + pyruvate
NAD-dependent malic enzyme: NAD + + malate → NADH + CO 2 + pyruvate
　本発明においては、ＮＡＤＰ型マリックエンザイムとＮＡＤ型マリックエンザイムの両方の活性を低下させることがより好ましく、特に、本発明の微生物がエシェリヒア属、エンテロバクター属、パントエア属、クレブシエラ属、又はセラチア属に属する細菌である場合に、両方の型のマリックエンザイムの活性を低下させることが好ましい。 In the present invention, it is more preferable to reduce the activities of both NADP-type and NAD-type malic enzymes. In particular, the microorganism of the present invention belongs to the genus Escherichia, Enterobacter, Pantoea, Klebsiella, or Serratia. When the bacterium belongs, it is preferable to reduce the activity of both types of malic enzyme.
　また、本発明の微生物は、ピルビン酸シンターゼ、又はピルビン酸：ＮＡＤＰ+オキシドレダクターゼの活性の増強に加えて、ピルビン酸デヒドロゲナーゼ活性が低下していることが好ましい。 In addition to the enhanced activity of pyruvate synthase or pyruvate: NADP + oxidoreductase, the microorganism of the present invention preferably has reduced pyruvate dehydrogenase activity.
　本発明において、ピルビン酸デヒドロゲナーゼ（以下、「ＰＤＨ」ということがある）活性とは、ピルビン酸を酸化的に脱炭酸し、アセチル-CoA(acetyl-CoA)を生成する反応を触媒する活性を意味する。この反応は、PDH（E1p：pyruvate dehydrogenase, EC:1.2.4.1、aceE遺伝子によってコードされる）、ジヒドロリポイルトランスアセチラーゼ（E2p：dihydrolipoyltransacetylase, EC:2.3.1.12、aceF遺伝子によってコードされる）、ジヒドロリポアミドデヒドロゲナーゼ（E3：dihydrolipoamide dehydrogenase; EC:1.8.1.4、lpdA遺伝子によってコードされる）の３種の酵素によって触媒される。すなわち、これらの３種類のサブユニットはそれぞれ以下の反応を触媒し、これら３つの反応を合わせた反応を触媒する活性をPDH活性という。PDH活性の確認は、VisserとStratingの方法（Visser, J. and Strating, M. 1982. Methods Enzymol. 89: 391-399）に従って測定することができる。 In the present invention, pyruvate dehydrogenase (hereinafter sometimes referred to as “PDH”) activity means an activity that catalyzes a reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA (acetyl-CoA). To do. This reaction is PDH (E1p: pyruvate dehydrogenase, EC: 1.2.4.1, encoded by aceE gene), dihydrolipoyltransacetylase (E2p: dihydrolipoyltransacetylase, EC: 2.3.1.12, encoded by aceF gene), It is catalyzed by three enzymes: dihydrolipoamide dehydrogenase (E3: dihydrolipoamide dehydrogenase; EC: 1.8.1.4, encoded by the lpdA gene). That is, these three types of subunits each catalyze the following reaction, and the activity of catalyzing the combined reaction of these three reactions is called PDH activity. Confirmation of PDH activity can be measured according to the method of Visser and Strating (Visser, J. and Strating, M. 1982. Methods Enzymol. 89: 391-399).
　E1p: pyruvate + [dihydrolipoyllysine-residue succinyltransferase] lipoyllysine → [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO2
　E2p：CoA + enzyme N6-(S-acetyldihydrolipoyl)lysine → acetyl-CoA + enzyme N6-(dihydrolipoyl)lysine
　E3: protein N6-(dihydrolipoyl)lysine + NAD+ → protein N6-(lipoyl)lysine + NADH + H+ E1p: pyruvate + [dihydrolipoyllysine-residue succinyltransferase] lipoyllysine → [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO 2
E2p: CoA + enzyme N6- (S-acetyldihydrolipoyl) lysine → acetyl-CoA + enzyme N6- (dihydrolipoyl) lysine
E3: protein N6- (dihydrolipoyl) lysine + NAD + → protein N6- (lipoyl) lysine + NADH + H +
　また、本発明の細菌は、マレートシンターゼ・イソシトレートリアーゼ・イソシトレートデヒドロゲナーゼキナーゼ／フォスファターゼオペロン（aceオペロン）が構成的に発現するか、又は同オペロンの発現が強化されるように改変された菌株であってもよい。マレートシンターゼ・イソシトレートリアーゼ・イソシトレートデヒドロゲナーゼキナーゼ／フォスファターゼオペロン（aceオペロン）が構成的に発現するとは、aceオペロンのプロモーターが、リプレッサータンパク質であるiclRにより抑制を受けないこと、抑制が解除されていることを意味する。 The bacterium of the present invention is modified so that malate synthase, isocitrate lyase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) is constitutively expressed, or expression of the operon is enhanced. Strains may be used. The constitutive expression of malate synthase, isocitrate triase, isocitrate dehydrogenase kinase / phosphatase operon (ace operon) means that the ace operon promoter is not repressed by the repressor protein iclR. It means that it has been released.
　aceオペロンを構成的に発現していること、また同オペロンの発現が強化していることは、aceオペロンがコードするタンパク質であるマレートシンターゼ(aceB)、イソシトレートリアーゼ(aceA)、イソシトレートデヒドロゲナーゼキナーゼ／フォスファターゼ（aceK）の酵素活性が非改変株、あるいは野生株と比べて増大していることによって確認出来る。 The constitutive expression of the ace operon and the enhanced expression of the operon are the proteins encoded by the ace operon, malate synthase (aceB), isocitrate triase (aceA), and isocyto This can be confirmed by the fact that the enzyme activity of rate dehydrogenase kinase / phosphatase (aceK) is increased compared to the unmodified strain or the wild strain.
　酵素活性の測定は、マレートシンターゼに関してはグリオキシル酸に依存するアセチルCoAのチオエステル結合の分解をA232の減少で測定する方法(Dixon,G.H.,Kornberg,H.L., 1960, Biochem.J, 1;41:p217-233）、イソシトレートリアーゼに関してはイソシトレートから生じるグリオキシル酸を２，４－ジニトロフェニルヒドラゾン誘導体として測定する方法(Roche,T.E..Williams J.O., 1970, Biochim.Biophys.Acta, 22;206(1):p193-195)、イソシトレートデヒドロゲナーゼキナーゼに関してはイソシトレートデヒドロゲナーゼに対するリン酸の脱着を32Pを使用して測定する方法（Wang, J.Y.J. and Koshland, D.E., Jr., 1982, Arch  Biochem. Biophys., 218, p59-67）などで確認出来る。 Enzyme activity was determined by measuring glyoxylic acid-dependent degradation of the thioester bond of acetyl-CoA by reducing A232 for malate synthase (Dixon, GH, Kornberg, HL, 1960, Biochem. J, 1; 41: p217-233), for isocitrate lyase, a method for measuring glyoxylic acid generated from isocitrate as a 2,4-dinitrophenylhydrazone derivative (Roche, TE. Williams JO, 1970, Biochim. Biophys. Acta, 22; 206 (1 ): p193-195), with respect to isocitrate dehydrogenase kinase, a method of measuring desorption of phosphate to isocitrate dehydrogenase using 32 P (Wang, JYJ and Koshland, DE, Jr., 1982, Arch Biochem. Biophys., 218, p59-67).
　抑制を解除するためには、例えば、aceオペロン上のリプレッサー（iclR）の結合部位を、iclRが結合できないように改変すればよい。また、同オペロンのプロモーターを、iclRによって発現抑制を受けない強力なプロモーター（lacプロモーターなど）に置換することによって、抑制を解除することもできる。 In order to release the suppression, for example, the binding site of the repressor (iclR) on the ace operon may be modified so that iclR cannot bind. In addition, the suppression can be released by replacing the promoter of the operon with a strong promoter (such as the lac promoter) that is not subject to expression suppression by iclR.
　また、iclR遺伝子の発現が低下又は欠失するように細菌を改変することによって、aceオペロンの発現を構成的にすることもできる。具体的には、iclRをコードする遺伝子の発現調節配列を同遺伝子が発現しないように改変するか、同リプレッサーの機能が失われるようにコード領域を改変することによって、aceオペロンの発現の抑制を解除することができる。 Also, the ace operon expression can be made constitutive by modifying the bacterium so that the expression of the iclR gene is reduced or deleted. Specifically, the expression control sequence of the gene encoding iclR is modified so that the gene does not express, or the coding region is modified so that the function of the repressor is lost, thereby suppressing the expression of the ace operon. Can be released.
＜１－２＞AldBタンパク質の活性を低下させる改変
　本発明の細菌は、上述したようなＬ－アミノ酸生産能を有する腸内細菌を、AldBタンパク質の活性が低下するように改変することによって得できる。また、本発明の細菌は、AldBタンパク質の活性が低下するように改変された細菌に、Ｌ－アミノ酸生産能を付与することによっても、取得できる。
　「AldBタンパク質」とは、エシェリヒア・コリK-12株のaldB遺伝子によってコードされるタンパク質、並びにそのホモログ及び人為的改変体等、保存的変異を有するタンパク質をいう。このような保存的変異を有するタンパク質を、保存的バリアントと記載する。保存的バリアントについては後述する。
　「AldBタンパク質の活性」とは、AldBタンパク質の持つアセトアルデヒドデヒドロゲナーゼ活性、すなわちアセトアルデヒドを酢酸に変換する反応を触媒する活性を意味し、特にNADP依存型又はNAD依存型のアセトアルデヒドデヒドロゲナーゼ活性を意味する。このような活性を有するタンパク質は、その名称は問わず、AldBタンパク質に包含される。例えば、パントエア・アナナティスのaldA遺伝子によってコードされるタンパク質は、NAD依存型のアセトアルデヒドデヒドロゲナーゼ活性を有しており、「AldBタンパク質」に包含される。AldBタンパク質は、Co-A非依存型アルデヒドデヒドロゲナーゼ（Co-A independent aldehyde dehydrogenase）とも呼ばれる。AldBタンパクの活性が低下したことの確認はK Hoら（Journal of Bacteriology Feb 2005. Vol 187 No.3 p1067-1073）の方法を用いて確認できる。 <1-2> Modification that reduces AldB protein activity The bacterium of the present invention can be obtained by modifying the enterobacteria having L-amino acid-producing ability as described above so that the activity of AldB protein is reduced. . The bacterium of the present invention can also be obtained by imparting L-amino acid-producing ability to a bacterium modified so that the activity of the AldB protein is reduced.
“AldB protein” refers to a protein having a conservative mutation, such as a protein encoded by the aldb gene of Escherichia coli K-12 strain, and homologs and artificially modified forms thereof. A protein having such a conservative mutation is described as a conservative variant. Conservative variants will be described later.
The “activity of AldB protein” means an acetaldehyde dehydrogenase activity of AldB protein, that is, an activity that catalyzes a reaction of converting acetaldehyde into acetic acid, and particularly an NADP-dependent or NAD-dependent acetaldehyde dehydrogenase activity. The protein having such activity is included in the AldB protein regardless of its name. For example, a protein encoded by the aldA gene of Pantoea ananatis has NAD-dependent acetaldehyde dehydrogenase activity and is included in the “AldB protein”. AldB protein is also called Co-A independent aldehyde dehydrogenase (Co-A independent aldehyde dehydrogenase). Confirmation that the activity of the AldB protein has decreased can be confirmed by the method of K Ho et al. (Journal of Bacteriology Feb 2005. Vol 187 No. 3 p1067-1073).
　尚、「AldBタンパク質の活性が低下する」とは、親株又は野生株等の非改変株に比べてAldBタンパク質の活性が相対的に低下している場合、及び、AldBタンパク質の活性を完全に欠損している場合の両方が含まれる。 “AldB protein activity is reduced” means that the activity of AldB protein is relatively reduced compared to the non-modified strain such as the parent strain or wild strain, and the activity of AldB protein is completely lost. If both are included.
　細菌を、AldBタンパク質の活性が低下するように改変するには、例えば、AldBタンパク質をコードするaldB遺伝子の発現を低下させればよい。 In order to modify bacteria so that the activity of the AldB protein is reduced, for example, the expression of the aldB gene encoding the AldB protein may be reduced.
　aldB遺伝子としては、具体的には、エシェリヒア・コリK-12株のaldB遺伝子（GenBank Accession NC_000913.2 GI:49175990 complement(3752996..3754534)）が挙げられる。同遺伝子の塩基配列を配列番号１に示した。配列番号２には、同遺伝子がコードするAldBタンパク質のアミノ酸配列を示した。また、パントエア・アナナティスLMG 20103株（GenBank Accession NC_013956.1 GI:291617493 complement(2166733..2168205)）、ペクトバクテリウム・アトロセプティカム（Pectobacterium atrosepticum）（旧名、エルビニア・カロトボーラ(Erwinia carotovora)）SCRI1043株（GenBank Accession NC_004547.2 GI:50119055 111626..113161）、サルモネラ・エンテリカ（Salmonella enterica）CT18株（NC_003198.1 GI:16762629 3978586..3980124）のaldB遺伝子がコードするAldBタンパク質のアミノ酸配列を、配列番号９～１１に、各々示した。尚、パントエア・アナナティスでは、AldBタンパク質ホモログをコードする遺伝子はaldA遺伝子と呼ばれているが、この遺伝子はaldB遺伝子、及び同遺伝子がコードするタンパク質はAldBタンパク質として記載する。これらのAldBタンパク質のアラインメントを図１、２に示す。パントエア・アナナティス、ペクトバクテリウム・アトロセプティカム、及び、サルモネラ・エンテリカのAldBは、エシェリヒア・コリのAldBと各々64.7%、81.4%、95.8%の相同性を有している。 Specific examples of the aldB gene include the aldB gene of the Escherichia coli K-12 strain (GenBank Accession NC_000913.2 GI: 49175990 complement (3752996..3754534)). The base sequence of this gene is shown in SEQ ID NO: 1. SEQ ID NO: 2 shows the amino acid sequence of the AldB protein encoded by the same gene. In addition, Pantoea Ananatis LMG 20103 strain (GenBank Accession NC_013956.1 GI: 291617493 complement (2166733..2168205)), Pectobacterium atrosepticum (formerly Erwinia carotovora) SCRI1043 The amino acid sequence of the AldB protein encoded by the aldB gene of the strain (GenBank Accession NC_004547.2 GI: 50119055 111626..113161), Salmonella enterica CT18 strain (NC_003198.1 GI: 16762629 3978586..3980124), These are shown in SEQ ID NOs: 9 to 11, respectively. In Pantoea Ananatis, the gene encoding the AldB protein homolog is called the aldA gene. This gene is described as the aldB gene, and the protein encoded by the gene is described as the AldB protein. The alignment of these AldB proteins is shown in FIGS. The AldB of Pantoea ananatis, Pectobacterium atrocepticam, and Salmonella enterica have 64.7%, 81.4%, and 95.8% homology with those of Escherichia coli, respectively.
　AldBタンパク質は、アセトアルデヒドデヒドロゲナーゼ活性を有する限り、それらのホモログや人為的改変体等、保存的変異を有するタンパク質であってもよい。このような保存的変異を有するタンパク質を、保存的バリアントと記載する。 As long as the AldB protein has acetaldehyde dehydrogenase activity, it may be a protein having a conservative mutation such as a homologue or artificially modified product thereof. A protein having such a conservative mutation is described as a conservative variant.
　AldBタンパク質の保存的バリアントとしては、例えば配列番号２、又は９～１１のアミノ酸配列において、１若しくは数個の位置での１若しくは数個のアミノ酸の置換、欠失、挿入又は付加等を含む配列を有するタンパク質であってもよい。 Examples of conservative variants of AldB protein include, for example, a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of SEQ ID NO: 2 or 9-11 It may be a protein having
　「１若しくは数個」とは、アミノ酸残基のタンパク質の立体構造における位置やアミノ酸残基の種類によっても異なるが、具体的には好ましくは1～20個、より好ましくは1～10個、さらに好ましくは1～5個を意味する。また、保存的変異の代表的なものは、保存的置換である。保存的置換とは、置換部位が芳香族アミノ酸である場合には、Phe、Trp、Tyr間で、置換部位が疎水性アミノ酸である場合には、Leu、Ile、Val間で、極性アミノ酸である場合には、Gln、Asn間で、塩基性アミノ酸である場合には、Lys、Arg、His間で、酸性アミノ酸である場合には、Asp、Glu間で、ヒドロキシル基を持つアミノ酸である場合には、Ser、Thr間でお互いに置換する変異である。保存的置換とみなされる置換としては、具体的には、AlaからSer又はThrへの置換、ArgからGln、His又はLysへの置換、AsnからGlu、Gln、Lys、His又はAspへの置換、AspからAsn、Glu又はGlnへの置換、CysからSer又はAlaへの置換、GlnからAsn、Glu、Lys、His、Asp又はArgへの置換、GluからGly、Asn、Gln、Lys又はAspへの置換、GlyからProへの置換、HisからAsn、Lys、Gln、Arg又はTyrへの置換、IleからLeu、Met、Val又はPheへの置換、LeuからIle、Met、Val又はPheへの置換、LysからAsn、Glu、Gln、His又はArgへの置換、MetからIle、Leu、Val又はPheへの置換、PheからTrp、Tyr、Met、Ile又はLeuへの置換、SerからThr又はAlaへの置換、ThrからSer又はAlaへの置換、TrpからPhe又はTyrへの置換、TyrからHis、Phe又はTrpへの置換、及び、ValからMet、Ile又はLeuへの置換が挙げられる。また、上記のようなアミノ酸の置換、欠失、挿入、付加、または逆位等には、遺伝子が由来する微生物の個体差、種の違いに基づく場合などの天然に生じる変異（mutant又はvariant）によって生じるものも含まれる。このようなタンパク質は、例えば、部位特異的変異法によって、コードされるタンパク質の特定の部位のアミノ酸残基が置換、欠失、挿入または付加を含むように野生型aldB遺伝子の塩基配列を改変することによって取得することができる。 “One or several” amino acid residues vary depending on the position of the protein in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1-20, more preferably 1-10, Preferably 1 to 5 are meant. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the microorganism from which the gene is derived. Also included by Such a protein is modified by, for example, site-directed mutagenesis so that the amino acid residue at a specific site of the encoded protein contains a substitution, deletion, insertion, or addition, so that the nucleotide sequence of the wild-type aldB gene is modified. Can be obtained by
　さらに、上記のような保存的変異を有するタンパク質は、アミノ酸配列全体に対して、例えば80%以上、好ましくは90%以上、より好ましくは95%以上、より好ましくは97%以上、より好ましくは98%以上、特に好ましくは99%以上の相同性を有し、かつ、野生型タンパク質と同等の機能を有するタンパク質であってもよい。尚、本明細書において、「相同性」（homology）」は、「同一性」（identity）を指すことがある。 Furthermore, the protein having the conservative mutation as described above is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more with respect to the entire amino acid sequence. It may be a protein having a homology of at least%, particularly preferably at least 99%, and having a function equivalent to that of a wild-type protein. In the present specification, “homology” may refer to “identity”.
　野生型aldB遺伝子は、上記のようなアミノ酸配列をコードするものであれば、エシェリヒア・コリ、パントエア・アナナティス、及びエンテロバクター・アエロゲネス等の遺伝子に限らず、任意のコドンをそれと等価のコドンに置換したものであってもよい。 The wild-type aldB gene is not limited to genes such as Escherichia coli, Pantoea ananatis, and Enterobacter aerogenes, as long as it encodes the amino acid sequence as described above. It may be what you did.
　また、野生型aldB遺伝子は、配列番号１の塩基配列と相補的な配列、又はその相補的な配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、アセトアルデヒドデヒドロゲナーゼ活性を有するタンパク質をコードするDNAであってもよい。ここで、「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。一例を示せば、相同性が高いDNA同士、例えば80%以上、好ましくは90%以上、より好ましくは95%以上、より好ましくは97%以上、より好ましくは98%以上、特に好ましくは99%以上の相同性を有するDNA同士がハイブリダイズし、それより相同性が低いDNA同士がハイブリダイズしない条件、あるいは通常のサザンハイブリダイゼーションの洗いの条件である60℃、1×SSC、0.1% SDS、好ましくは、0.1×SSC、0.1% SDS、さらに好ましくは、68℃、0.1×SSC、0.1% SDSに相当する塩濃度、温度で、１回、より好ましくは２～３回洗浄する条件が挙げられる。 The wild-type aldB gene is a protein that hybridizes under stringent conditions with a probe complementary to the nucleotide sequence of SEQ ID NO: 1, or a probe that can be prepared from the complementary sequence, and that has acetaldehyde dehydrogenase activity. It may be DNA encoding. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, particularly preferably 99% or more. 60 ° C, 1 × SSC, 0.1% SDS, which is a condition in which DNAs having homology with each other hybridize and DNAs having lower homology do not hybridize with each other, or normal Southern hybridization washing conditions, preferably The conditions include washing once, more preferably 2 to 3 times at a salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS.
　プローブとしては、aldB遺伝子の相補配列の一部を用いることもできる。そのようなプローブは、公知の遺伝子配列に基づいて作製したオリゴヌクレオチドをプライマーとし、これらの塩基配列を含むDNA断片を鋳型とするPCRによって作製することができる。例えば、プローブとして、300 bp程度の長さのDNA断片を用いる場合には、ハイブリダイゼーションの洗いの条件は、50℃、2×SSC、0.1% SDSが挙げられる。 As the probe, a part of the complementary sequence of the aldB gene can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.
　上記のようなタンパク質の保存的バリアントをコードする遺伝子は、野生型遺伝子の「保存的バリアント」である。 A gene encoding a conservative variant of a protein as described above is a “conservative variant” of a wild-type gene.
　上記した保存的バリアント及びそれをコードする遺伝子に関する記載は、前記のＬ－アミノ酸生産菌について記載した他の遺伝子、及び後述のadhE遺伝子についても同様に適用される。 The above description concerning the conservative variant and the gene encoding the same applies to the other genes described above for the L-amino acid-producing bacteria and the adhE gene described later.
　以下、AldBタンパク質の活性を低下させる改変について、具体的に説明する。
　AldBタンパク質の活性を低下させるような改変は、例えば、aldB遺伝子の発現を低下させることによって達成される。具体的には例えば、染色体上のaldB遺伝子のコード領域の一部又は全部を欠損させることによって、AldBタンパク質の細胞内の活性を低下させることができる。 Hereinafter, the modification which reduces the activity of AldB protein is demonstrated concretely.
Modifications that reduce the activity of the AldB protein are achieved, for example, by reducing the expression of the aldB gene. Specifically, for example, the intracellular activity of the AldB protein can be reduced by deleting part or all of the coding region of the aldB gene on the chromosome.
　またAldBタンパク質活性の低下は、aldB遺伝子のプロモーターやシャインダルガノ（SD）配列等の発現調節配列を改変することなどによって、同遺伝子の発現を低下させることによっても、達成することができる。また、発現調節配列以外の非翻訳領域の改変によっても、遺伝子の発現量を低下させることができる。さらには、染色体上のaldB遺伝子の前後の配列を含めて、同遺伝子全体を欠失させてもよい。また、染色体上のaldB遺伝子のコード領域にアミノ酸置換（ミスセンス変異）を導入すること、また終始コドンを導入すること（ナンセンス変異）、あるいは一～二塩基付加・欠失するフレームシフト変異を導入することによっても、遺伝子の発現を低下させることが出来る（J. Biol. Chem. 272:8611-8617（1997）, Proc. Natl. Acad. Sci. USA, 95:5511-5515 (1998), J. Biol. Chem. 266:20833-20839 (1991)）。 Moreover, the reduction in AldB protein activity can also be achieved by reducing the expression of the gene by modifying the expression regulatory sequence such as the promoter of aldB gene and Shine-Dalgarno (SD) sequence. In addition, the expression level of a gene can also be reduced by modifying an untranslated region other than the expression regulatory sequence. Furthermore, the entire gene including the sequence before and after the aldB gene on the chromosome may be deleted. Also, introduce amino acid substitutions (missense mutations) into the coding region of the aldB gene on the chromosome, introduce stop codons (nonsense mutations), or introduce frameshift mutations that add or delete one or two bases. The gene expression can also be reduced (J. Biol. Chem. 272: 8611-8617 (1997), Proc. Natl. Acad. Sci. USA, 95: 5511-5515 (1998), J. Biol. Chem. 266: 20833-20839 (1991)).
　また、AldBタンパク質の活性が低下するような改変であれば、Ｘ線もしくは紫外線照射、またはＮ-メチル-Ｎ'-ニトロ-Ｎ-ニトロソグアニジン等の変異剤による通常の変異処理による改変であってもよい。 In addition, if the modification reduces the activity of the AldB protein, the modification may be performed by X-ray or ultraviolet irradiation, or a normal mutation treatment with a mutation agent such as N-methyl-N′-nitro-N-nitrosoguanidine. Also good.
　発現調節配列の改変は、好ましくは１塩基以上、より好ましくは２塩基以上、特に好ましくは３塩基以上である。また、コード領域を欠失させる場合は、AldBタンパク質の活性が低下するのであれば、欠失させる領域は、Ｎ末端領域、内部領域、Ｃ末端領域のいずれの領域であってもよく、コード領域全体であってよい。通常、欠失させる領域は長い方が確実に遺伝子を不活化することができる。また、欠失させる領域の上流と下流のリーディングフレームは一致しないことが好ましい。 The modification of the expression regulatory sequence is preferably 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. When the coding region is deleted, the region to be deleted may be any of the N-terminal region, the internal region, and the C-terminal region as long as the activity of the AldB protein is reduced. It may be the whole. Usually, the longer region to be deleted can surely inactivate the gene. Further, it is preferable that the reading frames upstream and downstream of the region to be deleted do not match.
　aldB遺伝子のコード領域に他の配列を挿入する場合も、挿入の箇所は遺伝子のいずれの領域であってもよいが、挿入する配列は長い方が、確実に遺伝子を不活化することができる。挿入部位の前後の配列は、リーディングフレームが一致しないことが好ましい。他の配列としては、コードされるAldBタンパク質の機能を低下させるものであれば特に制限されないが、例えば、抗生物質耐性遺伝子やＬ-アミノ酸生産に有用な遺伝子を搭載したトランスポゾン等が挙げられる。 When another sequence is inserted into the coding region of the aldB gene, the insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. The sequences before and after the insertion site preferably do not match the reading frame. Other sequences are not particularly limited as long as they reduce the function of the encoded AldB protein, and examples thereof include antibiotic resistance genes and transposons carrying genes useful for L-amino acid production.
　例えば、aldBタンパク質の活性を低下させるような変異を遺伝子組換えにより導入する為には、以下のような方法が用いられる。aldB遺伝子の部分配列を改変し、正常に機能するAldBタンパク質を産生しないようにした変異型遺伝子を作製し、該遺伝子を含むＤＮＡで腸内細菌科に属する微生物に形質転換し、変異型遺伝子とゲノム上の遺伝子で組換えを起こさせることにより、ゲノム上のaldB遺伝子を変異型に置換することが出来る。このような相同組換えを利用した遺伝子置換は、「Redドリブンインテグレーション(Red-driven integration)」と呼ばれる方法（Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97:6640-6645 (2000)）、及び、Redドリブンインテグレーション法とλファージ由来の切り出しシステム（Cho, E. H., Gumport, R. I., Gardner, J. F. J. Bacteriol. 184: 5200-5203 (2002)）と組合わせた方法（WO2005/010175号参照）等の直鎖状ＤＮＡを用いる方法や、温度感受性複製起点を含むプラスミドを用いる方法などがある（Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97:6640-6645(2000); 米国特許第6303383号; 特開平05-007491号公報）。また、上述のような相同組換えを利用した遺伝子置換により部位特異的変異導入は、宿主上で複製能力を持たないプラスミドを用いても行うことが出来る。 For example, in order to introduce a mutation that reduces the activity of the aldB protein by genetic recombination, the following method is used. A partial gene of the aldB gene is modified to produce a mutant gene that does not produce a normally functioning AldB protein, transformed into a microorganism belonging to the family Enterobacteriaceae with a DNA containing the gene, By causing recombination with a gene on the genome, the aldB gene on the genome can be replaced with a mutant. The gene replacement using such homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)) and Red-driven integration method and excision system derived from λ phage (Cho, E. H., Gumport, R. I., Gardner, J. F. J.olBacteriol 184: 5200-5203 (2002)) (Methods using linear DNA such as WO2005 / 010175) and methods using plasmids containing temperature-sensitive replication origins (Datsenko, KA) , AndnerWanner, B. L. Proc. Natl.tlAcad. Sci. U S A. 97: 6640-6645 (2000); U.S. Pat. No. 6,303,383; In addition, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host.
　上記したAldBタンパク質の活性を低下させる方法は、前記のＬ－アミノ酸生産能の付与又は増強における酵素活性の低下においても、同様に適用することができる。 The above-described method for reducing the activity of the AldB protein can be similarly applied to the reduction of the enzyme activity in imparting or enhancing the L-amino acid producing ability.
＜１－３＞adhEタンパク質の活性を増強する改変
　本発明の細菌は、AdhEタンパク質の活性が増強されていることが好ましい。
　エシェリヒア・コリに関しては、嫌気条件でエタノールを生成する酵素として、以下の反応を可逆的に触媒する、アセトアルデヒドデヒドロゲナーゼ活性とアルコールデヒドロゲナーゼ活性を有するAdhEの存在が知られている。AdhEタンパク質の活性とは、これらの反応を触媒する活性を意味し、「AdhEタンパク質の活性が増強されている」とは、特に、少なくともアセトアルデヒドから直接アセチル-CoAを生成する反応を触媒する活性が増強されていることを意味する。AdhEタンパク質は、Co-A依存型アルデヒドデヒドロゲナーゼ（Co-A dependent aldehyde dehydrogenase）とも呼ばれる。
　エシェリヒア・コリのAdhEをコードするadhE遺伝子の配列は、WO2009/031565、米国特許出願公開第2009068712号に開示されている。また、エシェリヒア・コリK-12株のadhE遺伝子（NC_000913.2 GI:49175990 1294669..1297344）の塩基配列を配列番号３に、アミノ酸配列を配列番号４に示す。また、パントエア・アナナティスLMG 20103株（GenBank Accession NC_013956.1 GI:291617642 complement(4631008..4632396) ）、ペクトバクテリウム・アトロセプティカム（Pectobacterium atrosepticum）（旧名、エルビニア・カロトボーラ(Erwinia carotovora)）SCRI1043株（GenBank Accession NC_004547.2 GI:50121254 2634501..2637176）、及び、サルモネラ・エンテリカ（Salmonella enterica）CT18株（NC_003198.1 GI:16760134 complement(1259893..1262571)）のadhE遺伝子がコードするAdhEタンパク質のアミノ酸配列を、配列番号１２～１４に、各々示した。これらのAdhEタンパク質のアラインメントを図３～５に示す。 <1-3> Modification that enhances the activity of adhE protein The bacterium of the present invention preferably has enhanced activity of the AdhE protein.
Regarding Escherichia coli, the presence of AdhE having acetaldehyde dehydrogenase activity and alcohol dehydrogenase activity, which reversibly catalyzes the following reaction, is known as an enzyme that produces ethanol under anaerobic conditions. The activity of AdhE protein means the activity of catalyzing these reactions, and “the activity of AdhE protein is enhanced” means that the activity of catalyzing at least the reaction of directly producing acetyl-CoA from acetaldehyde. It means being strengthened. AdhE protein is also called Co-A dependent aldehyde dehydrogenase.
The sequence of the adhE gene encoding AdhE of Escherichia coli is disclosed in WO2009 / 031565, US Patent Application Publication No. 2009068712. In addition, the base sequence of the adhE gene (NC_000913.2 GI: 49175990 1294669..1297344) of Escherichia coli K-12 strain is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. In addition, Pantoea Ananatis LMG 20103 strain (GenBank Accession NC_013956.1 GI: 291617642 complement (4631008..4632396)), Pectobacterium atrosepticum (former name: Erwinia carotovora) SCRI1043 Strain (GenBank Accession NC_004547.2 GI: 50121254 2634501..2637176) and AdhE protein encoded by the adhE gene of Salmonella enterica CT18 strain (NC_003198.1 GI: 16760134 complement (1259893..1262571)) Are shown in SEQ ID NOs: 12 to 14, respectively. The alignment of these AdhE proteins is shown in FIGS.
アセチル-CoA + NADH + H+ → アセトアルデヒド + NAD+ + CoA
アセトアルデヒド + NADH + H+ → エタノール + NAD+ Acetyl-CoA + NADH + H + → Acetaldehyde + NAD + + CoA
Acetaldehyde + NADH + H + → Ethanol + NAD +
　AdhEタンパク質の活性は、例えば、adhE遺伝子の発現を増強することによって、増強することができる。
　以下に、adhE遺伝子の発現を増強する方法について説明する。これらの方法は、前記のＬ－アミノ酸生産菌について記載した遺伝子についても、適用することができる。 The activity of the AdhE protein can be enhanced, for example, by enhancing the expression of the adhE gene.
Hereinafter, a method for enhancing the expression of the adhE gene will be described. These methods can also be applied to the genes described above for L-amino acid producing bacteria.
　１つ目の方法は、目的遺伝子のコピー数を高める方法である。例えば、目的遺伝子を適当なベクター上にクローニングし、得られたベクターを用いて宿主細菌を形質転換することにより、該遺伝子のコピー数を高めることができる。
　形質転換に用いるベクターとしては、使用する微生物で自律複製可能なプラスミドが挙げられる。例えば、腸内細菌群に属する微生物の中で自律複製可能なプラスミドとして、pUC19、pUC18、pBR322、RSF1010、pHSG299、pHSG298、pHSG399、pHSG398、pSTV28、pSTV29（pHSG、pSTVはタカラバイオ社より入手可能）、pMW119、pMW118、pMW219、pMW218（pMWはニッポンジーン社より入手可能）等が挙げられる。なお、プラスミドの代わりにファージDNAをベクターとして用いてもよい。 The first method is to increase the copy number of the target gene. For example, the copy number of the gene can be increased by cloning the target gene on an appropriate vector and transforming the host bacterium with the obtained vector.
Examples of the vector used for transformation include a plasmid capable of autonomous replication with the microorganism used. For example, pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV are available from Takara Bio Inc.) , PMW119, pMW118, pMW219, pMW218 (pMW is available from Nippon Gene). In addition, phage DNA may be used as a vector instead of a plasmid.
　形質転換法としては、例えば、エシェリヒア・コリ K-12について報告されているような、受容菌細胞を塩化カルシウムで処理してDNAの透過性を増す方法（Mandel, M. and Higa, A.,J. Mol. Biol. 1970, 53, 159-162）、及び、電気パルス法（特開平2-207791号公報）等が挙げられる。 As a transformation method, for example, a method in which a recipient cell is treated with calcium chloride to increase DNA permeability as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), and electric pulse method (Japanese Patent Laid-Open No. 2-207791).
　遺伝子のコピー数を高めることは、目的遺伝子を微生物の染色体DNA上に多コピー導入することによっても達成できる。微生物の染色体DNA上に遺伝子を多コピーで導入するには、染色体DNA上に多コピー存在する配列を標的に利用して、相同組換え法（MillerI, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory）により行うことができる。染色体DNA上に多コピー存在する配列としては、レペティティブDNA、転移因子の端部に存在するインバーテッド・リピートが利用できる。あるいは、特開平2-109985号公報に開示されているように、目的遺伝子をトランスポゾンに搭載してこれを転移させて染色体DNA上に多コピー導入することも可能である。さらに、Muファージを用いる方法（特開平2-109985号）で宿主染色体に目的遺伝子を組み込むこともできる。染色体上に目的遺伝子が転移したことの確認は、その遺伝子の一部をプローブとして、サザンハイブリダイゼーションを行うことによって確認出来る。 Increase in gene copy number can also be achieved by introducing multiple copies of the target gene onto the chromosomal DNA of the microorganism. In order to introduce a gene in multiple copies on the chromosomal DNA of a microorganism, a homologous recombination method (MillerI, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in JP-A-2-109985, a target gene can be mounted on a transposon, transferred, and introduced in multiple copies on chromosomal DNA. Furthermore, the target gene can also be incorporated into the host chromosome by a method using Mu phage (Japanese Patent Laid-Open No. 2-109985). Confirmation that the target gene has been transferred onto the chromosome can be confirmed by Southern hybridization using a part of the gene as a probe.
　尚、遺伝子のコピー数を高める場合、目的遺伝子の産物の活性を増強できれば、コピー数は特に制限されないが、微生物がもともと目的遺伝子を有している場合は、２以上であることが好ましい。また、微生物が本発明の遺伝子をもともと有していない場合は、導入される遺伝子のコピー数は１であってもよいが、２以上であってもよい。 In addition, when increasing the copy number of the gene, the copy number is not particularly limited as long as the activity of the target gene product can be enhanced. However, when the microorganism originally has the target gene, it is preferably 2 or more. Further, when the microorganism originally does not have the gene of the present invention, the number of copies of the introduced gene may be 1 or 2 or more.
　２つ目の方法は、染色体DNA上またはプラスミド上において、目的遺伝子のプロモーター等の発現調節配列を適切な強さのものに置換することによって目的遺伝子の発現を増強させる方法である。例えば、thrプロモーター、lacプロモーター、trpプロモーター、trcプロモーター、pLプロモーター、tacプロモーター等がよく用いられるプロモーターとして知られている。プロモーターの強度の評価法および強力なプロモーターの例は、GoldsteinとDoiの論文（Goldstein, M. A. and Doi R. H. 1995. Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128）等に記載されている。 The second method is a method for enhancing expression of a target gene by substituting an expression regulatory sequence such as a promoter of the target gene with an appropriate strength on a chromosomal DNA or a plasmid. For example, thr promoter, lac promoter, trp promoter, trc promoter, pL promoter, tac promoter and the like are known as frequently used promoters. Methods for assessing promoter strength and examples of strong promoters are described in Goldstein and Doi (Goldstein, M. A. and Doi R. H. 1995. Prokaryotic promoters in biotechnology. Biotechnol.techAnnu. Rev., 1, 105- 128).
　また、国際公開WO00/18935に開示されているように、遺伝子のプロモーター領域に数塩基の塩基置換を導入し、適切な強度のものに改変することも可能である。発現調節配列の置換は、例えば、温度感受性プラスミドを用いた遺伝子置換と同様にして行うことができる。エシェリヒア・コリや、パントエア・アナナティスに用いることが出来る、温度感受性複製起点を有するベクターとしては、例えばWO 99/03988号国際公開パンフレットに記載の温度感受性プラスミドpMAN997やその誘導体等が挙げられる。また、λファージのレッド・リコンビナーゼ（Red recombinase）を利用した「Redドリブンインテグレーション(Red-driven integration)」と呼ばれる方法（Datsenko, K. A. and Wanner, B. L., 2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645）や、Redドリブンインテグレーション法とλファージ由来の切り出しシステム（Cho, E. H. et al. Bacteriol. 184: 5200-5203 (2002)）とを組合わせた方法（WO2005/010175号参照）等の直鎖状ＤＮＡを用いる方法によっても、発現調節配列の置換を行うことができる。なお、発現調節配列の改変は、上述したような遺伝子のコピー数を高める方法と組み合わせてもよい。 Also, as disclosed in International Publication WO00 / 18935, it is possible to introduce a base substitution of several bases into the promoter region of a gene and modify it to have an appropriate strength. The replacement of the expression regulatory sequence can be performed, for example, in the same manner as the gene replacement using a temperature sensitive plasmid. Examples of vectors having a temperature-sensitive replication origin that can be used for Escherichia coli and Pantoea ananatis include the temperature-sensitive plasmid pMAN997 and derivatives thereof described in WO99 / 03988 International Publication Pamphlet. In addition, a method called “Red-driven integration” using red recombinase of λ phage (Datsenko, K. A. and Wanner, B. L., 2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645) and Red driven integration method and λ phage-derived excision system (Cho, E. H. et al. Bacteriol. 184: 5200-5203 (2002)) The expression regulatory sequence can also be replaced by a method using linear DNA, such as the other methods (see WO2005 / 010175). The modification of the expression regulatory sequence may be combined with the method for increasing the gene copy number as described above.
　さらに、リボソーム結合部位（ＲＢＳ）と開始コドンとの間のスペーサ、特に開始コドンのすぐ上流の配列における数個のヌクレオチドの置換がmRNAの翻訳効率に非常に影響を及ぼすことが知られており、これらを改変することによって、翻訳量を向上させることが可能である。 Furthermore, it is known that the substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, greatly affects the translation efficiency of mRNA, It is possible to improve the amount of translation by modifying these.
　adhE遺伝子の発現が親株、例えば野生株や非改変株と比べて向上していることの確認は、同遺伝子のmRNAの量を野生型、あるいは非改変株と比較することによって確認出来る。発現量の確認方法としては、ノーザンハイブリダイゼーション法、RT-PCR法が挙げられる（Molecular cloning（Cold spring Harbor Laboratory Press,Cold spring Harbor(USA),2001））。発現量については、野生株あるいは非改変株と比較して、上昇していればいずれでもよいが、例えば野生株、非改変株と比べて１．５倍以上、より好ましくは２倍以上、さらに好ましくは３倍以上上昇していることが望ましい。 Confirmation that the expression of the adhE gene is improved compared to the parent strain, for example, a wild strain or an unmodified strain, can be confirmed by comparing the amount of mRNA of the same gene with a wild type or an unmodified strain. Examples of the expression level confirmation method include Northern hybridization and RT-PCR (Molecularolecloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The expression level may be any as long as it is increased compared to the wild strain or the unmodified strain, but for example, 1.5 times or more, more preferably 2 times or more compared to the wild strain or the non-modified strain, It is desirable that it rises 3 times or more.
　また、本発明の細菌は、好気的にエタノールを資化できることが好ましい。エシェリヒア・コリは、好気条件ではエタノールは資化できないが、好気的にエタノールを資化できるように改変された株を用いてもよい。元来好気的にエタノールを資化できない細菌を、好気的にエタノールを資化できるように改変するには、例えば、好気条件で機能する非天然型プロモーターの制御下で発現するように改変されたadh遺伝子を保持させること、又は、好気的にエタノールを資化できることを可能にする変異をコード領域内に有する変異型adhE遺伝子を保持させることが挙げられる（Clark, D. P., and Cronan, J. E. Jr. 1980. J. Bacteriol. 144: 179-184; Membrillo-Hernandez, J. et al. 2000. J. Biol. Chem. 275: 33869-33875）。さらに、この変異型adhE遺伝子は、好気条件で機能する非天然型プロモーターの制御下で発現するものであってもよい。 Moreover, it is preferable that the bacterium of the present invention can assimilate ethanol aerobically. Although Escherichia coli cannot assimilate ethanol under aerobic conditions, a strain modified so as to assimilate ethanol aerobically may be used. In order to modify bacteria that are not aerobically assimilating ethanol aerobically so that they can assimilate ethanol, for example, expression under the control of a non-native promoter that functions under aerobic conditions. Examples include retaining a modified adh gene or retaining a mutant adhE gene having a mutation in the coding region that enables aerobic assimilation of ethanol (Clark, D. P. , And Cronan, J. E. Jr. 1980. J. Bacteriol. 144: 179-184; Membrillo-Hernandez, J. et al. 2000. J. Biol. Chem. 275: 33869-33875). Furthermore, this mutant adhE gene may be expressed under the control of a non-natural promoter that functions under aerobic conditions.
　エシェリヒア・コリは、アルコールデヒドロゲナーゼをコードする遺伝子の上流のプロモーターを好気的に機能するプロモーターに置換することによって、好気条件でアルコールデヒドロゲナーゼが発現し、好気的にエタノールを資化できるようになる（WO2008/010565号パンフレット）。好気条件で機能する非天然型プロモーターとして、好気条件で或る特定レベルを超えてadhE遺伝子を発現することができる任意のプロモーターを用いることができる。好気条件は、振盪、通気及び撹拌等の方法によって酸素が供給される細菌の培養に通常用いられるものであり得る。具体的には、好気条件で遺伝子を発現することが知られている任意のプロモーターを用いることができる。例えば、解糖系、ペントースリン酸経路、ＴＣＡサイクル、アミノ酸生合成経路等に関与する遺伝子のプロモーターを用いることができる。さらに、Ptacプロモーター、lacプロモーター、trpプロモーター、trcプロモーター、PRプロモーター、又はλファージのPLプロモーターは全て、好気条件で機能する強いプロモーターであることが知られており、これらを用いることが好ましい。 In Escherichia coli, alcohol dehydrogenase can be expressed under aerobic conditions and ethanol can be assimilated aerobically by replacing the promoter upstream of the gene encoding alcohol dehydrogenase with a promoter that functions aerobically. (WO2008 / 010565 pamphlet). As a non-natural promoter that functions under aerobic conditions, any promoter that can express the adhE gene beyond a certain level under aerobic conditions can be used. Aerobic conditions can be those normally used for culturing bacteria that are supplied with oxygen by methods such as shaking, aeration and agitation. Specifically, any promoter known to express a gene under aerobic conditions can be used. For example, promoters of genes involved in glycolysis, pentose phosphate pathway, TCA cycle, amino acid biosynthesis pathway, etc. can be used. Furthermore, it is known that the Ptac promoter, lac promoter, trp promoter, trc promoter, PR promoter, or PL promoter of lambda phage are all strong promoters that function under aerobic conditions, and these are preferably used.
　前記のような変異を有するAdhE変異体として具体的には、エシェリヒア・コリのAdhEの568位のグルタミン酸残基がグルタミン酸及びアスパラギン酸以外のアミノ酸残基、例えばリジンで置換された変異体（Glu568Lys、E568K）がある（国際公開パンフレットWO2008/010565号公報）。 Specifically, as an AdhE mutant having the mutation as described above, a mutant in which the glutamic acid residue at position 568 of AdhE of Escherichia coli is substituted with an amino acid residue other than glutamic acid and aspartic acid, for example, lysine (Glu568Lys, E568K) (International Publication Pamphlet WO2008 / 010565).
　さらに、前記AdhE変異体は、以下の追加的変異を含んでいてもよい。
Ａ）560位のグルタミン酸残基の他のアミノ酸残基、例えばリジン残基への置換
Ｂ）566位のフェニルアラニン残基の他のアミノ酸残基、例えばバリン残基への置換、
Ｃ）22位のグルタミン酸残基、236位のメチオニン残基、461位のチロシン残基、554位のイソロイシン残基、及び786位のアラニン残基の他のアミノ酸残基、例えばそれぞれグリシン残基、バリン残基、システイン残基、セリン残基、及びバリン残基への置換、又は
Ｄ）上記変異の組合わせ。 Furthermore, the AdhE mutant may contain the following additional mutations.
A) Substitution of a glutamic acid residue at position 560 with another amino acid residue, such as a lysine residue B) Substitution of a phenylalanine residue at position 566 with another amino acid residue, such as a valine residue,
C) Glutamic acid residue at position 22, methionine residue at position 236, tyrosine residue at position 461, isoleucine residue at position 554, and other amino acid residues at position 786, such as a glycine residue, respectively. Substitution to valine residue, cysteine residue, serine residue, and valine residue, or D) Combination of the above mutations.
　他の微生物のAdhEについても、上記と同様の変異を持つ変異体を用いることができる。パントエア・アナナティス、ペクトバクテリウム・アトロセプティカム、及び、サルモネラ・エンテリカのAdhEは、エシェリヒア・コリのAdhEと各々89.0%、89.1%、97.2%の相同性を有している。各々のAdhEにおいて、上記の変異の位置に相当するアミノ酸残基は、図２に示したアラインメントにしたがって特定することできる。他の微生物のadhE遺伝子を用いる場合も、コードされるAdhEのアミノ酸配列と公知のAdhEのアミノ酸配列とのアラインメントを作成することにより、変異を導入する位置を特定することができる。 For AdhE of other microorganisms, mutants having the same mutations as described above can be used. Pantoea ananatis, Pectobacterium atrocepticam, and Salmonella enterica AdhE have 89.0%, 89.1%, and 97.2% homology, respectively, with Escherichia coli AdhE. In each AdhE, the amino acid residue corresponding to the position of the mutation can be identified according to the alignment shown in FIG. Even when the adhE gene of another microorganism is used, the position where the mutation is introduced can be specified by creating an alignment between the encoded amino acid sequence of AdhE and the known amino acid sequence of AdhE.
　「好気的にエタノールを資化できる」とは、エタノールを単一炭素源とする最少液体培地もしくは固体培地にて、好気条件で生育可能であることを意味する。「好気条件」は前記と同様に、振盪、通気及び撹拌等の方法によって酸素が供給される細菌の培養に通常用いられるものであり得る。また、「好気的にエタノールを資化できる」とは、ＡｄｈＥタンパク質のレベルに関して、Clark及びCronan（J. Bacteriol., 141, 177-183 (1980））の方法によって測定された無細胞抽出物におけるアルコールデヒドロゲナーゼ活性が、タンパク質１ｍｇ当たり１．５ユニット以上、好ましくは５ユニット以上、及びより好ましくは１０ユニット以上であることを意味する。 “Aerobically assimilate ethanol” means that it can grow in aerobic conditions in a minimal liquid medium or solid medium using ethanol as a single carbon source. “Aerobic conditions” can be those commonly used for culturing bacteria to which oxygen is supplied by methods such as shaking, aeration and agitation, as described above. “Aerobic assimilation of ethanol” means a cell-free extract measured by the method of Clark and Cronan (J. Bacteriol., 141, 177-183 (1980)) with respect to the level of AdhE protein. It means that the alcohol dehydrogenase activity in is 1.5 units or more, preferably 5 units or more, and more preferably 10 units or more per mg of protein.
　また、本発明の細菌は、リボヌクレアーゼＧの活性が低下するように改変されていてもよい。 In addition, the bacterium of the present invention may be modified so that the activity of ribonuclease G is reduced.
＜２＞Ｌ－アミノ酸の製造法
　本発明の細菌を、エタノールを炭素源として含む培地で培養し、該培地からＬ－アミノ酸を採取することにより、Ｌ－アミノ酸を製造することができる。 <2> Method for Producing L-Amino Acid L-amino acid can be produced by culturing the bacterium of the present invention in a medium containing ethanol as a carbon source and collecting L-amino acid from the medium.
　本発明の方法で使用する培地に含まれるエタノールの量は、本発明の方法に使用する細菌が炭素源として資化できる限り幾らでもよいが、培地中に単独の炭素源として添加する場合、１０w/v%以下、好ましくは５w/v%以下、さらに好ましくは２w/v%以下含まれることが好ましい。また、培地中に単独の炭素源として添加する場合、０．２w/v%以上、好ましくは０．５w/v%以上、さらに好ましくは１．０w/v%以上含まれていることが望ましい。 The amount of ethanol contained in the medium used in the method of the present invention may be any amount as long as the bacterium used in the method of the present invention can assimilate as a carbon source. / v% or less, preferably 5 w / v% or less, more preferably 2 w / v% or less. In addition, when added as a single carbon source in the medium, it is desirable that it be contained at 0.2 w / v% or more, preferably 0.5 w / v% or more, more preferably 1.0 w / v% or more.
　さらに、本発明の方法に使用する培地には、エタノールに加え、他の炭素源を添加してもよい。好ましいのは、グルコース、フラクトース、スクロース、ラクトース、ガラクトース、廃糖蜜、澱粉加水分解物やバイオマスの加水分解により得られた糖液などの糖類、フマール酸、クエン酸、コハク酸等の有機酸類である。なお他の炭素源を用いる場合には、炭素源中のエタノールの比率が１０重量％以上、好ましくは３０重量％以上、より好ましくは５０重量％以上であることが好ましい。 Furthermore, other carbon sources may be added to the medium used in the method of the present invention in addition to ethanol. Preferred are sugars such as glucose, fructose, sucrose, lactose, galactose, molasses, starch hydrolyzate and molasses obtained by hydrolysis of biomass, and organic acids such as fumaric acid, citric acid and succinic acid. . When other carbon sources are used, the ethanol ratio in the carbon source is preferably 10% by weight or more, preferably 30% by weight or more, more preferably 50% by weight or more.
　また、流加培地として使用する場合は、流加培地に単独の炭素源として添加する場合、流加後の培地中の濃度が５w/v%以下、好ましくは２w/v%以下、さらに好ましくは１w/v%以下で含まれることが好ましい。また、流加培地に単独の炭素源として添加する場合、０．０１w/v%以上、好ましくは０．０２w/v%以上、さらに好ましくは０．０５w/v%以上の量にて制御することが好ましい。 When used as a fed-batch medium, when added as a single carbon source to the fed-batch medium, the concentration in the medium after fed-batch is 5 w / v% or less, preferably 2 w / v% or less, more preferably It is preferably contained at 1 w / v% or less. In addition, when added as a single carbon source to the fed-batch medium, it should be controlled in an amount of 0.01 w / v% or more, preferably 0.02 w / v% or more, more preferably 0.05 w / v% or more. Is preferred.
　なお、本発明において、エタノールは、培養の全工程において一定濃度含まれてもよいし、流加培地のみあるいは初発培地のみに添加されていてもよく、その他の炭素源が充足していれば、一定時間エタルールが不足している期間があってもよい。一定時間とは、例えば発酵全体の時間のうち１０％、２０％、最大で３０％の時間でエタノールが不足していてもよい。このように一時的にエタノールの濃度が０になることがあっても、エタノールを含む培地での培養期間が存在する場合は、本発明の「エタノールを含む培地で培養する」との文言に含まれる。 In the present invention, ethanol may be contained at a constant concentration in all the steps of the culture, may be added only to the fed-batch medium or only to the initial medium, and if other carbon sources are satisfied, There may be a period where the etarule is insufficient for a certain period of time. For example, ethanol may be deficient in the time of 10%, 20%, and 30% at the maximum of the entire fermentation time. Thus, even if the ethanol concentration temporarily becomes 0, if there is a culture period in a medium containing ethanol, it is included in the phrase “cultivate in a medium containing ethanol” of the present invention. It is.
　培地中に添加する炭素源以外の成分としては、窒素源、無機イオン及び必要に応じその他の有機成分を用いることができる。本発明の培地中に含まれる窒素源としては、アンモニア、硫酸アンモニウム、炭酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、酢酸アンモニウム、ウレア等のアンモニウム塩または硝酸塩等が使用することができ、pH調整に用いられるアンモニアガス、アンモニア水も窒素源として利用できる。また、ペプトン、酵母エキス、肉エキス、麦芽エキス、コーンスティープリカー、大豆加水分解物等も利用出来る。培地中にこれらの窒素源が１種のみ含まれていてもよいし、２種以上含まれてもよい。これらの窒素源は、初発培地にも流加培地にも用いることができる。また、初発培地、流加培地とも、同じ窒素源を用いてもよいし、流加培地の窒素源を初発培地の窒素源と変更してもよい。 As a component other than the carbon source to be added to the medium, a nitrogen source, inorganic ions, and other organic components as required can be used. As the nitrogen source contained in the culture medium of the present invention, ammonium salts such as ammonia, ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate, urea and the like can be used, and used for pH adjustment. Ammonia gas and aqueous ammonia can also be used as a nitrogen source. Moreover, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean hydrolyzate and the like can also be used. Only one of these nitrogen sources may be included in the medium, or two or more thereof may be included. These nitrogen sources can be used for both the initial medium and the fed-batch medium. Further, the same nitrogen source may be used for both the initial culture medium and the fed-batch medium, or the nitrogen source of the fed-feed medium may be changed to the nitrogen source of the initial culture medium.
　本発明の培地には、炭素源、窒素源の他にリン酸源、硫黄源が含まれていることが好ましい。リン酸源としては、リン酸２水素カリウム、リン酸水素２カリウム、ピロリン酸などのリン酸ポリマー等が利用出来る。また、硫黄源とは、硫黄原子を含んでいるものであればいずれでもよいが、硫酸塩、チオ硫酸塩、亜硫酸塩等の硫酸塩、システイン、シスチン、グルタチオン等の含硫アミノ酸が望ましく、なかでも硫酸アンモニウムが望ましい。 The medium of the present invention preferably contains a phosphate source and a sulfur source in addition to a carbon source and a nitrogen source. As the phosphoric acid source, phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and pyrophosphoric acid can be used. The sulfur source may be any one containing sulfur atoms, but sulfates such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione are desirable. However, ammonium sulfate is desirable.
　また、培地には、上記成分の他に、増殖促進因子（増殖促進効果を持つ栄養素）が含まれていてもよい。増殖促進因子とは、微量金属類、アミノ酸、ビタミン、核酸、更にこれらのものを含有するペプトン、カザミノ酸、酵母エキス、大豆たん白分解物等が使用できる。微量金属類としては、鉄、マンガン、マグネシウム、カルシウム等が挙げられ、ビタミンとしては、ビタミンB1、ビタミンB2、ビタミンB6、ニコチン酸、ニコチン酸アミド、ビタミンB12等が挙げられる。これらの増殖促進因子は初発培地に含まれていてもよいし、流加培地に含まれていてもよい。 The medium may contain a growth promoting factor (a nutrient having a growth promoting effect) in addition to the above components. As the growth-promoting factor, trace metals, amino acids, vitamins, nucleic acids, peptone, casamino acid, yeast extract, soybean protein degradation products and the like containing these can be used. Examples of trace metals include iron, manganese, magnesium, calcium and the like, and examples of vitamins include vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. These growth promoting factors may be contained in the initial culture medium or in the fed-batch medium.
　また、生育にアミノ酸などを要求する栄養要求性変異株を使用する場合には、培地に要求される栄養素を補添することが好ましい。Ｌ-リジン生産菌は、後述のようにＬ-リジン生合成経路が強化されており、Ｌ-リジン分解能が弱化されているものが多いので、Ｌ-スレオニン、Ｌ-ホモセリン、Ｌ-イソロイシン、Ｌ-メチオニンから選ばれる１種又は２種以上を添加することが望ましい。初発培地と流加培地は、培地組成が同じであってもよく、異なっていてもよい。また、初発培地と流加培地は、硫黄濃度が同じであってもよく、異なっていてもよい。さらには、流加培地の流加が多段階で行われる場合、各々の流加培地の組成は同じであってもよく、異なっていてもよい。 Moreover, when using an auxotrophic mutant strain that requires an amino acid or the like for growth, it is preferable to supplement nutrients required for the medium. Many L-lysine producing bacteria have enhanced L-lysine biosynthetic pathways as described later, and L-lysine resolution is weakened. Therefore, L-threonine, L-homoserine, L-isoleucine, L -It is desirable to add one or more selected from methionine. The initial medium and fed-batch medium may have the same or different medium composition. The initial culture medium and the fed-batch medium may have the same or different sulfur concentration. Furthermore, when the feeding of the feeding medium is performed in multiple stages, the composition of each feeding medium may be the same or different.
　培養は、発酵温度２０～４５℃、特に好ましくは３３～４２℃で通気培養を行うことが好ましい。ここで酸素濃度は、５～５０％に、望ましくは１０％程度に調節して行う。また、ｐＨを５～９に制御し、通気培養を行うことが好ましい。培養中にｐＨが下がる場合には、例えば、炭酸カルシウムを加えるか、アンモニアガス、アンモニア水等のアルカリで中和することができる。このような条件下で、好ましくは１０時間～１２０時間程度培養することにより、培養液中に著量のＬ-アミノ酸が蓄積される。 The culture is preferably carried out by aeration culture at a fermentation temperature of 20 to 45 ° C, particularly preferably 33 to 42 ° C. Here, the oxygen concentration is adjusted to 5 to 50%, preferably about 10%. Further, it is preferable to perform aeration culture while controlling the pH to 5 to 9. When the pH falls during the culture, for example, calcium carbonate can be added or neutralized with an alkali such as ammonia gas or ammonia water. By culturing preferably for about 10 to 120 hours under such conditions, a significant amount of L-amino acid is accumulated in the culture solution.
　本発明においては、Ｌ-アミノ酸蓄積を一定以上に保つために、細菌の培養を種培養と本培養とに分けて行ってもよく、種培養をフラスコ等を用いたしんとう培養、又は回分培養で行い、本培養を流加培養、又は連続培養で行ってもよく、種培養、本培養ともに回分培養で行ってもよい。 In the present invention, in order to keep L-amino acid accumulation at a certain level or more, bacterial culture may be divided into seed culture and main culture. The main culture may be performed by fed-batch culture or continuous culture, and both seed culture and main culture may be performed by batch culture.
　本発明において、流加培養あるいは、連続培養を行う際には、一時的にエタノールまたはその他の炭素源の流加が停止するように間欠的に流加培地を流加してもよい。また、流加を行う時間の最大で30％以下、望ましくは20％以下、特に望ましくは10％以下で流加培地の供給を停止することが好ましい。流加培養液を間欠的に流加させる場合には、流加培地を一定時間添加し、2回目以降の添加はある添加期に先行する添加停止期において発酵培地中の炭素源が枯渇するときのpH上昇または溶存酸素濃度の上昇がコンピューターで検出されるときに開始するように制御を行い、培養槽内の基質濃度を常に自動的に低レベルに維持してもよい（米国特許5,912,113号明細書）。 In the present invention, when fed-batch culture or continuous culture is performed, the feed medium may be intermittently fed so that ethanol or other carbon sources are temporarily stopped. In addition, it is preferable to stop the feeding of the fed-batch medium at a maximum of 30% or less, desirably 20% or less, particularly desirably 10% or less of the feeding time. When the fed-batch culture is fed intermittently, the fed-batch medium is added for a certain period of time, and the second and subsequent additions are performed when the carbon source in the fermentation medium is depleted in the addition stop period preceding the addition stage. Control to start when a rise in pH or an increase in dissolved oxygen concentration is detected by the computer, so that the substrate concentration in the culture tank may always be automatically maintained at a low level (US Pat. No. 5,912,113). book).
　流加培養に用いられる流加培地は、エタノールとその他の炭素源及び増殖促進効果を持つ栄養素（増殖促進因子）を含む培地が好ましく、発酵培地中のエタノール濃度が一定以下になるように制御してもよい。
　流加培地に加えるその他の炭素源としては、グルコース、スクロース、フルクトースが好ましく、増殖促進因子としては、窒素源、リン酸、アミノ酸等が好ましい。窒素源としては、アンモニア、硫酸アンモニウム、炭酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、酢酸アンモニウム、ウレア等のアンモニウム塩または硝酸塩等を使用することができる。またリン酸源としては、リン酸２水素カリウム、リン酸水素２カリウムが使用でき、アミノ酸としては、栄養要求性変異株を使用する場合には要求される栄養素を補添することが好ましい。また、流加培地は１種でもよく、２種以上の培地を混合してもよい。２種以上の流加培地を用いる場合、それらの培地は混合して１つのフィード缶により流加させてもよいし、複数のフィード缶で流加させてもよい。 The fed-batch medium used for fed-batch culture is preferably a medium containing ethanol and other carbon sources and nutrients that have a growth-promoting effect (growth-promoting factor), and the ethanol concentration in the fermentation medium is controlled to be below a certain level. May be.
As other carbon sources to be added to the feed medium, glucose, sucrose, and fructose are preferable, and as a growth promoting factor, a nitrogen source, phosphoric acid, amino acid and the like are preferable. As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, ammonium acetate, and urea, nitrates, and the like can be used. Further, as the phosphate source, potassium dihydrogen phosphate and dipotassium hydrogen phosphate can be used, and as the amino acid, it is preferable to supplement the required nutrients when an auxotrophic mutant is used. In addition, the feed medium may be one kind or a mixture of two or more kinds. When two or more types of fed-batch media are used, the media may be mixed and fed with a single feed can, or fed with a plurality of feed cans.
　本発明で連続培養法を用いる場合には、引き抜きは流加と同時に行ってもよいし、一部引き抜いたあとで流加を行ってもよい。また培養液をＬ-アミノ酸と細胞を含んだまま引き抜いて、細胞だけ発酵槽に戻す菌体を再利用する連続培養法でもよい（フランス特許2669935号明細書参照）。連続的あるいは間欠的に栄養源を流加する方法は流加培養と同様の方法が用いられる。 In the case of using the continuous culture method in the present invention, the withdrawal may be performed simultaneously with the feeding, or the feeding may be performed after a part of the withdrawal is performed. Further, a continuous culture method may be used in which the culture medium is drawn out while containing L-amino acid and cells, and the cells are returned to the fermenter and reused (see French Patent No. 2669935). As a method for feeding the nutrient source continuously or intermittently, the same method as the fed-batch culture is used.
　菌体を再利用する連続培養法とは、予定したアミノ酸濃度に達したときに、発酵培地を間欠的にあるいは連続して引き抜き、Ｌ-アミノ酸のみを取り出し、菌体を含むろ過残留物を発酵槽中に再循環させる方法であり、例えばフランス特許2669935号明細書を参照にして実施することができる。 The continuous culture method that recycles bacterial cells means that when a predetermined amino acid concentration is reached, the fermentation medium is withdrawn intermittently or continuously, only L-amino acids are extracted, and the filtration residue containing the bacterial cells is fermented. This is a method of recirculation in a tank, and can be carried out with reference to, for example, French Patent No. 2669935.
　ここで、培養液を間欠的に引き抜く場合には、予定したＬ-アミノ酸濃度に到達したときに、Ｌ-アミノ酸を一部引き抜いて、新たに培地を流加して培養を行うとよい。また、添加する培地の量は、最終的に引き抜く前の培養液量と同量になるように設定することが好ましい。ここで同量とは、引き抜く前の培養液量と９３～１０７％の程度の量を意味する。 Here, when the culture solution is withdrawn intermittently, when a predetermined L-amino acid concentration is reached, a part of the L-amino acid is withdrawn, and a culture medium is newly fed to carry out the culture. Moreover, it is preferable that the amount of the medium to be added is set so as to be the same as the amount of the culture solution before the final withdrawal. Here, the same amount means an amount of about 93 to 107% of the amount of the culture solution before drawing.
　培養液を連続的に引き抜く場合には、栄養培地を流加させると同時に、あるいは流加させたあとに引き抜きを開始することが望ましく、例えば引き抜き開始時間としては流加を始めてから5時間以内、望ましくは3時間以内、さらに望ましくは1時間以内である。また引き抜く培養液量としては、流加させる量と同量で引き抜くことが好ましい。 When the culture medium is continuously withdrawn, it is desirable to start withdrawal at the same time as or after feeding the nutrient medium.For example, the withdrawal start time is within 5 hours from the start of feeding. Preferably it is within 3 hours, more preferably within 1 hour. The amount of the culture solution to be withdrawn is preferably with the same amount as that to be fed.
　また、Ｌ－リジン等の塩基性アミノ酸を製造する際には、培養中のｐＨが６．５～９．０、培養終了時の培地のｐＨが７．２～９．０となるように制御し、培地中の重炭酸イオン及び／又は炭酸イオンが少なくとも２０ｍＭ以上存在する培養期があるようにし、前記重炭酸イオン及び／又は炭酸イオンを塩基性アミノ酸のカウンタイオンとする方法で発酵し、目的の塩基性アミノ酸を回収する方法で製造を行ってもよい（特開2002-65287、US2002-0025564A、EP 1813677A）。 In addition, when producing a basic amino acid such as L-lysine, the pH during the cultivation is controlled to 6.5 to 9.0, and the pH of the medium at the end of the cultivation is controlled to 7.2 to 9.0. And so that there is a culture phase in which at least 20 mM or more of bicarbonate ions and / or carbonate ions are present in the medium, and fermented by a method using the bicarbonate ions and / or carbonate ions as counter ions of basic amino acids, The basic amino acid may be recovered by a method (JP 2002-65287 A, US 2002-0025564A, EP 1813677A).
　塩基性アミノ酸を生産する能力を有する微生物を培地中で好気培養するに際して、炭酸イオンもしくは重炭酸イオン又はこれらの両方を、塩基性アミノ酸の主なカウンタイオンとして利用することができる。塩基性アミノ酸のカウンタイオンとして必要な量の重炭酸イオン及び／又は炭酸イオンを培地中に存在させる方法としては、培養中の培地のｐＨが６．５～９．０、好ましくは６．５～８．０、培養終了時の培地のｐＨが７．２～９．０となるように制御し、さらに、発酵中の発酵槽内圧力が正となるように制御するか、又は、炭酸ガスもしくは炭酸ガスを含む混合ガスを培地に供給することが知られている（特開2002-65287、米国特許出願公開第20020025564号、EP1813677A）。 When microorganisms having the ability to produce basic amino acids are aerobically cultured in a medium, carbonate ions or bicarbonate ions or both of them can be used as main counter ions of basic amino acids. As a method for allowing a bicarbonate ion and / or carbonate ion necessary for counter ions of basic amino acids to be present in the medium, the pH of the medium during the culture is 6.5 to 9.0, preferably 6.5 to 9.0. 8.0, the pH of the medium at the end of the culture is controlled to be 7.2 to 9.0, and further the pressure in the fermenter during the fermentation is controlled to be positive, or carbon dioxide or It is known to supply a mixed gas containing carbon dioxide gas to a culture medium (Japanese Patent Laid-Open No. 2002-65287, US Patent Application Publication No. 20020025564, EP1813677A).
　本発明においては、発酵中の発酵槽内の圧力が正となるように制御すること、及び、炭酸ガスもしくは炭酸ガスを含む混合ガスを培地に供給することの両方を行ってもよい。いずれの場合も、培地中の重炭酸イオン及び／又は炭酸イオンが、好ましくは20mM以上、より好ましくは30mM以上、特に好ましくは40mM以上存在する培養期があるようにすることが好ましい。発酵槽内圧力、炭酸ガス又は炭酸ガスを含む混合ガスの供給量、又は制限された給気量は、例えば培地中の重炭酸イオン又は炭酸イオンを測定することや、pHやアンモニア濃度を測定することによって、決定することができる。 In the present invention, both control so that the pressure in the fermenter during fermentation may be positive and carbon dioxide or a mixed gas containing carbon dioxide may be supplied to the culture medium. In any case, it is preferable that there is a culture period in which bicarbonate ions and / or carbonate ions in the medium are preferably present at 20 mM or more, more preferably 30 mM or more, and particularly preferably 40 mM or more. The pressure in the fermenter, the supply amount of carbon dioxide or a mixed gas containing carbon dioxide, or the limited supply amount is measured, for example, by measuring bicarbonate ions or carbonate ions in the medium, or by measuring pH or ammonia concentration. Can be determined.
　上記態様においては、培養中の培地のｐＨが６．０～９．０、好ましくは６．５～８．０、培養終了時の培地のｐＨが７．２～９．０となるように制御する。上記態様によれば、従来の方法に比べて、カウンタイオンとして必要な量の重炭酸イオン及び／又は炭酸イオンを培地中に存在させるための培地のpHを低く抑えることが可能となる。アンモニアでｐＨを制御する場合、ｐＨを高めるためにアンモニアが供給され、塩基性アミノ酸のＮ源となり得る。培地に含まれる塩基性アミノ酸以外のカチオンとしては、培地成分由来のＫ、Ｎａ、Ｍｇ、Ｃａ等が挙げられる。これらは、好ましくは総カチオンの５０％以下であることが好ましい。 In the above embodiment, the pH of the medium during the culture is controlled to 6.0 to 9.0, preferably 6.5 to 8.0, and the pH of the medium at the end of the culture is controlled to 7.2 to 9.0. To do. According to the said aspect, compared with the conventional method, it becomes possible to hold down the pH of the culture medium for making the quantity of bicarbonate ion and / or carbonate ion which are required as a counter ion exist in a culture medium. When the pH is controlled with ammonia, ammonia is supplied to increase the pH, which can be an N source of basic amino acids. Examples of cations other than basic amino acids contained in the medium include K, Na, Mg, Ca and the like derived from medium components. These are preferably 50% or less of the total cations.
　また、発酵中の発酵槽内圧力が正となるようにするには、例えば、給気圧を排気圧より高くすればよい。発酵槽内圧力を正にすることによって、発酵により生成する炭酸ガスが培養液に溶解し、重炭酸イオン又は炭酸イオンを生じ、これらは塩基性アミノ酸のカウンタイオンとなり得る。発酵槽内圧力として具体的には、ゲージ圧（大気圧に対する差圧）で、０．０３～０．２ＭＰａ、好ましくは０．０５～０．１５ＭＰａ、さらに好ましくは０．１～０．３ＭＰａが挙げられる。また、培養液に炭酸ガス、又は炭酸ガスを含む混合ガスを供給することによって、培養液に炭酸ガスを溶解させてもよい。さらには、培養液に炭酸ガス又は炭酸ガスを含む混合ガスを供給しつつ、発酵槽内圧力が正となるように調節してもよい。 Moreover, in order to make the fermenter internal pressure during fermentation positive, for example, the supply air pressure may be set higher than the exhaust pressure. By making the pressure in the fermenter positive, the carbon dioxide gas generated by fermentation dissolves in the culture solution to produce bicarbonate ions or carbonate ions, which can be counter ions of basic amino acids. Specifically, the pressure in the fermenter is 0.03 to 0.2 MPa, preferably 0.05 to 0.15 MPa, more preferably 0.1 to 0.3 MPa in terms of gauge pressure (differential pressure with respect to atmospheric pressure). Can be mentioned. In addition, carbon dioxide gas may be dissolved in the culture solution by supplying carbon dioxide gas or a mixed gas containing carbon dioxide gas to the culture solution. Furthermore, you may adjust so that a fermenter internal pressure may become positive, supplying carbon dioxide or the mixed gas containing carbon dioxide to a culture solution.
　発酵槽内圧力を正に調節するには、例えば、給気圧を排気圧よりも高くするように設定すればよい。また、培養液に炭酸ガスを供給する場合は、例えば、純炭酸ガス、又は炭酸ガスを５体積％以上含む混合ガスを吹き込めばよい。 In order to positively adjust the fermenter pressure, for example, the supply air pressure may be set to be higher than the exhaust pressure. In addition, when carbon dioxide is supplied to the culture solution, for example, pure carbon dioxide or a mixed gas containing 5% by volume or more of carbon dioxide may be blown.
　尚、培地に重炭酸イオン及び／又は炭酸イオンを溶解させる上記の方法は、単独でもよいし、複数を組み合わせてもよい。 In addition, the above-mentioned method for dissolving bicarbonate ions and / or carbonate ions in the medium may be used alone or in combination.
　従来法では、通常、生成する塩基牲アミノ酸のカウンタアニオンとすべく、十分量の硫酸アンモニウムや塩化アンモニウムが、又、栄養成分として蛋白等の硫酸分解物もしくは塩酸分解物が培地に添加され、これらから与えられる硫酸イオン、塩化物イオンが培地に含まれる。従って、弱酸性である炭酸イオン濃度は培養中極めて低く、ppm単位である。上記態様では、これら硫酸イオン、塩化物イオンを減じ、微生物が発酵中に放出する炭酸ガスを上記発酵環境にて培地中に溶解せしめ、カウンタイオンとすることに特徴がある。したがって、上記態様においては、硫酸イオンや塩化物イオンを生育に必要な量以上培地に添加する必要はない。好ましくは、培養当初は硫酸アンモニウム等を培地に適当量フィードし、培養途中でフィードを止める。あるいは、培地中の炭酸イオン又は重炭酸イオンの溶存量とのバランスを保ちつつ、硫酸アンモニウム等をフィードしてもよい。また、塩基性アミノ酸の窒素源として、アンモニアを培地にフィードしてもよい。アンモニアは、単独で、又は他の気体とともに培地に供給することができる。 In the conventional method, a sufficient amount of ammonium sulfate or ammonium chloride is usually added to the medium as a counter anion of the basic amino acid to be produced, and a sulfate or hydrolyzate of protein or the like as a nutrient component is added to the medium. The culture medium contains sulfate ions and chloride ions. Therefore, the concentration of carbonate ion, which is weakly acidic, is extremely low during the culture, and is in ppm. The above aspect is characterized in that the sulfate ions and chloride ions are reduced, and carbon dioxide released by the microorganisms during fermentation is dissolved in the medium in the fermentation environment to form counter ions. Therefore, in the above-described embodiment, it is not necessary to add sulfate ions or chloride ions to the culture medium in an amount necessary for growth. Preferably, an appropriate amount of ammonium sulfate or the like is fed to the medium at the beginning of the culture, and the feed is stopped during the culture. Or you may feed ammonium sulfate etc., maintaining the balance with the dissolved amount of the carbonate ion or bicarbonate ion in a culture medium. Alternatively, ammonia may be fed to the medium as a nitrogen source for basic amino acids. Ammonia can be supplied to the medium alone or with other gases.
　培地に含まれる重炭酸イオン及び／又は炭酸イオン以外の他のアニオンの濃度は、微生物の生育に必要な量であれば、低いことが好ましい。このようなアニオンには、塩化物イオン、硫酸イオン、リン酸イオン、イオン化した有機酸、及び水酸化物イオン等が挙げられる。これらの他のイオンのモル濃度の合計は、好ましくは通常は９００ｍＭ以下、より好ましくは７００ｍＭ以下、特により好ましくは５００ｍＭ以下、さらに好ましくは３００ｍＭ以下、特に好ましくは２００ｍＭ以下である。 The concentration of bicarbonate ions and / or other anions other than carbonate ions contained in the medium is preferably low as long as it is an amount necessary for the growth of microorganisms. Such anions include chloride ions, sulfate ions, phosphate ions, ionized organic acids, hydroxide ions, and the like. The total molar concentration of these other ions is preferably usually 900 mM or less, more preferably 700 mM or less, particularly preferably 500 mM or less, still more preferably 300 mM or less, and particularly preferably 200 mM or less.
　上記態様においては、硫酸イオン、及び／又は、塩化物イオンの使用量を削減することが目的の一つであり、培地に含まれる硫酸イオンもしくは塩化物イオン、又はこれらの合計は、通常、７００ｍＭ以下、好ましくは５００ｍＭ以下、より好ましくは３００ｍＭ以下、さらに好ましくは２００ｍＭ以下、特に好ましくは１００ｍＭ以下である。 In the above embodiment, one of the purposes is to reduce the amount of sulfate ion and / or chloride ion used, and the sulfate ion or chloride ion contained in the medium, or the total of these, is usually 700 mM. Hereinafter, it is preferably 500 mM or less, more preferably 300 mM or less, still more preferably 200 mM or less, and particularly preferably 100 mM or less.
　通常は、塩基性アミノ酸のカウンタイオン源として培地に硫酸アンモニウムを添加すると、硫酸イオンによって培養液中の炭酸ガスが放出してしまう。それに対して、上記態様においては、過剰量の硫酸アンモニウムを培地に添加する必要がないので、炭酸ガスを発酵液中に容易に溶解させることができる。 Normally, when ammonium sulfate is added to the medium as a counter ion source for basic amino acids, carbon dioxide in the culture medium is released by sulfate ions. On the other hand, in the above embodiment, it is not necessary to add an excessive amount of ammonium sulfate to the medium, so that carbon dioxide gas can be easily dissolved in the fermentation broth.
　また、上記態様においては、「塩基性アミノ酸の生産を阻害しない」程度に培地中の総アンモニア濃度を制御することが好ましい。そのような条件としては、例えば、最適な条件において塩基性アミノ酸を生産する場合の収率及び／又は生産性に比べて、好ましくは５０％以上、より好ましくは７０％以上、特に好ましくは９０％以上の収率及び／又は生産性が得られる条件が含まれる。具体的には、培地中の総アンモニア濃度としては、好ましくは３００ｍＭ以下、より好ましくは２５０ｍＭ、特に好ましくは２００ｍＭ以下の濃度が挙げられる。アンモニアの解離度はｐＨが高くなると低下する。解離していないアンモニアは、アンモニウムイオンよりも菌に対して毒性が強い。そのため、総アンモニア濃度の上限は、培養液のｐＨにも依存する。すなわち、培養液のｐＨが高いほど、許容される総アンモニア濃度は低くなる。したがって、前記「塩基性アミノ酸の生産を阻害しない」総アンモニア濃度は、ｐＨ毎に設定することが好ましい。しかし、培養中の最も高いｐＨにおいて許容される総アンモニア濃度範囲を、培養期間を通じての総アンモニア濃度の上限値範囲としてもよい。 In the above embodiment, it is preferable to control the total ammonia concentration in the medium to such an extent that “the production of basic amino acids is not inhibited”. Such conditions include, for example, preferably 50% or more, more preferably 70% or more, particularly preferably 90%, as compared to the yield and / or productivity in the case of producing a basic amino acid under optimum conditions. Conditions for obtaining the above yield and / or productivity are included. Specifically, the total ammonia concentration in the medium is preferably 300 mM or less, more preferably 250 mM, and particularly preferably 200 mM or less. The degree of ammonia dissociation decreases with increasing pH. Undissociated ammonia is more toxic to bacteria than ammonium ions. Therefore, the upper limit of the total ammonia concentration also depends on the pH of the culture solution. That is, the higher the pH of the culture solution, the lower the allowable total ammonia concentration. Therefore, the total ammonia concentration that “does not inhibit the production of basic amino acids” is preferably set for each pH. However, the total ammonia concentration range allowed at the highest pH during the culture may be the upper limit range of the total ammonia concentration throughout the culture period.
　一方、微生物の生育及び塩基性物質の生産に必要な窒素源としての総アンモニア濃度としては、培養中にアンモニアが継続して枯渇しない窒素源が不足することにより微生物による目的物質の生産性を低下させない限り特に制限されず、適宜設定することができる。例えば、培養中にアンモニア濃度を経時的に測定し、培地中のアンモニアが枯渇したら少量のアンモニアを培地に添加してもよい。アンモニアを添加したときの濃度としては、特に制限されないが、例えば、総アンモニア濃度として好ましくは１ｍＭ以上、より好ましくは１０ｍＭ以上、特に好ましくは２０ｍＭ以上の濃度が挙げられる。 On the other hand, the total ammonia concentration as a nitrogen source necessary for the growth of microorganisms and the production of basic substances decreases the productivity of target substances by microorganisms due to the lack of a nitrogen source that does not continuously deplete ammonia during culture. There is no particular limitation as long as it is not set, and it can be set as appropriate. For example, the ammonia concentration may be measured over time during the culture, and a small amount of ammonia may be added to the medium when the ammonia in the medium is depleted. The concentration when ammonia is added is not particularly limited. For example, the total ammonia concentration is preferably 1 mM or more, more preferably 10 mM or more, and particularly preferably 20 mM or more.
　発酵液からのＬ－アミノ酸の回収は通常イオン交換樹脂法（Nagai,H.et al., Separation Science and Technology, 39(16),3691-3710）、沈殿法、膜分離法（特開平9-164323号、特開平9-173792号）、晶析法(WO2008/078448、WO2008/078646）、その他の公知の方法を組み合わせることにより実施できる。なお、菌体内にＬ－アミノ酸が蓄積する場合には、例えば菌体を超音波などにより破砕し、遠心分離によって菌体を除去して得られる上清からイオン交換樹脂法などによって、Ｌ－アミノ酸を回収することができる。 Recovery of L-amino acids from fermentation broth is usually performed by ion exchange resin method (Nagai, H. et al., Separation, Science and Technology, 39 (16), 3691-3710), precipitation method, membrane separation method No. 164323, JP-A-9-173792), crystallization methods (WO2008 / 078448, WO2008 / 078646), and other known methods can be combined. In the case where L-amino acid accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves, etc., and the microbial cells are removed by centrifugation. Can be recovered.
　尚、回収されるＬ－アミノ酸は、Ｌ－アミノ酸以外に細菌菌体、培地成分、水分、及び細菌の代謝副産物を含んでいてもよい。採取されたＬ－アミノ酸の純度は、50％以上、好ましくは85％以上、特に好ましくは95%以上である (JP1214636B, USP 5,431,933, 4,956,471, 4,777,051, 4946654, 5,840,358, 6,238,714, US2005/0025878))。 The recovered L-amino acid may contain bacterial cells, medium components, water, and bacterial metabolic byproducts in addition to the L-amino acid. The purity of the collected L-amino acid is 50% or more, preferably 85% or more, particularly preferably 95% or more (JP1214636B, USP 5,431,933, 4,956,471, 4,777,051, 4946654, 45,840,358, 6,238,714, US2005 / 0025878)).
　また、Ｌ－アミノ酸が培地中に析出する場合は、遠心分離又は濾過等により回収することができる。また、培地中に析出したＬ－アミノ酸は、培地中に溶解しているＬ－アミノ酸を晶析した後に、併せて単離してもよい。 If L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration. The L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.
　以下、本発明を実施例により更に具体的に説明する。 Hereinafter, the present invention will be described more specifically with reference to examples.
〔実施例１〕AldB活性が低下したＬ－リジン生産菌の構築
＜１－１＞エタノール資化性が付与されたＬ－リジン生産菌の構築
　L-リジン生産菌にエタノール資化性を付与するため、変異型アルコールデヒドロゲナーゼ遺伝子（adhE*）の導入を行った。変異型アルコールデヒドロゲナーゼ遺伝子として、MG1655::PL-tacadhE*（WO2008/010565）由来の遺伝子を用いた。MG1655::PL-tacadhE*株は、クロラムフェニコール耐性遺伝子（cat）と、PL-tacプロモーターにより制御される変異型adhE遺伝子が連結したＤＮＡ断片を、エシェリヒア・コリMG1655株のゲノムに挿入して得た株である。前記変異型adhE遺伝子は、568位のグルタミン酸残基がリジンで置換された変異体をコードしている。この変異型アルコールデヒドロゲナーゼを保持するエシェリヒア・コリは、好気条件でエタノールを資化することができる。 [Example 1] Construction of L-lysine-producing bacterium with reduced AldB activity <1-1> Construction of L-lysine-producing bacterium with ethanol-assimilating ability L-lysine-producing bacterium is imparted with ethanol-assimilating ability Therefore, a mutant alcohol dehydrogenase gene (adhE *) was introduced. As the mutant alcohol dehydrogenase gene, a gene derived from MG1655 :: PL-tacadhE * (WO2008 / 010565) was used. In the MG1655 :: PL-tacadhE * strain, a DNA fragment in which a chloramphenicol resistance gene (cat) and a mutant adhE gene controlled by the PL-tac promoter are linked is inserted into the genome of Escherichia coli MG1655 strain. This is the stock obtained. The mutant adhE gene encodes a mutant in which the glutamic acid residue at position 568 is replaced with lysine. Escherichia coli that retains this mutant alcohol dehydrogenase can assimilate ethanol under aerobic conditions.
　cat遺伝子をMG1655::PL-tacadhE*株のゲノムから除去できるようにするため、cat遺伝子を、ラムダファージのアタッチメントサイトとテトラサイクリン耐性遺伝子を連結したＤＮＡ断片（att-tet）へ置き換えた。 In order to be able to remove the cat gene from the genome of the MG1655 :: PL-tacadhE * strain, the cat gene was replaced with a DNA fragment (att-tet) linking the attachment site of lambda phage and the tetracycline resistance gene.
　cat遺伝子のatt-tet遺伝子への置き換えは、WO2005/010175に記載の、DatsenkoとWannerによって最初に開発された「Red-driven integration」と呼ばれる方法（Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645）によって行った。「Red-driven integration」方法によれば、目的とする遺伝子の一部を合成オリゴヌクレオチドの5’側に、抗生物質耐性遺伝子の一部を３’側にデザインした合成オリゴヌクレオチドをプライマーとして用いて得られたPCR産物を用いて、一段階で遺伝子破壊株を構築することができる。さらにλファージ由来の切り出しシステム（Cho, E.H. et al., J. Bacteriol. 2002 Sep; 184(18): 5200-5203）を組み合わせることにより、遺伝子破壊株に組み込んだ抗生物質耐性遺伝子を除去することが出来る。cat遺伝子をatt-tet遺伝子で置換えるためのプライマーとして、配列番号５及び６のプライマーを使用して行った。こうして、MG1655::PL-tacadhE*のcat遺伝子がatt-tet遺伝子に置き換えられたMG1655-att-tet-PL-tacadhE*株を得た。
　L-リジン生産菌にエタノール資化性を付与するため、MG1655-att-tet-PL-tacadhE*をドナーとして、L-リジン生産菌WC196ΔcadAΔldcC株（「WC196LC」とも記載する）にP1トランスダクションを行い、WC196LC-att-tet-PL-tacadhE*株を得た。 Replacement of the cat gene with the att-tet gene is a method called “Red-driven integration” originally developed by Datsenko and Wanner described in WO2005 / 010175 (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p6640-6645). According to the “Red-driven integration” method, a synthetic oligonucleotide designed with a part of the target gene on the 5 ′ side of the synthetic oligonucleotide and a part of the antibiotic resistance gene on the 3 ′ side is used as a primer. Using the obtained PCR product, a gene-disrupted strain can be constructed in one step. Furthermore, the antibiotic resistance gene incorporated into the gene-disrupted strain is removed by combining the excision system derived from λ phage (Cho, EH et al., J. Bacteriol. 2002 Sep; 184 (18): 5200-5203) I can do it. The primers of SEQ ID NOs: 5 and 6 were used as primers for replacing the cat gene with the att-tet gene. Thus, the MG1655-att-tet-PL-tacadhE * strain in which the cat gene of MG1655 :: PL-tacadhE * was replaced with the att-tet gene was obtained.
In order to confer ethanol assimilation to L-lysine-producing bacteria, P1 transduction was performed on L-lysine-producing bacteria WC196ΔcadAΔldcC (also referred to as “WC196LC”) using MG1655-att-tet-PL-tacadhE * as a donor , WC196LC-att-tet-PL-tacadhE * strain was obtained.
　次に、PL-tacプロモーター上流に導入されたatt-tet遺伝子を除去するために、ヘルパープラスミドpMW-intxis-ts（米国特許出願公開20060141586）を使用した。pMW-intxis-tsは、λファージのインテグラーゼ（Int）をコードする遺伝子、エクシジョナーゼ(Xis)をコードする遺伝子を搭載し、温度感受性の複製能を有するプラスミドである。 Next, a helper plasmid pMW-intxis-ts (US Patent Application Publication 20060141586) was used to remove the att-tet gene introduced upstream of the PL-tac promoter. pMW-intxis-ts is a plasmid carrying a gene encoding λ phage integrase (Int) and gene encoding excisionase (Xis) and having temperature-sensitive replication ability.
　上記で得られたWC196LC-att-tet-PL-tacadhE*株のコンピテントセルを常法に従って作製し、ヘルパープラスミドpMW-intxis-tsにて形質転換し、30℃で50 mg/Lのアンピシリンを含むLB寒天培地上にて平板培養し、アンピシリン耐性株を選択した。pMW-intxis-tsプラスミドを除去するために、LB寒天培地上、42℃で培養し、得られたコロニーのアンピシリン耐性、及びテトラサイクリン耐性を試験し、att-tet及びpMW-intxis-tsが脱落しているPL-tacadhE*導入株であるテトラサイクリン、アンピシリン感受性株を取得した。この株をWC196LC PL-tacadhE*株と名づけた。 Competent cells of the WC196LC-att-tet-PL-tacadhE * strain obtained above were prepared according to a conventional method, transformed with the helper plasmid pMW-intxis-ts, and 50 mg / L ampicillin at 30 ° C. Plated on an LB agar medium containing ampicillin resistant strains. In order to remove the pMW-intxis-ts plasmid, it was cultured on LB agar medium at 42 ° C, and the resulting colonies were tested for ampicillin resistance and tetracycline resistance. Att-tet and pMW-intxis-ts were removed. We obtained tetracycline and ampicillin sensitive strains, which are PL-tacadhE * introduced strains. This strain was named WC196LC PL-tacadhE * strain.
　WC196LC PL-tacadhE*株を、dapA、dapB及びlysC遺伝子を搭載したLys生産用プラスミドpCABD2（国際公開第WO01/53459号パンフレット）で常法に従い形質転換し、WC196LC PL-tacadhE*/pCABD2株を得た。pCABD2は、Ｌ－リジンによるフィードバック阻害が解除された変異を有するE. coli由来のジヒドロジピコリン酸合成酵素（DDPS）をコードする変異型dapA遺伝子と、Ｌ－リジンによるフィードバック阻害が解除された変異を有するE. coli由来のアスパルトキナーゼIIIをコードする変異型lysC遺伝子と、E. coli由来のジヒドロジピコリン酸レダクターゼをコードするdapB遺伝子と、ブレビバクテリウム・ラクトファーメンタム由来ジアミノピメリン酸デヒドロゲナーゼをコードするddh遺伝子を含んでいる。 The WC196LC PL-tacadhE * strain was transformed with the Lys production plasmid pCABD2 (International Publication No. WO01 / 53459 pamphlet) carrying the dapA, dapB and lysC genes according to a conventional method to obtain the WC196LC PL-tacadhE * / pCABD2 strain. It was. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from E. coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine. A mutant lysC gene encoding aspartokinase III derived from E. coli, dapB gene encoding dihydrodipicolinate reductase derived from E. coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.
＜１－２＞WC196LC PL-tacadhE*/pCABD2株からのAldB非産生株の構築
　MG1655株に対して、「Red-driven integration」方法により、aldB遺伝子の欠失を行った。aldB遺伝子の欠失用プライマーとして、配列番号７及び８のプライマーを使用した。これによって、MG1655ΔaldB::Tet株を得た。MG1655ΔaldB::Tet株をドナーとして、L-リジン生産菌WC196LC PL-tacadhE*株にP1トランスダクションを行い、AldB非産生株WC196LC PL-tacadhE*ΔaldB::Tet/pCABD2株を得た。これらの株を20mg/Lのストレプトマイシンを含むＬ培地にて終ＯＤ600≒0.6となるように37℃にて培養した後、培養液と等量の40％グリセロール溶液を加えて攪拌した後、適当量ずつ分注し-80℃に保存した。これをグリセロールストックと呼ぶ。 <1-2> Construction of AldB non-producing strain from WC196LC PL-tacadhE * / pCABD2 strain The aldB gene was deleted from the MG1655 strain by the “Red-driven integration” method. As primers for deletion of the aldB gene, primers of SEQ ID NOs: 7 and 8 were used. As a result, the MG1655ΔaldB :: Tet strain was obtained. MG1655ΔaldB :: Tet strain was used as a donor, P1-transduction was performed on L-lysine-producing WC196LC PL-tacadhE * strain, and AldB non-producing strain WC196LC PL-tacadhE * ΔaldB :: Tet / pCABD2 strain was obtained. After culturing these strains in L medium containing 20 mg / L streptomycin at 37 ° C. so that the final OD600≈0.6, an equal amount of 40% glycerol solution and the same amount as the culture solution were added and stirred. Aliquots were stored at -80 ° C. This is called glycerol stock.
〔実施例２〕AldB非産生株のＬ－リジン生産能の評価
　前記の株のグリセロールストックを融解し、各100μLを、20mg/Lのストレプトマイシンを含むLプレートに均一に塗布し、37℃にて15時間培養した。得られたプレートのおよそ1/8の菌体を、太試験管(内径18 mm)の、20mg/Lのストレプトマイシンを含む発酵培地の5 mLに接種し、往復振とう培養装置で、攪拌120rpmの条件下、37℃において培養した。
　AldB非改変株WC196LC PL-tacadhE*/pCABD2株についても、同様の培養を行った。
　エタノールを炭素源とする発酵培地の組成を以下に示す。 [Example 2] Evaluation of L-lysine-producing ability of non-AldB-producing strain Melt the glycerol stock of the above-mentioned strain, apply 100 μL of each uniformly onto an L plate containing 20 mg / L of streptomycin, at 37 ° C. Cultured for 15 hours. Approximately 1/8 of the cells on the obtained plate was inoculated into 5 mL of a fermentation medium containing 20 mg / L of streptomycin in a large test tube (inner diameter: 18 mm) and stirred at 120 rpm with a reciprocating shaker. Cultured at 37 ° C under conditions.
The same culture was performed for the AldB non-modified strain WC196LC PL-tacadhE * / pCABD2 strain.
The composition of the fermentation medium using ethanol as a carbon source is shown below.
［Ｌ－リジン発酵培地組成］
エタノール                            10 ml/L
(NH4)2SO4                              24 g/L
K２HPO４                              1.0 g/L
MgSO4・7H2O                            1.0 g/L
FeSO4・7H2O                            0.01 g/L
MnSO4・5H2O                            0.082 g/L
イーストエキストラクト                2.0 g/L
CaCO3（日本薬局方）                   30 g/L
蒸留水　　　　　　　　　　　　　　　　最終量1L
　KOHでpH5.7に調整し、115℃で10分オートクレーブを行った。但しMgSO4・7H2Oは別に殺菌し、エタノールはフィルターろ過により滅菌した。CaCO3は、180℃で２時間乾熱滅菌したものを入れた。 [L-lysine fermentation medium composition]
Ethanol 10 ml / L
(NH 4 ) 2 SO 4 24 g / L
K2HPO4 1.0 g / L
MgSO 4・ 7H 2 O 1.0 g / L
FeSO 4・ 7H 2 O 0.01 g / L
MnSO 4・ 5H 2 O 0.082 g / L
Yeast Extract 2.0 g / L
CaCO 3 (Japanese Pharmacopoeia) 30 g / L
Distilled water Final volume 1L
The pH was adjusted to 5.7 with KOH and autoclaved at 115 ° C. for 10 minutes. However, MgSO 4 · 7H 2 O was sterilized separately, and ethanol was sterilized by filter filtration. CaCO 3 was sterilized by dry heat at 180 ° C. for 2 hours.
　16時間培養後20μLのエタノールを添加し、24時間培養後40μLのエタノールを添加した。41時間培養後、培地中に蓄積したL-リジンの量を公知の方法（サクラ精機  バイオテックアナライザーAS210）により測定し、培養液中のエタノール濃度を公知の方法（王子計測機器バイオセンサBF-5）により測定した。結果を表１に示す。AldB非産生株（WC196LC PL-tacadhE*ΔaldB::Tet/pCABD2株）は、対照株（WC196LC PL-tacadhE*/pCABD2株）と比較して、有意に高いＬ-リジン生産を示した。 After culturing for 16 hours, 20 μL of ethanol was added, and after culturing for 24 hours, 40 μL of ethanol was added. After culturing for 41 hours, the amount of L-lysine accumulated in the medium was measured by a known method (Sakura Seiki Biotech Analyzer AS210), and the ethanol concentration in the culture solution was measured by a known method (Oji Scientific Instruments Biosensor BF-5 ). The results are shown in Table 1. The AldB non-producing strain (WC196LC PL-tacadhE * ΔaldB :: Tet / pCABD2 strain) showed significantly higher L-lysine production than the control strain (WC196LC PL-tacadhE * / pCABD2 strain).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
〔配列表の説明〕
配列番号１：E. coliのaldB遺伝子の塩基配列
配列番号２：E. coliのAldBアミノ酸配列
配列番号３：E. coliのadhE遺伝子の塩基配列
配列番号４：E. coliのAdhEアミノ酸配列
配列番号５：cat遺伝子をatt-tet遺伝子で置換えるためのプライマー
配列番号６：cat遺伝子をatt-tet遺伝子で置換えるためのプライマー
配列番号７：aldB遺伝子の欠失用プライマー
配列番号８：aldB遺伝子の欠失用プライマー
配列番号９：P. ananatisのAldBアミノ酸配列
配列番号10：P. atrosepticumのAldBのアミノ酸配列
配列番号11：S. entericaのAldBのアミノ酸配列
配列番号12：P. ananatisのAdhEのアミノ酸配列
配列番号13：P. atrosepticumのAdhEのアミノ酸配列
配列番号14：S. entericaのAdhEのアミノ酸配列 [Explanation of Sequence Listing]
SEQ ID NO: 1: E. coli aldb gene base sequence SEQ ID NO: 2: E. coli AldB amino acid sequence SEQ ID NO: 3: E. coli adhE gene base sequence SEQ ID NO: 4: E. coli AdhE amino acid sequence SEQ ID NO: 5: Primer for replacing cat gene with att-tet gene SEQ ID NO: 6: Primer for replacing cat gene with att-tet gene SEQ ID NO: 7: primer for deletion of aldB gene SEQ ID NO: 8: of aldB gene Primer for deletion SEQ ID NO: 9: AldB amino acid sequence of P. ananatis SEQ ID NO: 10: AldB amino acid sequence of P. atrosepticum SEQ ID NO: 11: AldB amino acid sequence of S. enterica SEQ ID NO: 12: AdhE amino acid of P. ananatis SEQ ID NO: 13: AdhE amino acid sequence of P. atrosepticum SEQ ID NO: 14: AdhE amino acid sequence of S. enterica
　本発明により、エタノールを炭素源として、Ｌ－アミノ酸を効率よく発酵生産することができる。 According to the present invention, L-amino acid can be efficiently fermented and produced using ethanol as a carbon source.

Claims (11)

  1. 　Ｌ－アミノ酸生産能を有する腸内細菌科に属する細菌を、エタノールを含む培地で培養し、該培地からＬ－アミノ酸を採取する、Ｌ－アミノ酸の製造法であって、
    　前記細菌は、ＡｌｄＢタンパク質の活性が低下するように改変されていることを特徴とする方法。     A method for producing an L-amino acid, wherein a bacterium belonging to the family Enterobacteriaceae having L-amino acid producing ability is cultured in a medium containing ethanol, and L-amino acid is collected from the medium,
    The method wherein the bacterium is modified so that the activity of the AldB protein is reduced.
  2. 　ａｌｄＢ遺伝子のコード領域及び／又は同遺伝子の発現制御領域に変異が導入されたことにより、前記ＡｌｄＢタンパク質の活性が低下した、請求項１に記載の方法。 The method according to claim 1, wherein the activity of the AldB protein is reduced by introducing a mutation into the coding region of the aldB gene and / or the expression control region of the gene.
  3. 　前記細菌は染色体上のａｌｄＢ遺伝子が破壊されたことを特徴とする、請求項１又は２に記載の方法。 3. The method according to claim 1 or 2, wherein the bacterium has a chromosomal aldB gene disrupted.
  4. 　前記ＡｌｄＢタンパク質は、下記（Ａ）又は（Ｂ）のいずれかのタンパク質である、請求項１～３のいずれか一項に記載の方法。
    　（Ａ）配列番号２に示すアミノ酸配列を有するタンパク質、又は、
    　（Ｂ）配列番号２に示すアミノ酸配列において、１又は数個のアミノ酸の置換、欠失、挿入、または付加を含むアミノ酸配列を有し、かつ、アセトアルデヒドデヒドロゲナーゼ活性を有するタンパク質。     The method according to any one of claims 1 to 3, wherein the AldB protein is any one of the following proteins (A) and (B).
    (A) a protein having the amino acid sequence shown in SEQ ID NO: 2, or
    (B) A protein having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids and having acetaldehyde dehydrogenase activity in the amino acid sequence shown in SEQ ID NO: 2.
  5. 　前記ａｌｄＢ遺伝子は、下記（ａ）又は（ｂ）のＤＮＡである請求項２～４のいずれか一項に記載の方法。
    　（ａ）配列番号１の塩基配列を有するＤＮＡ、又は、
    　（ｂ）配列番号１の塩基配列に相補的な配列、又は同配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズし、かつ、アセトアルデヒドデヒドロゲナーゼ活性を有するタンパク質をコードするＤＮＡ。     The method according to any one of claims 2 to 4, wherein the aldB gene is the following DNA (a) or (b):
    (A) DNA having the base sequence of SEQ ID NO: 1, or
    (B) A DNA that hybridizes under stringent conditions with a sequence that is complementary to the base sequence of SEQ ID NO: 1 or that can be prepared from the same sequence, and that encodes a protein having acetaldehyde dehydrogenase activity.
  6. 　前記細菌がエシェリヒア属、エンテロバクター属、又はパントエア属に属する細菌である、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the bacterium belongs to the genus Escherichia, Enterobacter or Pantoea.
  7. 　前記細菌がエシェリヒア・コリである、請求項６に記載の方法。 The method according to claim 6, wherein the bacterium is Escherichia coli.
  8. 　前記細菌は、さらにＡｄｈＥタンパク質の活性が増強された、請求項１～７のいずれか一項に記載の方法。 The method according to any one of claims 1 to 7, wherein the bacterium further has enhanced activity of the AdhE protein.
  9. 　前記細菌は、好気的にエタノールを資化できるように改変された、請求項１～８に記載の方法。 The method according to any one of claims 1 to 8, wherein the bacterium has been modified so as to assimilate ethanol aerobically.
  10. 　前記Ｌ－アミノ酸がＬ－リジンである、請求項１～８のいずれか一項に記載の方法。 The method according to any one of claims 1 to 8, wherein the L-amino acid is L-lysine.
  11. 　前記細菌は、ジヒドロジピコリン酸レダクターゼ、ジアミノピメリン酸デカルボキシラーゼ、ジアミノピメリン酸デヒドロゲナーゼ、フォスフォエノールピルビン酸カルボキシラーゼ、アスパルテートアミノトランスフェラーゼ、ジアミノピメリン酸エピメラーゼ、アスパルテートセミアルデヒドデヒドロゲナーゼ、テトラヒドロジピコリン酸スクシニラーゼ、及び、スクシニルジアミノピメリン酸デアシラーゼからなる群より選択される１種または２種以上の酵素の活性が増強されている、及び／または、リジンデカルボキシラーゼの活性が弱化されている、請求項９に記載の方法。 The bacterium is dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and succinyl diaminopiminase. 10. The method according to claim 9, wherein the activity of one or more enzymes selected from the group consisting of melinic acid deacylase is enhanced and / or the activity of lysine decarboxylase is attenuated.
PCT/JP2011/065027 2010-07-01 2011-06-30 Method for producing l-amino acid WO2012002486A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2010150988A JP2013176301A (en) 2010-07-01 2010-07-01 Method for producing l-amino acid
JP2010-150988 2010-07-01

Publications (1)

Publication Number Publication Date
WO2012002486A1 true WO2012002486A1 (en) 2012-01-05

Family

ID=45402189

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2011/065027 WO2012002486A1 (en) 2010-07-01 2011-06-30 Method for producing l-amino acid

Country Status (2)

Country Link
JP (1) JP2013176301A (en)
WO (1) WO2012002486A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015060314A1 (en) * 2013-10-21 2015-04-30 味の素株式会社 Method for producing l-amino acid
US10563234B2 (en) 2014-06-03 2020-02-18 Ajinomoto Co., Inc. Method for producing L-amino acids
CN111850010A (en) * 2020-06-08 2020-10-30 黑龙江伊品生物科技有限公司 dapB gene modified recombinant strain and construction method and application thereof
WO2021248890A1 (en) * 2020-06-08 2021-12-16 黑龙江伊品生物科技有限公司 Recombinant strain producing l-lysine and construction methods therefor and use thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2013147882A (en) 2013-10-28 2015-05-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") METHOD FOR PRODUCING L-AMINO ACIDS USING BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE PUTRESCIN DEGRADATION WAY IS BROKEN

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006025477A1 (en) * 2004-09-01 2006-03-09 Kyowa Hakko Kogyo Co., Ltd. Industrially useful microorganism
WO2007037301A1 (en) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Process for producing useful substance
WO2007037300A1 (en) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Process for producing useful substance
JP2009543543A (en) * 2006-07-19 2009-12-10 味の素株式会社 Method for producing L-amino acid using bacteria of Enterobacteriaceae

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006025477A1 (en) * 2004-09-01 2006-03-09 Kyowa Hakko Kogyo Co., Ltd. Industrially useful microorganism
WO2007037301A1 (en) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Process for producing useful substance
WO2007037300A1 (en) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Process for producing useful substance
JP2009543543A (en) * 2006-07-19 2009-12-10 味の素株式会社 Method for producing L-amino acid using bacteria of Enterobacteriaceae

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XU J ET AL.: "aldB, an RpoS-Dependent Gene in Escherichia coli Encoding an Aldehyde Dehydrogenase That Is Repressed by Fis and Activated by Crp", J BACTERIOL., vol. 177, no. 11, 1995, pages 3166 - 3175 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015060314A1 (en) * 2013-10-21 2015-04-30 味の素株式会社 Method for producing l-amino acid
JPWO2015060314A1 (en) * 2013-10-21 2017-03-09 味の素株式会社 Method for producing L-amino acid
US10563234B2 (en) 2014-06-03 2020-02-18 Ajinomoto Co., Inc. Method for producing L-amino acids
CN111850010A (en) * 2020-06-08 2020-10-30 黑龙江伊品生物科技有限公司 dapB gene modified recombinant strain and construction method and application thereof
WO2021248890A1 (en) * 2020-06-08 2021-12-16 黑龙江伊品生物科技有限公司 Recombinant strain producing l-lysine and construction methods therefor and use thereof

Also Published As

Publication number Publication date
JP2013176301A (en) 2013-09-09

Similar Documents

Publication Publication Date Title
US8512987B2 (en) Method of producing L-amino acid
CN107893089B (en) Method for producing L-amino acid
EP1999266B1 (en) Method for producing l-amino acid
US8293505B2 (en) L-amino acid-producing microorganism and a method for producing an L-amino acid
US8951760B2 (en) Method for producing an L-amino acid
CN108690856B (en) Process for producing L-amino acid
US8394612B2 (en) Method for production of an L-amino acid
JP2007185184A (en) L-amino acid-productive microorganism and method for producing l-amino acid
EP1979486A1 (en) L-amino acid producing bacterium and method of producing l-amino acid
EP2382320B1 (en) Process for producing l-amino acids employing bacteria of the enterobacteriacea family in a culture medium with controlled glycerol concentration
EP3686216A1 (en) Method for producing l-amino acid
WO2012002486A1 (en) Method for producing l-amino acid
WO2015186749A1 (en) Method for producing l-amino acids
US8313933B2 (en) L-amino acid producing bacterium and method for producing L-amino acid
WO2011096554A1 (en) MANUFACTURING METHOD FOR MUTANT rpsA GENE AND L-AMINO ACID
JP2010200645A (en) Method for producing l-amino acid
JP2010246483A (en) Method for producing l-amino acid
WO2015060314A1 (en) Method for producing l-amino acid
WO2010101053A1 (en) Method for producing l-amino acid
JP2008086305A (en) L-amino acid-producing bacterium and method for producing l-amino acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11800948

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11800948

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP