WO2014027702A1 - Procédé de production de l-arginine à l'aide d'une bactérie de la famille des entérobactériacées présentant une n-acétylornithine désacétylase dotée d'une activité réduite - Google Patents

Procédé de production de l-arginine à l'aide d'une bactérie de la famille des entérobactériacées présentant une n-acétylornithine désacétylase dotée d'une activité réduite Download PDF

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WO2014027702A1
WO2014027702A1 PCT/JP2013/072341 JP2013072341W WO2014027702A1 WO 2014027702 A1 WO2014027702 A1 WO 2014027702A1 JP 2013072341 W JP2013072341 W JP 2013072341W WO 2014027702 A1 WO2014027702 A1 WO 2014027702A1
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gene
bacterium
amino acid
activity
arge
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Mikhail Markovich Gusyatiner
Yulia Georgievna Rostova
Elvira Borisovna Voroshilova
Mikhail Yurievich Kiryukhin
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Ajinomoto Co.,Inc.
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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01035—Glutamate N-acetyltransferase (2.3.1.35)

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids such as L-arginine by fermentation of a bacterium of the family Enterobacteriaceae which has been modified to contain the argJ gene, encoding an enzyme having at least an ornithine acetyltransferase activity, and having downregulated the argE gene encoding N-acetylornithine deacetylase.
  • L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
  • Another method for enhancing L-amino acids production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, and phosphate fluxes, and genes encoding toxins, etc.
  • the biosynthesis is initiated by acetylating L-glutamate with amino acid N-acetyltransferase (EC 2.3.1.1 , also referred to as N-acetyl-L- glutamate synthetase) encoded by the argA gene.
  • N-acetyltransferase EC 2.3.1.1 , also referred to as N-acetyl-L- glutamate synthetase
  • the follow-up biosynthetic reactions are catalyzed by the enzymes commonly referred to as N- acetylglutamate kinase, N-acetyl-y-glutamylphosphate reductase, N- acetylornithine aminotransferase, N-acetylornithine deacetylase, ornithine carbamoy transferase, argininosuccinate synthase, and argininosuccinate lyase encoded by the argB, argC, argD, argE, argF, argG and argH genes, respectively.
  • the enzymes commonly referred to as N- acetylglutamate kinase, N-acetyl-y-glutamylphosphate reductase, N- acetylornithine aminotransferase, N-acetylornithine deacetylase, orni
  • the acetyl group cleaved off by N-acetylornithine deacetylase (ArgE) from N-acetylornithine to produce ornithine, is recruited further by the coenzyme A (HS-CoA, CoA) to form acetylated coenzyme A (AcS-CoA, acetyl- CoA) which acts as the acetyl group donor in the amino acid N- acetyl transferase ⁇ argA) catalyzed acetylation of L-glutamate.
  • coenzyme A HS-CoA, CoA
  • AcS-CoA acetylated coenzyme A
  • the monofunctional and bifunctional ArgJ enzymes can be distinguished, for example, by two means: (i) by enzymatic assay using two acetyl group donors such as N-acetylornithine and AcS-CoA; and (ii) by complementation test using argE and argA mutants of E. coli for the cloned argJ gene.
  • the monofunctional ArgJ enzyme transfers the acetyl group from N- acetylornithine to L-glutamate and complements the argE mutant, whereas the bifunctional ArgJ enzyme transfers the acetyl group from N- acetylornithine and AcS-CoA and complements argE and argA mutant strains.
  • An E. coli bacterium natively having the linear pathway has been modified to contain the argJ gene from Bacillus stearothermophilus or Thermotoga neapolitana (T. neapolitana), which encodes the bifunctional ArgJ enzyme, to initiate the less energy consuming cyclic pathway and thus to increase production of L-arginine by the modified bacterium (U.S. Patent No. 6,897,048 B2).
  • the amino acid N-acetyltransferase N-acetyl-L-glutamate synthetase
  • ArgA amino acid N-acetyltransferase
  • the number of copies of the argA gene can be increased by cloning the wild-type argA gene on plasmid vectors and incorporating them into the strain having the argJ gene cloned ( U.S. Patent No. 6,897,048 B2).
  • the argA gene encoding the mutant amino acid N-acetyltransferase resistant to the feedback inhibition by L-arginine can be introduced into E. coli strain to improve L-arginine production (EP1 170361 A2).
  • the inventors of the present invention have assumed that in a microorganism, for example, in a bacterium having the heterologous argJ gene, which encodes monofunctional ornithine acetyltransferase or bifunctional ornithine acetyltransferase/ N-acetylglutamate synthase, attenuation of expression of the argE gene or inactivation of the argE gene may retrieve additional energy from the processes related to the acetyl group transfer from N-acetylornithine to L-glutamate thus promoting increased L- arginine production by the modified strain as compared with a parent strain.
  • the ArgJ enzyme having mono or dual activity may rescue the L-arginine biosynthesis in argE or argA and argE mutants upon cloning of the arg J gene from a donor microorganism.
  • the artificial cyclic pathway may function through direct acetylation of L-glutamate by the acetyl group originated from N-acetylornithine rather than from energetically wealthy AcS-CoA.
  • An aspect of the present invention is to provide a bacterium belonging to the family Enterobacteriaceae, which may belong to the genus Escherichia, and more specifically, to the species Escherichia coli, which has the activity of ornithine acetyltransferase or ornithine acetyltransferase/ N-acetylglutamate synthase (ArgJ), and in which the N-acetylornithine deacetylase (ArgE) is inactive or the activity of ArgE is decreased.
  • Enterobacteriaceae which may belong to the genus Escherichia, and more specifically, to the species Escherichia coli, which has the activity of ornithine acetyltransferase or ornithine acetyltransferase/ N-acetylglutamate synthase (ArgJ), and in which the N-acetylornithine deacet
  • Another aspect of the present invention is to provide a method for producing L-amino acids such as L-arginine using a bacterium of the family Enterobacteriaceae as described herein.
  • An aspect of the present invention is to provide L-arginine-producing bacterium of the family Enterobacteriaceae having a recombinant DNA comprising the argJ gene encoding an enzyme having at least an ornithine acetyltransferase activity, wherein the bacterium has been modified to contain the downregulated N-acetylornithine deacetylase activity.
  • argJ gene is derived from a microorganism having an enzyme having the ornithine acetyltransferase or ornithine acetyltransferase/ N-acetylglutamate synthase activity.
  • argJ gene is derived from the microorganism belonging to the family selected from the group consisting of the families Thermotogaceae, Bacillaceae and Methanocaldococcaceae.
  • argJ gene encodes a protein selected from the group consisting of:
  • A a protein having the amino acid sequence of SEQ ID NO: 2 which has ornithine acetyltransferase/ N-acetylglutamate synthase activity
  • B a variant protein having the amino acid sequence of SEQ ID NO: 2, but which includes substitution, deletion, insertion, and/or addition of one or several amino acid residues and has ornithine acetyltransferase/N-acetylglutamate synthase activity according to the amino acid sequence of SEQ ID NO: 2.
  • N-acetylornithine deacetylase is a protein selected from the group consisting of:
  • (E) a variant protein having the amino acid sequence of SEQ ID NO: 4, but which includes substitution, deletion, insertion, and/ or addition of one or several amino acid residues and has N-acetylornithine deacetylase activity according to the amino acid sequence of SEQ ID NO: 4.
  • An aspect of the present invention is to provide a method for producing L-arginine or a salt thereof comprising: (i) cultivating the bacterium as described above in a culture medium to accumulate L-arginine or the salt thereof in the bacterium or culture medium, or both; and, if necessary,
  • FIG. 1 shows the scheme for integration of the argJ gene into the chromosome of the E. coli MG 1655 argAAargR strain.
  • FIG. 2 shows the scheme for construction of the E. coli MGl655 argAAargRli.artP ⁇ ::Pn ⁇ p& y ⁇ o rgJargEm24::Cm strain.
  • an L-amino acid-producing bacterium can mean a bacterium of the family Enterobacteriaceae which has an ability to produce, excrete or secrete, and cause accumulation of an L-amino acid in a culture medium or the bacterial cells when the bacterium is cultured in the medium.
  • L-amino acid-producing ability can mean the ability of the bacterium to produce, excrete or secrete, and cause accumulation of the L- amino acid in a medium or the bacterial cells to such a level that the L-amino acid can be collected from the medium or the bacterial cells, when the bacterium is cultured in the medium.
  • L-amino acid can mean L-alanine, L-arginine, L- asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
  • the bacterium may inherently have the L-amino acid-producing ability or may be modified to have an L-amino acid-producing ability by using a mutation method or DNA recombination techniques.
  • the bacteria belonging to the family Enterobacteriaceae can be from the genera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, and so forth, and can have the ability to produce an L- amino acid.
  • strains from the family Enterobacteriaceae which can be modified include a bacterium of the genus Escherichia, Enterobacter or Pantoea.
  • E. coli which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used (Bachmann, B.J., Derivations and genotypes of some mutant derivatives of E. coli K- 12, p. 2460-2488. In F.C. Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2 nd ed. ASM Press, Washington, D.C., 1996).
  • the species E. coli is a particular example. Specific examples of E. coli include E. coli W31 10 (ATCC 27325), E.
  • coli MG1655 ATCC 47076
  • E. coli K- 12 strain E. coli K- 12 strain.
  • These strains are available from, for example, the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to www.atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
  • Enterobacter bacteria examples include Enterobacter agglomerans, Enterobacter aerogenes, and so forth.
  • Pantoea bacteria include Pantoea ananatis, and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc.
  • a bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae.
  • Pantoea ananatis strain When a Pantoea ananatis strain is bred by genetic engineering techniques, Pantoea ananatis AJ 13355 strain (FERM BP-6614), AJ 13356 strain (FERM BP-6615), AJ 13601 strain (FERM BP-7207) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above.
  • an L-arginine-producing bacterium can mean a bacterium which has an ability to produce, excrete or secrete, and cause accumulation of L-arginine in a culture medium or the bacterial cells in an amount larger than a wild- type or a parent strain of E. coli such as E. coli K- 12 when the bacterium is cultured in the medium.
  • the L-arginine-producing ability can mean the ability of the bacterium to produce, excrete or secrete, and cause accumulation of L-arginine in a medium in an amount of not less than 0.5 g/L, or not less than 1.0 g/L, so that L-arginine can be collected from the medium and /or the bacterial cells when the bacterium is cultured in the medium.
  • parent strains which can be used to derive L-arginine- producing bacteria include, but are not limited to, strains belonging to the genus Escherichia such as E. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N- acetylglutamate synthase (EP 1170361 A2), E. coli strain 382 (VKPM B-7926) (EP1 170358 Al), an L-arginine-producing strain having the argA gene introduced (EP1 170361 Al), and the like.
  • E. coli strain 237 VKPM B-7925
  • E. coli strain 382 VKPM B-7926
  • EP1 170358 Al an L-arginine-producing strain having the argA gene introduced
  • the L-arginine-producing bacterium of the family Enterobacteriaceae can be modified further to contain the heterologous argJ gene which encodes monofunctional ornithine acetyltransferase or bifunctional ornithine acetyltransferase/N-acetylglutamate synthase (ArgJ).
  • the exemplary bacterium of the family Enterobacteriaceae may have the argJ gene encoding the mono- or bifunctional ArgJ enzyme resistant to feedback inhibition by L- arginine.
  • the argJ gene can be derived from a thermophilic microorganism such as a microorganism belonging to the family Thermotogaceae, and more specifically to the genus Thermotoga, for example, T. neapolitana (T. neapolitana) .
  • the argJ gene may also derive from other bacterial sources such as Bacillus stearothermophilus (or Geobacillus stearothermophilus) of the family Bacillaceae or Archaea sources such as Methanocaldococcus jannaschii (M, jannaschii) (formerly Methanococcus jannaschii) of the family Methanocaldococcaceae, and the like.
  • various bacterial sources of the argJ gene may be used as soon as the argJ gene encodes an enzyme having at least an ornithine acetyltransferase activity.
  • the L-arginine-producing bacterium of the family Enterobacteriaceae can be modified further to contain inactive argE gene or the argE gene with attenuated expression so that the N-acerylornithine deacetylase (ArgE) in the modified bacterium is not active or the activity of the N-acetylornithine deacetylase is decreased as compared with a non-modified bacterium.
  • ArgE N-acerylornithine deacetylase
  • an L-arginine-producing bacterium can also mean the bacterium of the family Enterobacteriaceae having the argA gene encoding the wild-type or a mutant amino acid N-acetyltransferase (ArgA).
  • the ArgA enzyme resistant to feedback inhibition by L-arginine may be referred to as the mutant amino acid N-acetyltransferase.
  • the exemplary mutant ArgA protein resistant to feedback inhibition by L-arginine may derive from the wild-type ArgA protein by substitution of the amino acid sequence at the position from 15 to 19 to another amino acid sequence as disclosed in EP 1170361 A2.
  • mutant ArgA protein having substitution, deletion, insertion, and/or addition of one or several amino acid residues at one or more positions other than from 15 to 19 in the amino acid sequence may also be referred to as the mutant ArgA protein provided that the three- dimensional structure of the mutant amino acid N-acetyltransferase is not significantly changed relative to the wild-type protein or an activity of the mutant enzyme is not deteriorated and it is resistant to feedback inhibition by L-arginine.
  • an L-arginine-producing bacterium can also mean the bacterium of the family Enterobacteriaceae as described above having enhanced, attenuated, and/ or inactivated the argA gene encoding the wild- type or a mutant amino acid N-acetyltransferase.
  • the phrase "an L-arginine-producing bacterium” can also mean the bacterium of the family Enterobacteriaceae modified further to be devoid of the ArgR-mediated transcriptional repression.
  • the DNA-binding transcriptional dual regulator ArgR involved in the negative control of L- arginine biosynthesis pathway in microorganisms can be inactivated by, for example, inactivation of the ArgR encoding gene (argR ⁇ .
  • a wild- type protein can mean a native protein naturally produced by a wild-type or a parent bacterial strain of the family Enterobacteriaceae, for example, by the wild-type E. coli MG1655 strain.
  • a wild-type protein can be encoded by the wild-type, or non-modified, gene naturally occurring in a genome of wild-type bacterium.
  • a bacterium modified to contain the argJ gene can mean that the bacterium belonging to the first bacterial species, naturally not containing the argJ gene and thus referred to as a recipient bacterium, has been modified to contain one or more recombinant DNA molecules, having the argJ gene, synthesized and/ or originated and introduced from a donor bacterium belonging to the second bacterial species, which is different from the first species.
  • the exemplary modification to introduce a recombinant DNA can include the heterologous gene expression.
  • the recipient bacterium may belong to the family Enterobacteriaceae, for example, to the species E. coli; the donor bacterium may belong to a thermophilic bacterium, for example, to the species T. neapolitana.
  • a bacterium modified to contain the recombinant DNA can mean the bacterium modified to contain an exogenous DNA by, for example, conventional methods such as, for example, transformation, transfection, infection, conjugation and mobilization. Transformation, transfection, infection, conjugation or mobilization of a bacterium with the DNA encoding a protein can impart the bacterium an ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation and mobilization include any known methods that have been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells of Escherichia coli K- 12 to DNA has been reported for efficient DNA transformation and transfection (Mandel M.
  • the heterologous expression of the argJ gene in host microorganisms can be achieved by substituting rare (low-usage in the host organism) codons for synonymous middle- or high-usage codons, where codon usage can be defined as the number of times (frequency) a codon is translated per unit time in the cell of an organism or an average codon frequency of the sequenced protein-coding reading frames of an organism (Zhang S.P. et al., Gene, 1991, 105(l):61-72).
  • the codon usage per organism can be found in the Codon Usage Database, which is an extended web-version of the CUTG (Codon Usage Tabulated from GenBank) (http:/ /www.kazusa.or.jp/codon/ ; Nakamura Y. et al., Codon usage tabulated from the international DNA sequence databases: status for the year 2000, Nucleic Acids Res., 2000, 28(1):292).
  • GenBank Codon Usage Tabulated from GenBank
  • such mutations can include, without limiting, the substitution of rare Arg codons AGA, AGG, CGG, CGA for CGT or CGC; rare He codon ATA for ATC or ATT; rare Leu codon CTA for CTG, CTC, CTT, TTA or TTG; rare Pro codon CCC for CCG or CCA; rare Ser codon TCG for TCT, TCA, TCC, AGC or AGT; rare Gly codons GGA, GGG for GGT or GGC; and so forth.
  • the substitution of low-usage codons for synonymous high-usage codons can be preferable.
  • the substituting rare and/ or low-usage codons for synonymous middle- or high-usage codons may be combined with co- expression of the genes which encode tRNAs recognizing rare codons.
  • a bacterium modified to contain the downregulated argE gene can mean that the bacterium has been modified in such a way that in the modified bacterium the expression of the argE gene is attenuated or the argE gene is inactivated.
  • expression of the argE gene is attenuated can mean that the amount of the ArgE protein in the modified bacterium in which expression of the argE gene is attenuated, is reduced as compared with a non-modified bacterium, for example, a wild-type or parent strain.
  • the phrase "expression of the argE gene is attenuated" can also mean that the modified bacterium includes a modified argE gene, which encodes a mutant ArgE protein having decreased activity as compared with the wild- type ArgE protein, or a region operably linked to the gene, including sequences controlling gene expression, such as promoters, enhancers, attenuators and transcription termination signals, ribosome-binding sites (RBS), and other expression control elements is modified resulting in a decrease in the expression level of the argE gene, and other examples (see, for example, W095/ 34672; Carrier T.A. and Keasling J.D., Biotechnol. Prog. 1999, 15:58-64).
  • a modified argE gene which encodes a mutant ArgE protein having decreased activity as compared with the wild- type ArgE protein, or a region operably linked to the gene, including sequences controlling gene expression, such as promoters, enhancers, attenuators and transcription termination signals
  • Expression of the argE, argA, and/ or argR gene(s) can be attenuated by replacing an expression control sequence of the gene(s), such as a promoter on the chromosomal DNA, with a weaker one.
  • the strength of a promoter is defined by the frequency of initiation acts of RNA synthesis. Examples of methods for evaluating the strength of promoters and strong promoters are described in Goldstein et al., Prokaryotic promoters in biotechnology, Biotechnol. Anna. Rev., 1995, 1 : 105- 128), and so forth.
  • nucleotide substitution for several nucleotides in a promoter region of a target gene and thereby modify the promoter to be weakened as disclosed in International Patent Publication WO00/ 18935.
  • substitution of several nucleotides in the SD sequence, and/ or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site (RBS) greatly affects the translation efficiency of mRNA.
  • This modification of the RBS may be combined with decreasing transcription of the argE, argA, and/ or argR gene(s).
  • Expression of the argE, argA, and/ or argR gene(s) can also be attenuated by insertion of a transposon or an IS (insertion sequence) into the coding region of the gene (U.S. Patent No. 5, 175, 107) or in the region controlling gene expression, or in the proximal part of the argE, argA, and/ or argR gene(s) structure, where the argE, argA, and/or argR gene(s) is/are the distal part, or by conventional methods such as mutagenesis with ultraviolet irradiation (UV) irradiation or nitrosoguanidine (N-methyl-N'-nitro-N- nitrosoguanidine).
  • UV ultraviolet irradiation
  • nitrosoguanidine N-methyl-N'-nitro-N- nitrosoguanidine
  • the incorporation of a site-specific mutation can be conducted by known chromosomal editing methods based, for example, on ⁇ Re
  • enzymatic activity of ArgE is decreased can mean that the enzymatic activity of N-acetylorni thine deacetylase (ArgE), amino acid N-acetyl transferase (ArgA), and/ or ArgR regulator, respectively, is lower than that in a non-modified strain, for example, a wild-type strain of the bacterium belonging to the family Enterobacteriaceae, or more specifically, the species E. coll Exemplary, the enzymatic activity of N-acetylornithine deacetylase (ArgE), amino acid N-acetyl transferase (ArgA), and/ or ArgR regulator can be decreased by the gene inactivation.
  • the enzymatic activity of enzymes encoded by the argE, argA, and/ or argR gene(s) can also be decreased by introducing a mutation into the chromosome so that intracellular activity of the proteins encoded the argE, argA, and/ or argR gene(s) is decreased as compared with a non-modified strain.
  • Such a mutation on the gene(s) or upstream the genes in the operon structure can be the replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene(s) (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug- resistance gene and/ or transcription termination signal, deletion of a part of the gene(s) or deletion of the entire gene(s) (Qiu Z. and Goodman M.F., J. Biol. Chem., 1997, 272:861 1- 8617; Kwon D.H. et al., J. Antimicrob. Chemother., 2000, 46:793-796).
  • the amount of the ArgE protein encoded by the argE gene can be decreased to such a level that the residual ArgE activity is less than about 3%, or less than about 2%, or less than about 1% of the initial activity but higher than zero, as compared with a non-modified bacterium.
  • the specific activity of ArgE encoded by the argE gene can be decreased to such a level that the residual specific activity of ArgE is not more than 1000 nmol/mg min, 750 nmol/mg min, 500 nmol/mg min, 250 nmol/mg min, 100 nmol/mg min, 50 nmol/mg min, 25 nmol/mg min, 10 nmol/mg min, 5 nmol/mg min, or about 3 nmol/mg min as resulted from the spread of experimental data.
  • the residual specific activity can be more than 0.5 nmol/mg min, 1 nmol/mg min, 2 nmol/mg min, or about 3 nmol/mg min as resulted from the spread of experimental data.
  • the specific activity of ArgE of the bacterium per mg crude protein can be determined in crude extracts of the sonicated bacterial cells by the described method (Takahara K. et al. FEBS J., 2005, 272:5353-5364).
  • the crude protein concentration can be determined by the Bradford protein assay (Bradford M.M., Anal. Biochem., 1976, 72:248-254) using bovine serum albumin as a standard.
  • the enzymatic activity of N-acetylornithine deacetylase may be decreased by varying the cultivation conditions such as the acidity of the culture medium (pH), the concentration of co-factor(s) such as metal-ions, temperature, ionic strength, and so forth.
  • the phrase "the argE gene is inactivated” can mean that the modified gene encodes a completely inactive or non-functional enzyme.
  • the modified DNA region is unable to naturally express the gene due to deletion of a part of or the entire gene, shifting of the reading frame of the gene, introduction of missense/ nonsense mutation(s), or modification of an adjacent region of the gene, including sequences controlling gene expression such as promo ter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
  • Inactivation of the gene can also be performed, for example, by conventional methods such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97(12):5978-83; Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y., et al., Nature Genet, 1998, 20: 123- 128) based on "Red/ET-driven integration" or " Red/ET-mediated integration".
  • the phrase "activity of ArgE is abolished” is equivalent to the phrase “the ArgE protein is inactive” and can mean that the activity of ArgE is completely absent, that is equal to zero, or the activity of ArgE is below the detection level when the activity is measured using the method described in Takahara K. et al. FEES J., 2005, 272:5353-5364.
  • Examples of a wild-type Enterobactenaceae bacterium which may serve as controls for the above comparison, include the E. coli MG1655 strain, and so forth.
  • the specific activity of ArgE encoded by the argE gene can be abolished to such a level that the residual specific activity of ArgE is zero or not more than 0.01 nmol/mg min, 0.05 nmol/mg min, 0.1 nmol/mg min, or about 0.5 nmol/mg min as resulted from the spread of experimental data and the detection level, when measuring activity as described above.
  • the activity of the ArgE protein (N-acetylornithine deacetylase) can be decreased and/ or abolished. Therefore, the phrase “activity of the ArgE is downregulated” can be understood as the phrase(s) "activity of the ArgE is decreased” and/ or "activity of ArgE is abolished” as specified above.
  • the phrase "enhanced expression of the argA gene” can mean that the total enzymatic activity of the corresponding gene protein product, ArgA, is increased by, for example, introducing and /or increasing the copy number of the argA gene in bacterial genome, or enhancing the activity per molecule (may be referred to as a specific activity) of the proteins encoded by said gene, as compared with a non-modified strain such as a wild-type or a parent strain.
  • the bacterium can be modified so that the activity of the ArgA protein per cell is increased to 150% or more, 200% or more, 300% or more, of the activity of a non-modified strain.
  • Examples of a non-modified strain serving as a reference for the above comparison include a wild-type strain of a microorganism belonging to the family Enterobactenaceae such as the E. coli MG1655 strain (ATCC 47076), W3110 strain (ATCC 27325), Pantoea ananatis AJ 13335 strain (FERM BP-6614), and so forth.
  • a wild-type strain of a microorganism belonging to the family Enterobactenaceae such as the E. coli MG1655 strain (ATCC 47076), W3110 strain (ATCC 27325), Pantoea ananatis AJ 13335 strain (FERM BP-6614), and so forth.
  • enhanced expression of the argA gene can also mean that the expression level of the argA gene is higher than that level in a non- modified strain, for example, a wild-type or parent strain.
  • Methods which can be used to enhance expression of the argA gene include, but are not limited to, increasing the argA gene copy number in bacterial genome (in the chromosome and/ or in the autonomously replicated plasmid) and/ or introducing the argA gene into a vector that is able to increase the copy number and/ or the expression level of the argA gene in a bacterium of the family Enterobactenaceae according to genetic engineering methods known to the one skilled in the art.
  • vectors include, but are not limited to, broad-host- range plasmids such as pCM HO, pRK310, pVKlO l, pBBR122, pBHRl, and the like.
  • Multiple copies of the argA gene can also be introduced into the chromosomal DNA of a bacterium by, for example, homologous recombination, Mu-driven integration, or the like. Homologous recombination can be carried out using a sequence multiple copies in the chromosomal DNA. Sequences with multiple copies in the chromosomal DNA include, but are not limited to, repetitive DNA or inverted repeats present at the end of a transposable element.
  • argA gene it is possible to incorporate the argA gene into a transposon and allow it to be transferred to introduce multiple copies of the argA gene into the chromosomal DNA.
  • Mu-driven integration more than 3 copies of the gene can be introduced into the chromosomal DNA during a single act (Akhverdyan V.Z. et al. , Biotechnol. ( Russian), 2007, 3:3- 20).
  • Enhancing of the argA gene expression can also be achieved by increasing the expression level of the argA gene by modification of adjacent regulatory regions of the argA gene or introducing native and /or modified foreign regulatory regions.
  • Regulatory regions or sequences can be exemplified by promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements (e.g., regions to which repressors or inducers bind and/ or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA).
  • RBS ribosome-binding sites
  • the exemplary promoters enhancing the argA gene expression can be the potent promoters.
  • the lac promoter, the trp promoter, the trc promoter, the tac promoter, the PR or the PL promoters of lambda phage are all known to be potent promoters.
  • Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of the argA gene to obtain a stronger promoter function, thus resulting in the increased transcription level of the argA gene located downstream of the promoter.
  • heterologous argJ gene can be enhanced using the same approaches as described above for the org A gene.
  • the copy number, presence or absence of the gene and/ or operon genes can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like.
  • the level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
  • the amount of the protein coded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), or mass spectrometry analysis of the protein samples, and the like.
  • Methods for manipulation with recombinant molecules of DNA, molecular cloning and heterologous gene expression such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, and the like may be ordinary methods well- known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2 nd ed., Cold Spring Harbor Laboratory Press (1989) or Green M.R. and Sambrook J.R., "Molecular Cloning: A Laboratory Manual", 4 th ed., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L.
  • the argJ gene may encode the monofunctional ornithine acetyltransferase protein ArgJ (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. MJ_0186; UniProtKB/Swiss-Prot, Protein Knowledgebase, entry No. Q57645).
  • the argJ gene (GenBank accession No. NC_000909.1; nucleotide positions: 184215 to 185423, complement; Gene ID: 1451033) may be located between the MJ_0187 gene on the same strand and the MJ_0185 gene on the opposite strand on the chromosome of M. jannaschii DSM 2661.
  • the nucleotide sequence of the argJ gene and the amino acid sequence of the monofunctional ArgJ protein encoded by the argJ gene may be shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
  • the argJ gene encodes the bifunctional ornithine acetyl transferase/ N- acetylglutamate synthase protein ArgJ (KEGG, entry No. CTN_1 181; UniProtKB/Swiss-Prot, entry No. Q9Z4S1).
  • the argJ gene (GenBank accession No. NC_01 1978.1 ; nucleotide positions: 1 146678 to 1147871, complement; Gene ID: 7377498) is located between the argC and CTN_1180 genes on the chromosome of T. neapolitana DSM 4359.
  • the nucleotide sequence of the argJ gene and the amino acid sequence of the bifunctional ArgJ protein encoded by the org J gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the argE gene encodes the N-acetylornithine deacetylase protein ArgE (KEGG, entry No. b3957).
  • the argE gene (GenBank accession No. NC_000913.2; nucleotide positions: 4151719 to 4152870, complement; Gene ID: 948456) is located between the ppc gene on the same strand and the argC gene on the opposite strand on the chromosome of E. coli strain K- 12.
  • the nucleotide sequence of the argE gene and the amino acid sequence of the ArgE protein encoded by the argE gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the argA gene encodes the amino acid N-acetyltransferase protein ArgA (KEGG, entry No. b2818).
  • the argA gene (GenBank accession No. NC_000913.2; nucleotide positions: 2947264 to 2948595; Gene ID: 947289) is located between the amiC and recD genes, both on the opposite strand, on the chromosome of E. coli strain K- 12.
  • the nucleotide sequence of the argA gene and the amino acid sequence of the ArgA protein encoded by the argA gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the argR gene encodes the DNA-binding transcriptional dual regulator, L-arginine-binding, ArgR (KEGG, entry No. b3237).
  • the argR gene (GenBank accession No. NC_000913.2; nucleotide positions: 3382725 to 3383195; Gene ID: 947861) is located between the yhcN gene on the same strand and the mdh gene on the opposite strand on the chromosome of E. coli strain K- 12.
  • the nucleotide sequence of the argR gene and the amino acid sequence of the ArgR protein encoded by the argR gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the argJ, argE, argA and argR genes are not limited to the genes shown in SEQ ID NOs: 1 , 3, 5, 7 and 29, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1 , 3, 5, 7 and 29, and which encode variants of the ArgJ, ArgE, ArgA and ArgR proteins.
  • a variant protein can mean a protein which has one or several changes in the sequence compared with SEQ ID NOs: 2, 4, 6, 8 and 30, whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but still maintain an activity similar to that of the ArgJ, ArgE, ArgA and ArgR proteins, respectively, or the three- dimensional structure of the ArgJ, ArgE, ArgA and ArgR proteins is not significantly changed relative to the wild-type or non-modified proteins.
  • the number of changes in the variant protein depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It can be, but is not strictly limited to, 1 to 30, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NOs: 2, 4, 6, 8 and 30.
  • the exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s) so that the activity and features of the variant protein are maintained, and are similar to those of the ArgJ, ArgE, ArgA and ArgR proteins.
  • the representative conservative mutation is a conservative substitution.
  • the conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, lie and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gin, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gin and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group.
  • conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution Asn, Glu, Lys, His, Asp or Arg for Gin, substitution Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for He, substitution of lie, Met, Val or Phe for Leu, substitution Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He or Leu for Val
  • the exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/ are compensated for by one or more secondary mutations in the different position(s) of amino acids sequence so that the activity and features of the variant protein are maintained, and are similar to those of the ArgJ, ArgE, ArgA and ArgR proteins.
  • BLAST search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci.
  • the computer program BLAST calculates three parameters: score, identity and similarity.
  • the FASTA search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods EnzymoL, 1990, 183:63-98).
  • the ClustalW method is described by Thompson J.D. et al. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res., 1994, 22:4673-4680).
  • argJ, argE, argA and argR genes can be variant nucleotide sequences.
  • the phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes "a variant protein” using any synonymous amino acid codons according to the standard genetic code table (see, e.g., Lewin B., Genes VIII, 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458).
  • a variant nucleotide sequence can also mean, but not be limited to, a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NOs: 1 , 3, 5, 7 and 29, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it encodes functional protein.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed.
  • stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of l xSSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate), or in another example, O.
  • Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequences shown in SEQ ID NOs: 1, 3, 5, 7 and 29, and a DNA fragment containing the nucleotide sequence as a template.
  • the length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • the washing conditions after hybridization can be exemplified by 2*SSC, 0.1% SDS at 50°C, 60°C or 65°C.
  • the variant nucleotide sequences encoding variant proteins of the ArgJ, ArgE, ArgA and ArgR proteins can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., Trends Genet, 1989, 5: 185- 189) utilizing primers prepared based on the nucleotide sequence of the argJ, argE, argA and argR genes, or chemically synthesized as full-length gene structure. Genes encoding the ArgJ, ArgE, ArgA and ArgR proteins or their variant proteins of other microorganisms can be obtained in a similar manner.
  • Activity of ornithine acetyl transferase (monofunctional ArgJ) (EC: 2.3.1.35; synonyms: glutamate N-acetyltransferase, N-acetyl-L-glutamate synthetase, etc.) means activity of catalyzing the following reaction: N2- acetyl-L-ornithine + L-glutamate * ⁇ L-ornithine + N-acetyl-L-glutamate.
  • Activity of ornithine acetyltransferase/N-acetylglutamate synthase (bifunctional ArgJ) (EC: 2.3.1.35/2.3.1.1) means activity of catalyzing the following reactions: N2-acetyl-L-ornithine + L-glutamate ⁇ L-ornithine + N- acetyl-L-glutamate, and acetyl-CoA + L-glutamate ⁇ CoA + N-acetyl-L- glutamate.
  • N-acetylornithine deacetylase (EC: 3.5.1.16; synonyms: 2-N-acetyl-L-ornithine amidohydrolase, N-acetylornithinase) means activity of catalyzing the following reaction: N2-acetyl-L-ornithine + H2O ⁇ acetate + L-ornithine.
  • N-acetyltransferase (EC: 2.3.1.1 ; synonyms: N-acetylglutamate synthase, N-acetyl-L-glutamate synthetase) means activity of catalyzing the following reaction: acetyl-CoA + L-glutamate ⁇ CoA + N-acetyl-L-glutamate.
  • operably linked to a gene can mean that the regulatory region(s) is linked to the nucleotide sequence of the nucleic acid molecule or gene of interest in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, an ti terminated, attenuated, decreased or repressed expression) of the nucleotide sequence, preferably expression of a gene product encoded by the nucleotide sequence.
  • an enzyme having at least an ornithine acetyltransferase activity can mean that the enzyme has the activity of ornithine acetyltransferase (monofunctional ArgJ) or activity of ornithine acetyltransferase /N-acetylglutamate synthase (bifunctional ArgJ) as described above.
  • the bacterium as described herein can be obtained by introduction of the aforementioned DNAs into a bacterium inherently having an ability to produce an L- amino acid.
  • the bacterium as described herein can be obtained by imparting an ability to produce an L-amino acid to a bacterium already harboring the aforementioned DNAs.
  • the bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence, without departing from the scope of the present invention.
  • the method for producing L-amino acid can include the steps of cultivating the bacterium in a culture medium to allow the L-amino acid to be produced, excreted, and accumulated in the culture medium, and collecting the L-amino acid from the culture medium and/ or the bacterial cells.
  • the cultivation of the bacterium, and collection and purification of an L-amino acid or a salt thereof from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an L-amino acid is produced using a microorganism.
  • the culture medium for an L-amino acid production may be a typical medium that contains a carbon source, a nitrogen source, inorganic ions, and other organic components as required.
  • the carbon source saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and the like can be used.
  • inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate
  • organic nitrogen such as of soy bean hydrolyzates
  • ammonia gas aqueous ammonia; and the like
  • Vitamins such as vitamin Bi, required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, or yeast extract, and the like may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions, and the like may be added, if necessary.
  • Cultivation can be performed under aerobic conditions for 16 to 96 hours, or for 48 to 72 hours; the culture temperature during cultivation is controlled within 30 to 45°C, or within 30 to 37°C; and the pH is adjusted between 5 and 8, or between 6.5 and 7.2.
  • the pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.
  • a 1 to 5-day cultivation leads to the accumulation of the target L- amino acid in the liquid medium.
  • solids such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid or a salt thereof can be recovered from the fermentation liquor by any combination of conventional techniques such as concentration, ion-exchange chromatography, and crystallization.
  • the mutant E. coli MG1655 strain having deleted the argR gene was constructed using known gene-knockout method (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) (Example
  • the chloramphenicol-resistance gene (cat, Cm R -marker) was used to mark the AargR mutation, and the kanamycin-resistance gene (kan, Km R - marker) was used to mark the AargE mutation.
  • Cells were cured from the Cm R -marker as described (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).
  • the pJ-T plasmid containing the argJ gene from T. neapolitana (U.S. Patent No. 6,897,048) was transferred to the obtained mutant E.
  • the E. coli MG1655AargR strain was constructed by the ARed-mediated integration method initially developed by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).
  • the DNA fragment containing the chloramphenicol resistance marker (Cm R ) was obtained by PCR using primers PI (SEQ ID NO: 9) and P2 (SEQ ID NO: 10), and the pMW1 18-aiiL-Cm-aiii? plasmid as the template (WO2005010175 Al).
  • Primer PI contains a region complementary to the region located at the 5'-end of the argR gene and a region complementary to the attR region.
  • Primer P2 contains a region complementary to the region located at the 3 '-end of the argR gene and a region complementary to the attL region.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final elongation for 5 min at 72°C.
  • the obtained PCR product (about 1.6 kbp) was purified by electrophoresis in agarose gel and used for electroporation of the E.
  • the pKD46 plasmid includes a 2, 154 nucleotide DNA fragment of phage ⁇ (nucleotide positions from 31088 to 33241 , GenBank accession No.: J02459), and contains genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , exo genes) under the control of the arabinose-inducible ParaB promoter.
  • the pKD46 plasmid is necessary for integration of the PCR product into the chromosome of the E. coli MG1655 strain (ATCC 47076).
  • the E. coli MG1655 strain containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA (Accession No. CGSC7669).
  • Electrocompetent cells were prepared as follows: the E. coli MG1655/pKD46 strain was grown at 30°C overnight in LB medium (Luria- Bertani broth, also referred to as lysogenic broth; Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (150 mg/L), then the culture was diluted 100 times with 5 mL of SOB medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (150 mg/L) and L-arabinose (1 mM).
  • the cells were grown with aeration (250 rpm) at 30°C to ODeoo of «0.6. Electrocompetent cells were made by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 ⁇ L of cells and «100 ng of the PCR product. Cells after electroporation were incubated with 1 mL of SOC medium (Sambrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001) at 37°C for 2.5 hours, plated onto L-agar containing chloramphenicol (25 mg/L) and grown at 37°C to select Cm R -recombinants.
  • SOC medium Standardbrook J. and Russell D.W., Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001
  • the E. coli MG1655 AargR strain having deleted the argE gene was constructed by the ARed-mediated integration method initially developed by Datsenko K.A. and Wanner B.L. ⁇ Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645).
  • the DNA fragment containing the Km R -marker was obtained by PCR using primers P3 (SEQ ID NO: 1 1) and P4 (SEQ ID NO: 12), and the pMWl 18- attL- Km- attR plasmid as the template (Katashkina Zh.I. et al., Mol. Biol (Mosk.), 2005, 39(5):823-831).
  • Example 2 E. coli MG1655Aargi?/pKD46 strain was used for electroporation by the obtained DNA fragment (Example 1.1).
  • the antibiotic resistant cells were selected using kanamycin (25 mg/L), and obtained Km R -recombinants were cured from the pKD46 plasmid as described in Example 1.1.
  • Km R -recombinants were cured from the pKD46 plasmid as described in Example 1.1.
  • the E. coli MG1655AargRAargE::Km was obtained.
  • Example 2 E. coli MG1655AargRAargE::Km was obtained.
  • the fermentation medium contained (g/L):
  • Glucose and magnesium sulfate were sterilized separately.
  • CaC03 was dry-heat sterilized at 180°C for 2 hours. The pH was adjusted to 7.0. Antibiotic was introduced into the medium after sterilization.
  • the argj gene from T. neapolitana was incorporated into the chromosome of the E. coli MG1655AargR strain to avoid the effect of instability of the pJ-T plasmid carrying the argj gene.
  • the effective artificial promoter ⁇ ⁇ ⁇ 8 ⁇ ⁇ was constructed and placed before the argj gene (Example 3.1).
  • the argA gene was deleted from the chromosome (Example 3.1).
  • the AargE mutation was marked by the kanamycin resistance gene (/can) (Example 1.2) and transferred into the chromosome of the E. coli MG1655AargAAargRAartP-J::P n i P 8 ⁇ ? ioargJ strain using the well-known method of general PI -transduction (Miller J.H., Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, 1972).
  • the colonies of Km-resistant transductants were selected on L-agar containing kanamycin (25 mg/L) (Example 1).
  • the E. coli MG1655 argAAargRAartP-J::P n ip8 ⁇ ( ,ioargJAargE::Km strain was obtained.
  • the E. coli MG1655 having deleted the argA gene marked with cai-gene was constructed by the ARed-mediated integration method initially developed by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Set USA, 2000, 97(12):6640-6645) (Example 1. 1).
  • the DNA fragment containing the Cm R - marker was obtained by PCR using primers P5 (SEQ ID NO: 13) and P6 (SEQ ID NO: 14), and the pMWl 18-attL-Cm-attR plasmid as the template (WO2005010175 Al).
  • P5 SEQ ID NO: 13
  • P6 SEQ ID NO: 14
  • the DNA fragment containing a promoter of the nlpD gene from E. coli was obtained by PCR using primers P7 (SEQ ID NO: 15) and P8 (SEQ ID NO: 16), chromosomal DNA of E. coli MG1655 as the template.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 20 sec at 94°C, 20 sec at 55°C, 15 sec at 72°C; final elongation for 5 min at 72°C.
  • the amplified DNA fragment (about 0.2 kbp) was purified by electrophoresis in agarose gel and treated with endonucleases Pael and SaR (Fermentas) (Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2 nd ed., Cold Spring Harbor Laboratory Press (1989); instructions of manufacturer).
  • the obtained DNA fragment was ligated with the pMIV-5JS plasmid (Russian patent application No. 2006132818 and EP 1942183), which had been previously treated with endonucleases Pael and Sail, using the manufacturer's instructions.
  • the ligation mixture was incubated at 4°C overnight and then was used to transform the E.
  • coli MG1655 strain ATCC 47076
  • ATCC 47076 electroporation as described in Example 1.
  • the resulting transformants were plated on L-agar containing ampicillin (50 mg/L), and the plates were incubated at 37°C overnight until individual colonies were visible. Plasmids were isolated from the obtained transformants and analyzed by the restriction analysis using Pael and Sail nucleases. Thus the pMIV-PnlpD plasmid containing the native PnipD promoter of the nlpD gene from E. coli was obtained.
  • the randomization of the - 10 region of the PnipD promoter and the selection of the P n ip8 promoter were performed.
  • the 3'-end of PnipD was obtained by PCR amplification using primers P7 (SEQ ID NO: 15) and P9 (SEQ ID NO: 17), and the pMIV-PnlpD plasmid as the template.
  • Primer P9 has random nucleotides, which are depicted in SEQ ID NO: 17 as "n", where n is A, G, C, or T.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 20 sec at 94°C, 20 sec at 60°C, 15 sec at 72°C; final elongation for 5 min at 72°C.
  • the 5'-end of P n i P D was obtained by PCR amplification using primers P8 (SEQ ID NO: 16) and P10 (SEQ ID NO: 18), and the pMIV-PnlpD plasmid as the template.
  • Primer P10 has random nucleotides, which are depicted in SEQ ID NO: 18 as "n", where n is A, G, C, or T.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 20 sec at 94°C, 20 sec at 60°C, 15 sec at 72°C; final elongation for 5 min at 72°C.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 12 cycles: 20 sec at 94°C, 20 sec at 60°C, 15 sec at 72°C; final elongation for 5 min at 72°C.
  • the amplified DNA fragment (about 0.2 kbp) was purified by electrophoresis in agarose gel.
  • the purified DNA fragment was treated with Klenow fragment (Fermentas) and ligated in equimolar ratio with the pMWl 18- ⁇ ⁇ - ⁇ . ⁇ plasmid (Russian patent application No. 2006134574), which had been previously treated with endonuclease Xbcd (Fermentas) followed by treatment with Klenow fragment.
  • the ligation mixture was incubated at 4°C overnight and then was used to transform the E. coli MG1655 by electroporation as described in Example 1.
  • the resulting transformants were plated on L-agar containing kanamycin (20 mg/L), and the plates were incubated at 37°C overnight until individual colonies were visible.
  • Plasmids were isolated from the obtained transformants and analyzed by the restriction analysis using Pstl and Hindill nucleases (Fermentas) (Sambrook J., Fritsch E.F. and Maniatis T., "Molecular Cloning: A Laboratory Manual", 2 nd ed., Cold Spring Harbor Laboratory Press (1989); instructions of manufacture). Thus the pMW-Km-Pnlp8 plasmid containing the P n i P 8 promoter was obtained.
  • the DNA fragment containing the argrJ gene was obtained by PCR using primers Pl l (SEQ ID NO: 19) and P12 (SEQ ID NO: 20), and the plasmid DNA pJ-T (U.S. Patent No. 6,897,048) as the template.
  • the conditions for PCR were as follows: initial denaturation for 1 min at 95°C; profile for 25 cycles: 1 min at 95°C, 30 sec at 55°C, 1 min at 72°C; final elongation for 2 min at 72°C.
  • the amplified DNA fragment (about 1.2 kbp) was purified by electrophoresis in agarose gel.
  • the DNA fragment containing the attL-Km-XattR-PnipS io cassette was obtained by PCR using primers P13 (SEQ ID NO: 21) and P14 (SEQ ID NO: 22), and the plasmid pMW-Km-Pnlp8 as the template.
  • the conditions for PCR were as follows: initial denaturation for 1 min at 95°C; profile for 25 cycles: 1 min at 95°C, 30 sec at 55°C, lmin 40 sec at 72°C; final elongation foe 5 min at 72°C.
  • the amplified DNA fragment (about 1.7 kbp) ( Figure 1) was purified by electrophoresis in agarose gel.
  • Primers PI 1 (SEQ ID NO: 19) and PI 4 (SEQ ID NO: 22) contain overlapping regions.
  • the obtained DNA fragments ( Figure 1) with overlapping regions were PCR amplified using primers P12 (SEQ ID NO: 20) and P13 (SEQ ID NO: 21), and the obtained DNA fragments as the templates.
  • the conditions for PCR were as follows: initial denaturation for 2 min at 95°C; profile for 30 cycles: 30 sec at 94°C, 30 sec at 55°C, 2 min at 72°C; final elongation for 5 min at 72 °C.
  • the amplified DNA fragment (about 2.9 kbp) was purified by electrophoresis in agarose gel.
  • the obtained DNA fragment contains the Km R -marker and the argJ gene under control of the ⁇ ⁇ 8 ⁇ ⁇ promoter.
  • This fragment was integrated in place of the artPIQMJ gene cluster into the chromosome of the E. coli MG1655 argAAargR strain by the ARed-mediated integration method as described above.
  • the E. coli MG1655AargAAargRAartP-J::Pn ⁇ p8 ⁇ p ioargJw&s obtained.
  • the E. coli MG1655AargAAargRAartP-J::P n ip8 ⁇ ? ioargJ strain was used as parent strain to construct the strain having the argE gene with attenuated expression.
  • the Cm R -marker was introduced into intergenic region between ppc and argE genes of MG1655 strain by the ARed-mediated integration method (Example 1.1).
  • the DNA fragment containing the Cm R - marker encoded by the cat gene was obtained by PCR using primers PI 5 (SEQ ID NO: 23) and P16 (SEQ ID NO: 24), and the pMWl 18- attL-C -attR plasmid as the template (WO2005010175 Al).
  • Primer PI 5 contains a region complementary to the region located downstream to the argE gene and a region complementary to the attR region.
  • Primer P16 contains a region complementary to the region located upstream to the ppc gene and a region complementary to the attL region.
  • the conditions for PCR were as follows: initial denaturation for 3 min at 95°C; profile for the initial 2 cycles: 1 min at 95°C, 30 sec at 50°C, 40 sec at 72°C; profile for the final 25 cycles: 30 sec at 95°C, 30 sec at 54°C, 40 sec at 72°C; final elongation for 5 min at 72°C.
  • the PCR product (about 1.6 kbp) was purified by electrophoresis in agarose gel and used for electroporation of the E. coli MG1655/pKD46 strain as described in Example 1.1. After electrotransformation of the E. coli MG1655/pKD46 strain by the obtained DNA fragment having cat gene, several colonies were selected on L-agar plates containing chloramphenicol (20 mg/L). Electrocompetent cells were prepared, electroporation was performed, and Cm R -recombinants were selected as described in Example 1. 1. Thus the E. coli MG1655-Cm- argE strain was obtained.
  • the L76K replacement in ArgE was obtained.
  • the first DNA fragment containing the Cm R -marker and the distal part of argE encoding mutation L76K was obtained by PCR using primers P17 (SEQ ID NO: 25) and P18 (SEQ ID NO: 26) containing ovelapping regions, and the chromosomal DNA of the E. coli MG1655-Cm- argE strain as the template.
  • the conditions for PCR were as follows: initial denaturation for 1 min at 95°C; profile for 25 cycles: 1 min at 95°C, 30 sec at 55°C, 2 min at 72°C; final elongation for 5 min at 72°C.
  • the amplified first DNA fragment (about 2.8 kbp) ( Figure 2) was purified by electrophoresis in agarose gel.
  • the second DNA fragment containing the proximal part of argE encoding mutation L76K was obtained by PCR using primers PI 9 (SEQ ID NO: 27) and P20 (SEQ ID NO: 28), and the chromosomal DNA of the E. coli MG1655-Cm- argE strain as the template.
  • the conditions for PCR were as follows: initial denaturation for 1 min at 95°C; profile for 25 cycles: 1 min at 95°C, 30 sec at 55°C, 30 sec at 72°C; final elongation for 5 min at 72°C.
  • the amplified second DNA fragment (about 0.3 kbp) ( Figure 2) was purified by electrophoresis in agarose gel.
  • the third DNA fragment was obtained by PCR using primers P17 (SEQ ID NO: 25) and P20 (SEQ ID NO: 28), and obtained the first and second DNA fragments with overlapping regions (Figure 2) as the template
  • the conditions for PCR were as follows: initial denaturation for 2 min at 95°C; profile for 30 cycles: 30 sec at 94°C, 30 sec at 55°C, 2 min 20 sec at 72°C; final elongation for 5 min at 72°C.
  • the amplified third DNA fragment (about 3.1 kbp) was purified by electrophoresis in agarose gel.
  • the mutant argE gene was named the argEm24 gene.
  • the third DNA fragment was integrated instead of the native argE gene into the chromosome of the E. coli MG1655AargAAargRAartP- J::Pn ⁇ ioargJ strain by the ARed-mediated integration method as described above.
  • the E. coli MG1655AargAAargRAartP-J::P n ip8 9 ioargJargEm24::Cm strain was obtained.
  • Enzymatic activity of the control and mutant ArgE L76K proteins encoded by the mutant argEm.24 gene was measured as described in Takahara K. et al. FEBS J., 2005, 272:5353-5364 using crude proteins extracts obtained after cells lysis by sonication.
  • the E. coli MG1655, MG1655Aargf£, and MG1655 argEm.24 strains were used as protein sources to evaluate attenuation level from the L76K mutation in ArgE.
  • the E. coli MG1655 argE and MG1655 argEm.24 strains were obtained as described in Auxiliary example 1.
  • the ArgE L76K protein showed specific activity of less than about 1% as compared with the non-modified ArgE protein.
  • the E. coli MG1655 AargE: :Km was obtained by the procedure described in Example 1.2 for the E. coli MG1655AargRAargE::Km strain.
  • the Km R -marker was eliminated using the pMW-Int/Xis helper plasmid (WO2005010175 Al) as described in Example 1.1 for Cm R -marker.
  • the E. coli MG1655AargrE strain was obtained.
  • the E. coli MG1655argEm24::Cm strain was obtained as described in Example 5 for the E. coli MG1655AargAAargRAartP-J::P n i P 8 v ioargJ argEm24::Cm strain.
  • the Cm R -marker was eliminated using the pMW-Int/Xis helper plasmid (WO2005010175 Al) as described in Example 1.1.
  • WO2005010175 Al pMW-Int/Xis helper plasmid

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Abstract

La présente invention concerne un procédé de production d'acide L-aminé comme la L-arginine par fermentation à l'aide d'une bactérie de la famille des Entérobactériacées, particulièrement une bactérie appartenant au genre Escherichia, qui a été modifiée pour contenir le gène argJ, codant pour une enzyme ayant au moins une activité ornithine acétyltransférase, et le gène argE atténué ou inactivé codant pour la N-acétylornithine désacétylase.
PCT/JP2013/072341 2012-08-17 2013-08-15 Procédé de production de l-arginine à l'aide d'une bactérie de la famille des entérobactériacées présentant une n-acétylornithine désacétylase dotée d'une activité réduite WO2014027702A1 (fr)

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WO2017146195A1 (fr) 2016-02-25 2017-08-31 Ajinomoto Co., Inc. Procédé de production d'acides l-aminés faisant appel à une bactérie de la famille des enterobacteriaceae surexprimant un gène codant pour un exporteur de fer
CN107922955A (zh) * 2015-07-20 2018-04-17 Cj第制糖株式会社 用于产生腐胺或鸟氨酸的微生物和使用其产生腐胺或鸟氨酸的方法
CN108642100A (zh) * 2018-05-16 2018-10-12 江南大学 一种在钝齿棒杆菌中合成l-鸟氨酸的方法
CN110964683A (zh) * 2019-12-02 2020-04-07 天津科技大学 生产l-精氨酸的基因工程菌及其构建方法与应用

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* Cited by examiner, † Cited by third party
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Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4278765A (en) 1978-06-30 1981-07-14 Debabov Vladimir G Method for preparing strains which produce aminoacids
US4346170A (en) 1979-07-23 1982-08-24 Ajinomoto Company, Incorporated Method for producing L-lysine by fermentation
US5175107A (en) 1988-10-25 1992-12-29 Ajinomoto Co., Inc. Bacterial strain of escherichia coli bkiim b-3996 as the producer of l-threonine
WO1995016042A1 (fr) 1993-12-08 1995-06-15 Ajinomoto Co., Inc. Procede de production de l-lysine par fermentation
EP0685555A1 (fr) 1994-05-30 1995-12-06 Ajinomoto Co., Inc. Bactérie productrice de L-isoleucine et procédé de préparation de L-isoleucine par fermentation
WO1995034672A1 (fr) 1994-06-14 1995-12-21 Ajinomoto Co., Inc. GENE A DESHYDROGENASE α-CETOGLUTARIQUE
WO1996015246A1 (fr) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Adn regulant l'expression genique dans des bacteries du genre corynebacterium
US5661012A (en) 1992-11-10 1997-08-26 Ajinomoto Co., Inc. Method for the production of L-threonine by fermentation, using mutated DNA encoding aspartokinase III
WO2000018935A1 (fr) 1998-09-25 2000-04-06 Ajinomoto Co.,Inc. Procede de construction d'une bacterie produisant des acides amines, et procede de production d'acides amines par une technique de fermentation utilisant ladite bacterie
EP1170358A1 (fr) 2000-07-06 2002-01-09 Ajinomoto Co., Ltd. Escherichia coli produisant de la L-arginine et procédé de préparation de L-arginine
EP1170361A2 (fr) 2000-06-28 2002-01-09 Ajinomoto Co., Inc. Nouveau mutant de la N-acetylglutamate synthase et procédé pour la production de la L-arginine
EP1201758A1 (fr) * 2000-10-27 2002-05-02 Ajinomoto Co., Inc. Procédé de préparation de L-arginine par fermentation
US20020058315A1 (en) 2000-09-26 2002-05-16 Ajinomoto Co., Inc. Bacterium having ability to produce L-glutamic acid, L-proline or L-arginine and method for producing L-glutamic acid, L-proline or L-arginine
WO2005010175A1 (fr) 2003-07-29 2005-02-03 Ajinomoto Co., Inc. Procede de production de l-lysine ou de l-threonine au moyen de bacteries escherichia a activite enzyme malique attenuee
US20060216796A1 (en) 2003-11-21 2006-09-28 Kenichi Hashiguchi Method for producing l-amino acid by fermentation
RU2006132818A (ru) 2006-09-13 2008-03-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) Мутантная ацетолактатсинтаза и способ продукции разветвленных l-аминокислот
RU2006134574A (ru) 2006-09-29 2008-04-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) Способ конструирования рекомбинантных бактерий, принадлежащих к роду pantoea и способ продукции l-аминокислот с использованием бактерий, принадлежащих к роду pantoea

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL1664318T3 (pl) * 2004-01-30 2010-03-31 Ajinomoto Kk Mikroorganizm wytwarzający L-aminokwas i sposób wytwarzania L-aminokwasu

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4278765A (en) 1978-06-30 1981-07-14 Debabov Vladimir G Method for preparing strains which produce aminoacids
US4346170A (en) 1979-07-23 1982-08-24 Ajinomoto Company, Incorporated Method for producing L-lysine by fermentation
US5175107A (en) 1988-10-25 1992-12-29 Ajinomoto Co., Inc. Bacterial strain of escherichia coli bkiim b-3996 as the producer of l-threonine
US5661012A (en) 1992-11-10 1997-08-26 Ajinomoto Co., Inc. Method for the production of L-threonine by fermentation, using mutated DNA encoding aspartokinase III
WO1995016042A1 (fr) 1993-12-08 1995-06-15 Ajinomoto Co., Inc. Procede de production de l-lysine par fermentation
US6040160A (en) 1993-12-08 2000-03-21 Ajinomoto Co., Inc. Method of producing L-lysine by fermentation
EP0685555A1 (fr) 1994-05-30 1995-12-06 Ajinomoto Co., Inc. Bactérie productrice de L-isoleucine et procédé de préparation de L-isoleucine par fermentation
WO1995034672A1 (fr) 1994-06-14 1995-12-21 Ajinomoto Co., Inc. GENE A DESHYDROGENASE α-CETOGLUTARIQUE
WO1996015246A1 (fr) 1994-11-11 1996-05-23 Forschungszentrum Jülich GmbH Adn regulant l'expression genique dans des bacteries du genre corynebacterium
WO2000018935A1 (fr) 1998-09-25 2000-04-06 Ajinomoto Co.,Inc. Procede de construction d'une bacterie produisant des acides amines, et procede de production d'acides amines par une technique de fermentation utilisant ladite bacterie
EP1170361A2 (fr) 2000-06-28 2002-01-09 Ajinomoto Co., Inc. Nouveau mutant de la N-acetylglutamate synthase et procédé pour la production de la L-arginine
EP1170358A1 (fr) 2000-07-06 2002-01-09 Ajinomoto Co., Ltd. Escherichia coli produisant de la L-arginine et procédé de préparation de L-arginine
US20020058315A1 (en) 2000-09-26 2002-05-16 Ajinomoto Co., Inc. Bacterium having ability to produce L-glutamic acid, L-proline or L-arginine and method for producing L-glutamic acid, L-proline or L-arginine
EP1201758A1 (fr) * 2000-10-27 2002-05-02 Ajinomoto Co., Inc. Procédé de préparation de L-arginine par fermentation
US6897048B2 (en) 2000-10-27 2005-05-24 Ajinomoto Co. Inc Microorganisms and method for L-arginine production by fermentation
WO2005010175A1 (fr) 2003-07-29 2005-02-03 Ajinomoto Co., Inc. Procede de production de l-lysine ou de l-threonine au moyen de bacteries escherichia a activite enzyme malique attenuee
US20060216796A1 (en) 2003-11-21 2006-09-28 Kenichi Hashiguchi Method for producing l-amino acid by fermentation
RU2006132818A (ru) 2006-09-13 2008-03-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) Мутантная ацетолактатсинтаза и способ продукции разветвленных l-аминокислот
EP1942183A1 (fr) 2006-09-13 2008-07-09 Ajinomoto Co., Inc. Synthase d'acétolactate mutante et procédé de production d'acides amino L à chaine branchée
RU2006134574A (ru) 2006-09-29 2008-04-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) Способ конструирования рекомбинантных бактерий, принадлежащих к роду pantoea и способ продукции l-аминокислот с использованием бактерий, принадлежащих к роду pantoea

Non-Patent Citations (54)

* Cited by examiner, † Cited by third party
Title
"Applications and statistics for multiple high-scoring segments in molecular sequences", PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
AKHVERDYAN ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 91, 2011, pages 857 - 871
AKHVERDYAN ET AL.: "Mu-driven integration/amplification", APPL. MICROBIOL. BIOTECHNOL., vol. 91, 2011, pages 857 - 871, XP019931851, DOI: doi:10.1007/s00253-011-3416-y
AKHVERDYAN V.Z. ET AL., APPL. MICROBIOL BIOTECHNOL., vol. 91, 2011, pages 857 - 871
AKHVERDYAN V.Z. ET AL., BIOTECHNOL. (RUSSIAN, vol. 3, 2007, pages 3 - 20
BACHMANN, B.J. ET AL.: "E. coli and Salmonella cellular and molecular biology", 1996, ASM PRESS, article "Derivations and genotypes of some mutant derivatives of E. coli K-12", pages: 2460 - 2488
BERNARD R. GLICK; JACK J. PASTERNAK; CHERYL L. PATTEN: "Molecular Biotechnology: principles and applications of recombinant DNA", 2009, ASM PRESS
BRADFORD M.M., ANAL. BTOCHOM., vol. 72, 1976, pages 248 - 254
CARRIER T.A.; KEASLING J.D., BIOTECHNOL PROG., vol. 15, 1999, pages 58 - 64
CUNIN R. ET AL., MICROBIOL. REV., vol. 50, 1986, pages 314 - 352
DATABASE UniProt [Online] 16 November 2011 (2011-11-16), "RecName: Full=Acetylornithine deacetylase; Short=AO; Short=Acetylornithinase; EC=3.5.1.16; AltName: Full=N-acetylornithinase;", XP002715801, retrieved from EBI accession no. UNIPROT:G2BGN4 Database accession no. G2BGN4 *
DATABASE UniProt [Online] 23 April 2003 (2003-04-23), "RecName: Full=Arginine biosynthesis bifunctional protein ArgJ; Includes: RecName: Full=Glutamate N-acetyltransferase; EC=2.3.1.35; AltName: Full=Ornithine acetyltransferase; Short=OATase; AltName: Full=Ornithine transacetylase;", XP002715800, retrieved from EBI accession no. UNIPROT:Q9Z4S1 Database accession no. Q9Z4S1 *
DATSENKO K.A.; WANNER B.L., PROC. NATL ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 6640 - 45
DATSENKO K.A.; WANNER B.L., PROC. NATL ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 6640 - 6645
DATSENKO K.A.; WANNER B.L., PROC. NATL. ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 6640 - 45
DATSENKO K.A.; WANNER B.L., PROC. NATL. ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 6640 - 6645
DATSENKO K.A.; WANNER B.L., PROC. NATL. ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 6640.6645
DE DEKEN R.H., BIOCHIRN. BIOPHYS. ACTA., vol. 78, 1962, pages 606 - 616
ECKHARDT T. ET AL., MOI. GEN. GENET., vol. 138, 1975, pages 225 - 232
EVANS JR., T.C.; XU M.-Q.: "Heterologous gene expression in E. colT", 2011, HUMANA PRESS
GOLD L. ET AL., ANNU. REV. MICROBIAL., vol. 35, 1981, pages 365 - 403
GOLDSTEIN ET AL.: "Prokaryotic promoters in biotechnology", BIOTECHNOL. ANNU. REV., vol. 1, 1995, pages 105 - 128, XP008038474
GREEN M.R.; SAMBROOK J.R.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
HAAS D. ET AL., EUR. J. BIOCHEM., vol. 31, 1972, pages 290 - 295
HUI A. ET AL., EMBO J., vol. 3, 1984, pages 623 - 629
KATASHKINA ZH.I. ET AL., MOL. BIOL. (MOSK., vol. 39, no. 5, 2005, pages 823 - 831
KOMA D. ET AL., APPL. MICROBIAL. BIOTECHNOL, vol. 93, no. 2, 2012, pages 815 - 829
KWON D.H. ET AL., J. ARTTIMICROB. CHEMOTHER., vol. 46, 2000, pages 793 - 796
LEWIN B.: "Genes VIII", 2004, PEARSON EDUCATION, INC.
MANDEL M.; HIGA A.: "Calcium-dependent bacteriophage DNA infection", J. MOL. BIOL, vol. 53, 1970, pages 159 - 162, XP024011744, DOI: doi:10.1016/0022-2836(70)90051-3
MARC F. ET AL., EUR. J. BIOCHEM., vol. 267, no. 16, 2000, pages 5217 - 5226
MILLER J.H.: "Experiments in Molecular Genetics", 1972, COLD SPRING HARBOR LA. PRESS
MILLER J.H.: "Experiments in molecular genetics", 1972, COLD SPRING HARBOR LABORATORY PRESS
MINAEVA N. ET AL., BMC BIOTECHNOLOGY, vol. 8, 2008, pages 63
MORSE M.L. ET AL.: "Transduction in Escherichia coli K- 12", GENETICS, vol. 41, no. 1, 1956, pages 142 - 156
NAKAMURA Y. ET AL.: "Codon usage tabulated from the international DNA sequence databases: status for the year 2000", NUCLEIC ACIDS RES., vol. 28, no. 1, 2000, pages 292, XP002941557, Retrieved from the Internet <URL:http://www.kazusa.orjp/codon> DOI: doi:10.1093/nar/28.1.292
PEARSON W.R.: "Rapid and sensitive sequence comparison with FASTP and FASTA", METHODS ENZYMOL, vol. 183, 1990, pages 63 - 98, XP000670614, DOI: doi:10.1016/0076-6879(90)83007-V
QIU Z.; GOODMAN M.F., J. BIOL. CHEM., vol. 272, 1997, pages 8611 - 8617
SAKANYAN V. ET AL., J. GEN. MICROBIAL, vol. 138, 1992, pages 125 - 130
SAKANYAN V. ET AL., MICROBIOLOGY, vol. 142, 1996, pages 99 - 108
SAMBROOK J.; FRITSCH E.F.; MANIATIS T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK J.; RUSSELL D.W.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SAMUEL K.; ALTSCHUL S.F.: "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes", PROC. NATL ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 2268
SHI D. ET AL., J. BIOL. CHEM., vol. 280, 2005, pages 14366 - 14369
TAKAHARA K. ET AL., FEBS J., vol. 272, 2005, pages 5353 - 5364
THOMPSON J.D. ET AL.: "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680, XP002956304
TYO K.E.J. ET AL., NATURE BIOTECHNOL, vol. 27, 2009, pages 760 - 765
TYO K.E.J. ET AL., NATURE BIOTECHNOL., vol. 27, 2009, pages 760 - 765
UDAKA S.; KINOSHITA S., J. GEN. APPI. MICROBIAL., vol. 4, 1958, pages 272 - 282
VOGEL H.J.; MACLELLAN W.L., METHODS ENZYMOL., vol. 17A, 1970, pages 265 - 269
WHITE T.J. ET AL., TRENDS GENET., vol. 5, 1989, pages 185 - 189
YU D. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 97, no. 12, 2000, pages 5978 - 83
ZHANG S.P. ET AL., GENE, vol. 105, no. 1, 1991, pages 61 - 72
ZHANG Y. ET AL., NATURE GENET., vol. 20, 1998, pages 123 - 128

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