WO2012002486A1 - Procédé de production d'acide aminé l - Google Patents

Procédé de production d'acide aminé l Download PDF

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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
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gene
amino acid
activity
strain
protein
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佑介 萩原
裕梨 益満
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • 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.

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Abstract

L'invention concerne un procédé pour produire efficacement un acide aminé L à partir d'éthanol comme matière première en utilisant une bactérie appartenant à la famille des Enterobacteriaceae, et concerne également une bactérie utilisée dans ce procédé. Une bactérie modifiée de telle sorte que l'activité de la protéine Aldb soit réduite, de préférence une bactérie également modifiée pour que l'activité de la protéine AdhE soit augmentée, est utilisée dans le procédé de production d'un acide aminé L dans lequel une bactérie appartenant à la famille des Enterobacteriaceae capable de produire un acide aminé L est cultivée dans un milieu contenant de l'éthanol et l'acide aminé L est récupéré à partir du milieu.
PCT/JP2011/065027 2010-07-01 2011-06-30 Procédé de production d'acide aminé l WO2012002486A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015060314A1 (fr) * 2013-10-21 2015-04-30 味の素株式会社 Procédé de production d'un acide aminé de forme l
US10563234B2 (en) 2014-06-03 2020-02-18 Ajinomoto Co., Inc. Method for producing L-amino acids
CN111850010A (zh) * 2020-06-08 2020-10-30 黑龙江伊品生物科技有限公司 一种dapB基因改造的重组菌株及其构建方法与应用
WO2021248890A1 (fr) * 2020-06-08 2021-12-16 黑龙江伊品生物科技有限公司 Souche recombinée produisant de la l-lysine, procédés de construction correspondants et leur utilisation

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
RU2013147882A (ru) 2013-10-28 2015-05-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") СПОСОБ ПОЛУЧЕНИЯ L-АМИНОКИСЛОТ С ИСПОЛЬЗОВАНИЕМ БАКТЕРИЙ СЕМЕЙСТВА Enterobacteriaceae, В КОТОРОЙ НАРУШЕН ПУТЬ ДЕГРАДАЦИИ ПУТРЕСЦИНА

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WO2007037301A1 (fr) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Procédé de production d'une substance utile
WO2007037300A1 (fr) * 2005-09-29 2007-04-05 Kyowa Hakko Kogyo Co., Ltd. Procédé de production d'une substance utile
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015060314A1 (fr) * 2013-10-21 2015-04-30 味の素株式会社 Procédé de production d'un acide aminé de forme l
JPWO2015060314A1 (ja) * 2013-10-21 2017-03-09 味の素株式会社 Ｌ−アミノ酸の製造法
US10563234B2 (en) 2014-06-03 2020-02-18 Ajinomoto Co., Inc. Method for producing L-amino acids
CN111850010A (zh) * 2020-06-08 2020-10-30 黑龙江伊品生物科技有限公司 一种dapB基因改造的重组菌株及其构建方法与应用
WO2021248890A1 (fr) * 2020-06-08 2021-12-16 黑龙江伊品生物科技有限公司 Souche recombinée produisant de la l-lysine, procédés de construction correspondants et leur utilisation

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